Melanins and Melanosomes: Biosynthesis, Biogenesis, Physiological, and Pathological Functions

Edited by Jan Borovanský and Patrick A. Riley Melanins and Melanosomes

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Edited by Jan Borovanský and Patrick A. Riley

Melanins and Melanosomes Biosynthesis, Biogenesis, Physiological, and Pathological Functions

The Editors Prof. Patrick A. Riley Em. Prof. of Cell Pathology University College London Grange Avenue London N20 8AB United Kingdom Prof. Jan Borovanský Charles University 1st Faculty of Medicine Institute of Biochemistry and Experimental Oncology U Nemocnice 5 128 53 Praha 2 Czech Republic Cover The cover design is intended to reflect the two principal elements of the book: melanins and melanosomes. The melanins are represented by the graduated background pigment spanning the range of colours from essentially black eumelanin, through reddish to paler shades, representing pheomelanin. The celluar organelles responsible for the production, containment and distribution of melanins, the melanosomes, are represented by symbolic images based on their electron microscopic appearance.

Limit of Liability/Disclaimer of Warranty: While the publisher and authors have used their best efforts in preparing this book, they make no representations or warranties with respect to the accuracy or completeness of the contents of this book and specifically disclaim any implied warranties of merchantability or fitness for a particular purpose. No warranty can be created or extended by sales representatives or written sales materials. The Advice and strategies contained herein may not be suitable for your situation. You should consult with a professional where appropriate. Neither the publisher nor authors shall be liable for any loss of profit or any other commercial damages, including but not limited to special, incidental, consequential, or other damages. Library of Congress Card No.: applied for British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library. Bibliographic information published by the Deutsche Nationalbibliothek The Deutsche Nationalbibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data are available on the Internet at . © 2011 Wiley-VCH Verlag & Co. KGaA, Boschstr. 12, 69469 Weinheim, Germany Wiley-Blackwell is an imprint of John Wiley & Sons, formed by the merger of Wiley’s global Scientific, Technical, and Medical business with Blackwell Publishing. All rights reserved (including those of translation into other languages). No part of this book may be reproduced in any form – by photoprinting, microfilm, or any other means – nor transmitted or translated into a machine language without written permission from the publishers. Registered names, trademarks, etc. used in this book, even when not specifically marked as such, are not to be considered unprotected by law. Composition Toppan Best-set Premedia Ltd., Hong Kong Printing and Binding Cover Design Grafik-Design Schulz, Fußgönheim Printed in the Federal Republic of Germany Printed on acid-free paper ISBN: 978-3-527-32892-5 ISBN oBook: 978-3-527-63615-0 ISBN ePDF: 978-3-527-63614-3

V

Dedication “All nature is but art, unknown to thee; All chance, direction which thou canst not see” Alexander Pope: An Essay on Man Were we to ascribe to chance the existence of this volume we should have to begin with the moment in July 1952 when Professor A.F. Richter, Head of the Second Institute of Medical Chemistry at Charles University in Prague, opened a dust-covered cabinet from which he took, apparently at random, a bottle containing a dark powder and handed it to a young assistant with the words: “Young man, study the contents of this flask.” The assistant was Jiri Duchon and the label on the flask read: “Human melanosarcoma, prepared by H. Waelsch.” Jiri Duchon was born on 27 July 1927, the only son of an eminent scientist. On graduating in medicine in 1952 he joined Richter’s laboratory and his careful analysis of the sample given to him set him upon the course of studies that were to occupy him for the rest of his life. He defended his PhD thesis in 1962 on the topic of “Urinary melanogens in melanoma” and he subsequently made many important contributions to quantitative analysis of the products of melanogenesis. In recognition of his early work, Jiri Duchon was awarded a Roosevelt Fellowship that enabled him to spend 15 months at Harvard in the laboratory of T.B. Fitzpatrick. This was in 1967–1968 when he met and established a friendship with Makoto Seiji who had just developed the methods for melanosome isolation. On his return to Prague, Jiri Duchon set about improving the isolation technique and analyzing these newly discovered organelles. Under his direction and inspiration the Prague laboratory became the leading European center for the detailed biochemical investigation of melanosomes. Jiri Duchon was Head of the Institute for 26 years and many of his collaborators have continued to contribute significantly to the field of study that he promoted. Professor Duchon was an internationally recognized and highly respected member of the pigment cell fraternity, and was elected an Honorary Member of the European Society for Pigment Cell Research in 1998. It was partly in his honor that the scientific session on “Melanin and Melanosomes” was arranged at the Federation of European Biochemical Societies (FEBS) Congress in 2009, but

VI

Dedication

tragically he was taken ill on the very day of the Symposium. He was full of encouragement for the project that grew out of the meeting, namely that of publishing a definitive volume devoted to the subject of his academic endeavors, but died on 2 November 2009, long before it was completed. In recognition of his seminal role in the events that led to the production of this book we dedicate this volume to Jiri Duchon with affectionate remembrance of a fine scientist, an inspirational teacher, a kindly and cultivated companion, and a true friend.

Professor Jiri Duchon MD, PhD, DrSc (1927–2009) (Photograph by K. Meister)

VII

Contents

Dedication V Preface XV List of Contributors 1 1.1 1.2 1.3 1.3.1 1.3.2 1.3.3 1.4 1.5

2 2.1 2.2 2.2.1 2.2.1.1 2.2.1.2 2.2.1.3 2.2.2 2.2.2.1 2.2.2.2 2.2.2.3 2.2.2.4 2.2.2.5 2.3

XIX

History of Melanosome Research 1 Jan Borovanský Introduction 1 Melanosome Research in the Pre-Seiji Era 1 Melanosome Research in the Seiji Era 5 Terminology of Melanosomes 5 Ultrastructural and Histochemical Studies 6 Biochemical Studies 7 Melanosome Research in the Post-Seiji Era 9 Other Historical Aspects 11 Acknowledgments 12 References 13 Classical and Nonclassical Melanocytes in Vertebrates 21 Sophie Colombo, Irina Berlin, Véronique Delmas, and Lionel Larue Definition of Melanogenic Cells 21 Distribution and Function of Melanogenic Cells 24 Classical Melanocytes 25 Melanocytes in the Epidermis 25 Melanocytes in the Dermis 27 Melanophores in Lower Vertebrates 28 Nonclassical Melanocytes 28 Melanocytes of the Eye 28 Melanocytes of the Inner Ear 31 Melanocytes of the Heart 33 Melanocytes of the Brain and Neuromelanins 36 Melanin in Adipose Tissue 37 Embryonic Development of Melanogenic Cells 37

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2.3.1 2.3.1.1 2.3.1.2 2.3.2 2.3.2.1 2.3.2.2 2.3.2.3 2.3.2.4 2.4 2.4.1 2.4.2 2.4.2.1 2.4.2.2

3 3.1 3.1.1 3.1.2 3.1.3 3.1.4 3.1.5 3.2 3.2.1 3.2.2 3.2.3 3.2.4 3.2.5 3.2.6 3.3 3.3.1 3.3.1.1 3.3.2 3.3.3 3.3.4 3.3.5

4 4.1 4.2

Classical Melanocytes 38 Early Determined Melanoblasts: The Dorsolateral Pathway 38 Late Determined Melanoblasts: A Common Origin with SCPs and the Dorsoventral Migratory Pathway 40 Nonclassical Melanocytes 41 Melanocytes of the Murine Eye 42 Melanocytes of the Murine Heart 44 Other Nonclassical Murine Melanocytes 45 Other Organisms 45 Transfer of Melanin from Classical and Nonclassical Melanocytes 45 Melanosome Transport 46 Melanosome Transfer 46 Melanosome Transfer from Classical Melanocytes 47 Transfer of Melanin from Nonclassical Melanocytes 51 References 52 Biological Chemistry of o-Quinones 63 Patrick A. Riley, Christopher A. Ramsden, and Edward J. Land General Biological Significance of o-Quinones 63 Antibiosis 63 Defensive Secretions 64 Balanid Adhesion 64 Cuticular Hardening in Insects 65 Pigmentation 65 o-Quinone Reactivity 66 Structure and Reactivity 66 Reduction 68 Addition Reactions: Intermolecular addition 71 Polymerization 71 Intramolecular Addition (Cyclization) 72 Addition–Elimination (Substitution) Reactions 73 Role of o-Quinones in Melanogenesis 74 Nonenzymatic Formation of Melanogenic Intermediates 74 Contributions from Pulse Radiolysis to the Chemistry of Eumelanogenesis and Pheomelanogenesis 74 Balance between Eumelanogenesis and Pheomelanogenesis 78 Control of Melanogenesis: Phase I Melanogenesis 78 Tyrosinase Activation 78 Tyrosinase Inactivation 79 References 83 Biosynthesis of Melanins 87 José Carlos García-Borrón and M. Concepción Olivares Sánchez Introduction 87 Raper–Mason Pathway 88

Contents

4.2.1 4.2.2 4.2.3 4.3 4.3.1 4.3.2 4.3.2.1 4.3.2.2 4.3.3 4.3.4 4.3.5 4.4 4.4.1 4.4.2 4.4.2.1 4.4.2.2 4.5

5 5.1 5.1.1 5.1.1.1 5.1.1.2 5.1.1.3 5.1.2 5.1.3 5.2 5.2.1 5.2.1.1 5.2.1.2 5.2.1.3 5.2.2 5.2.2.1 5.2.2.2 5.2.2.3 5.2.2.4 5.2.2.5 5.2.3

Phase I Melanogenesis: The Proximal Raper–Mason Pathway – From L-tyrosine to L-dopachrome 88 Distal Melanogenic Steps: From l-Dopachrome to Eumelanins Biosynthesis of Pheomelanins 91 Structural and Functional Properties of the Melanogenic Enzymes 92 Structure of Tyrosinase and Related Proteins 92 Catalytic Cycle of Tyrosinase 95 Cresolase (Tyrosine Hydroxylase) Reaction Cycle 96 Catecholase (Dopa Oxidase) Reaction Cycle 98 Dct/Tyrp2 98 Tyrp1 100 Other Melanosomal Proteins 101 Regulation of the Melanogenic Pathway 102 Eumelanogenesis versus Pheomelanogenesis: Regulation of the Type of Melanin Pigments 102 Regulation of the Amount of Pigment 104 Regulation of Tyrosinase Levels 104 Control of Tyrosinase-Specific Activity 106 Conclusions and Perspectives 107 Acknowledgments 109 References 109

90

Inhibitors and Enhancers of Melanogenesis 117 Alain Taïeb, Muriel Cario-André, Stefania Briganti, and Mauro Picardo Introduction 117 Melanin Biochemistry 118 Melanin Biosynthesis 118 Tyrosinase Maturation and Degradation 118 Catalytic Site 119 Paracrine Signaling and Regulation of Epidermal Melanogenesis 119 Methods of Study 120 Depigmenting Agents 121 Agents Acting Prior to Melanin Synthesis 121 Transcriptional Inhibition of Melanogenic Enzymes 121 Post-Translational Modification of Melanogenic Enzymes 128 Increased Tyrosinase Ubiquination 129 Agents Acting During Melanin Synthesis 129 Interference with Tyrosinase 129 TRP-2 Modulation 136 Interference with Byproduct Production (Antioxidant and Reducing Agents) 136 Interference with the Melanogenic Pathway 138 Peroxidase Inhibitors 139 Agents Acting After Melanin Synthesis 139

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X

Contents

5.2.3.1 5.2.3.2 5.3 5.3.1 5.3.1.1 5.3.1.2 5.3.2 5.3.2.1 5.3.2.2 5.3.2.3 5.3.2.4 5.3.2.5 5.3.2.6 5.3.2.7

Inhibitors of Melanosome Transfer 139 Acceleration of Epidermal Turnover 141 Enhancers of Melanogenesis 143 Activation Through Receptor Mechanisms 144 Melanotropic Peptides 144 Cytokines and Growth Factors 145 Non-Receptor-Mediated Activation 147 Forskolin and cAMP 147 Oligonucleotides and p53 Activation 147 Piperin 147 Lipids (Sphingolipids and Prostaglandins) 147 Phospholipase A2 148 PPAR Activators 148 Psoralens and Photosensitizing Agents 148 References 149

6

Structure of Melanins 167 Shosuke Ito, Kazumasa Wakamatsu, Marco d’Ischia, Alessandra Napolitano, and Alessandro Pezzella Introduction 167 Classification and General Properties of Melanins 168 Biosynthetic Studies 169 Early Stages of Melanogenesis 169 Late Stages of Eumelanogenesis 171 Late Stages of Pheomelanogenesis 174 Concept of Mixed Melanogenesis 175 Degradative Studies 176 Eumelanins 176 Pheomelanins 178 Analysis of Eumelanins and Pheomelanins 180 Conclusions 180 References 181

6.1 6.2 6.3 6.3.1 6.3.2 6.3.3 6.3.4 6.4 6.4.1 6.4.2 6.5 6.6

7 7.1 7.2 7.3 7.3.1 7.3.2 7.3.3 7.4 7.4.1 7.4.2 7.4.2.1

Properties and Functions of Ocular Melanins and Melanosomes 187 Małgorzata Rózᠨanowska Introduction 187 Biogenesis of Ocular Melanosomes and Melanogenesis 187 Melanin Content in Pigmented Structures of the Eye 190 Melanin Content in the RPE 190 Melanin Content in the Choroid 193 Melanin Content in the Iris 193 Structure of Ocular Melanosomes 194 Morphology of Ocular Melanosomes 195 Molecular Composition of Ocular Melanosomes 196 Melanosomal Proteins 196

Contents

7.4.2.2 7.5 7.5.1 7.5.2 7.6 7.6.1 7.6.2 7.6.3 7.6.4 7.7 7.7.1 7.7.2 7.7.3 7.8 7.9

8

8.1 8.2 8.3 8.4 8.5 8.6 8.7 8.7.1 8.7.2 8.7.3 8.7.4 8.8

9 9.1 9.2

Melanosomal Lipids 197 Role of Ocular Melanin as a Broadband Optical Filter 198 Role of the Iris as a Filter of Light 198 Role of Melanin in Light Transmission Through the RPE and Choroid 200 Antioxidant Properties of Ocular Melanin 201 Scavenging of Free Radicals 201 Quenching of Electronically Excited States of Photosensitizers and Singlet Oxygen 203 Sequestration of Redox-Active Metal Ions 206 Testing Protective Effects of Ocular Melanin in Cultured Cells 207 Pro-Oxidant Properties of Ocular Melanosomes 209 Generation of ROS and Oxidation of Cellular Reductants 210 Pro-Oxidant Effects of Interactions of Melanosomes with Metal Ions 213 Cytotoxic Properties of Aged RPE Melanin Granules and Their Potential Consequences for Retinal Aging and AMD 214 Other Properties of Ocular Melanosomes and Their Implications 216 Conclusions 217 References 218 Biological Role of Neuromelanin in the Human Brain and Its Importance in Parkinson’s Disease 225 Kay L. Double, Wakako Maruyama, Makoko Naoi, Manfred Gerlach, and Peter Riederer What are Neuromelanins? 225 Phylogenetic Development of Neuromelanin 227 Development and Metabolism of Neuromelanin 227 Structure of Neuromelanin 231 Biological Role of Neuromelanin in the Human Brain 232 Is Neuromelanin Involved in Neurological Disease? 233 Effects of Neuromelanin In Vitro and In Vivo 235 Mechanisms of Neuromelanin Cytotoxicity 235 Neuromelanin Effects on Mitochondrial Function 237 Neuromelanin Effects on the UPS 239 Comparison of the Cytotoxicity of Neuromelanin with Synthetic DA-M 239 Conclusions 241 Acknowledgments 241 References 241 Biogenesis of Melanosomes 247 Cédric Delevoye, Francesca Giordano, Michael S. Marks, and Graça Raposo Introduction 247 Melanosomes: Intracellular Organelles Specialized in Melanin Synthesis 249

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9.2.1 9.2.2 9.3 9.3.1 9.3.2 9.3.3 9.3.3.1 9.3.3.2 9.3.3.3 9.3.3.4 9.3.4 9.3.5 9.3.5.1 9.3.5.2 9.3.5.3 9.3.5.4 9.4 9.4.1 9.4.2 9.4.2.1 9.4.2.2 9.4.3 9.4.4 9.4.5 9.5

10 10.1 10.2 10.2.1 10.2.2 10.2.3 10.3 10.3.1 10.3.1.1 10.3.2 10.3.2.1 10.3.2.2

Melanosomes Are Unique Organelles That Develop through Different Stages 249 Melanosomal Components 251 Endocytic System and Formation of Melanosomes 254 Organelles of the Endocytic Pathway 254 Melanosomes Are LROs but Are Distinct from Lysosomes 256 Pmel17 and Generation of Early-Stage Melanosomes 259 Pmel17 Structure 259 Pmel17 Forms the Fibrillar Matrix upon Which Melanins Deposit 259 Pmel17 Biosynthesis and Amyloid Formation 260 Functional Importance of Fibrillar Melanosomes 262 OA Type 1 and Melanosome Biogenesis 263 Origin of the Melanosome 263 Early-Stage Melanosomes Originate within the Endocytic Pathway 263 Melanosomes Do Not Originate from the ER 265 Melanosomes Segregate from the Endocytic Pathway beyond Stage I Melanosomes 266 Components of Mature Melanosomes Are Sorted from Distinct Endosomal Intermediates 267 Melanosome Maturation: Cargo Sorting to Mature Melanosomes 269 Griscelli Syndrome and CHS 269 HPS 271 Adaptor Protein (AP) Complexes 271 BLOC Complexes 273 Molecular Motors and the Cytoskeleton 276 SNAREs, Rabs, and Other Regulators 277 Lipids 279 Conclusions 279 Acknowledgments 280 References 281 Transport and Distribution of Melanosomes 295 Mireille Van Gele and Jo Lambert Introduction 295 Model Systems to Study Pigment Transport 296 Melanophores from Fish and Amphibians 296 Mammalian Melanocytes 298 RPE Cells 299 Intracellular Melanosome Transport 299 Microtubule-Based transport 300 Kinesin and Dynein 300 Actin-Based Transport 301 MYO5A 301 RAB27A 302

Contents

10.3.2.3 10.3.2.4 10.3.2.5 10.4 10.5 10.5.1 10.5.1.1 10.5.1.2 10.5.1.3 10.5.2 10.5.2.1 10.5.2.2 10.6 10.7

11 11.1 11.2 11.3 11.4 11.4.1 11.4.2 11.4.3 11.5 11.6 11.7 11.7.1 11.7.2 11.7.3 11.8

12 12.1 12.2

MLPH 303 RAB27A–MLPH–MYO5A Tripartite Protein Complex 304 RAB27A as a New MITF Target Gene 306 Melanosome Motility in RPE: The Rab27a–Myrip–Myo7a Tripartite Complex 307 Melanosome Transfer 309 Modes of Transfer 309 Cytophagocytosis 309 Exocytosis 310 Filopodial-Phagocytosis Model 311 Molecular Players 312 PAR-2 and KGF 312 Adhesion Molecules: Cadherins and Lectins 313 Fate of Melanin in the Keratinocyte 313 Conclusions 315 Acknowledgments 316 References 316 Genetics of Melanosome Structure and Function 323 Vincent J. Hearing Introduction 323 Genes Involved in Melanoblast Development, Migration, and Specification 324 Genes Involved in Melanocyte Differentiation, Survival, and Proliferation 325 Genes Involved in Regulating Melanocyte Function 327 Regulation of Constitutive Skin, Hair, and Eye Color 330 Hypopigmentation 332 Hyperpigmentation 332 Genes Involved in the Biogenesis of Melanosomes and Other Lysosome-Related Organelles 333 Genes Involved in Melanin Production 334 Genes Involved in Melanosome Movement, Transfer, and Distribution 336 Movement 336 Transfer 337 Distribution 337 Conclusions 338 References 338 Physiological and Pathological Functions of Melanosomes 343 Jan Borovanský and Patrick A. Riley Tissue Concentration of Melanosomes 343 Melanosome Properties and Functions Are Determined by Their Chemical Composition 344

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XIV

Contents

12.3 12.4 12.4.1 12.4.2 12.4.3 12.4.4 12.5 12.5.1 12.5.2 12.5.3 12.6 12.7 12.7.1 12.7.2 12.7.3 12.8 12.9

13 13.1 13.1.1 13.1.2 13.2 13.3

13.4

13.5 13.6 13.7

Functional Microanatomy of the Melanosome 346 Melanosomes as Centers of Free Radical Activity 350 Free Radical Nature of Melanins 350 Radicals and Reactive Species Associated with Melanogenesis 352 Possible Role of Protein-Bound Dopa 355 Melanosomes as a Therapeutic Target 356 Melanosomes as Energy Transducers 358 Photon/Phonon Conversion 359 Photochemical Reactions 360 Sound/Heat Conversion 360 Melanosomes and Metal Ions 360 Affinity of Melanosomes for Polycyclic and Other Compounds 364 Melanoma Detection and Treatment 365 Participation of Melanosomes in Chemoresistance 366 Long-Term Deposition of Compounds in Melanosomes 367 Exploitation of Melanosomal Proteins and Melanin as Specific Targets in Melanoma Therapy 368 Conclusions 370 Acknowledgments 370 References 371 Dysplastic Nevi as Precursor Melanoma Lesions 383 Stanislav Pavel, Nico P.M. Smit, and Karel Pizinger Nevi as Risk Factors for Melanoma 383 Development of Melanocytic Nevi 383 Description of Dysplastic Nevi 384 Dysplastic Nevi as Precursor Lesions of Melanoma 384 Cytological Differences between Normal Skin Melanocytes and Dysplastic Nevus Cells: Melanosomal and Mitochondrial Aberrations 386 Metabolic Differences between Normal Skin Melanocytes and Dysplastic Nevus Cells: Preference for Pheomelanogenesis in Dysplastic Nevus Cells 387 Pheomelanogenesis as a Possible Cause of Intracellular Oxidative Imbalance 388 Dysplastic Nevus Cells as Senescent Cells 389 Are Dysplastic Nevus Cells a Class of Cells Exhibiting a Mutator Phenotype? 389 References 391 Index 395

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Preface “To that small part of ignorance that we arrange and classify we give the name knowledge” Ambrose Bierce This book is entitled Melanin and Melanosomes, and is about pigment and pigmentation. It is important, however, that we bear in mind that, while the primary function of melanocytes is the production of pigment in melanosomes, these cells have other attributes and perform other significant functions. Some of these are well recognized, such as the involvement of the retinal pigment epithelium in photoreceptor physiology (detailed in Chapter 7). Another interesting possibility is that melanogenesis may be the source of some of the substrate for dopamine synthesis [1], and melanocytes may have other important neuroendocrine functions as pro-propriomelanocortin processing cells and a source of prostaglandin D synthase (reviewed by Takeda et al. [2]). Some of these actions may go some way to explaining the remarkable anatomical distribution of melanocytes, often in locations that are not illuminated, such as the leptomeninges. However, this volume is devoted to melanin and melanosomes, and is primarily concerned with vertebrate, especially human, pigmentation. We include melanin that is formed by oxidative processes that are enzymatically catalyzed in specialized cells, both the neural crest-derived dendritic melanocytes (named “classical” melanocytes in Chapter 2) and optic cup-derived retinal pigment epithelial cells (“nonclassical” melanocytes), as well as melanin generated by other oxidative pathways, such as the neuromelanin of the midbrain. The importance of this latter pigment, particularly in relation to Parkinson’s disease, is set out in Chapter 8. The enzymatically generated melanin in vertebrates is synthesized and deposited in specialized intracellular organelles, the melanosomes, and this book concentrates on the many aspects of the formation and functions of these organelles. The melanosome is a highly specialized organelle, the history of which owes much to the early work at Charles University under the aegis of Jiri Duchon (1927–2009), to whom this book is dedicated. Many of the important properties of melanosomes were established in Prague in the early 1970s by a series of investigations on isolated and purified preparations of this organelle, and investigators at Charles University have continued to

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Preface

contribute significantly to the advancement of this field, and to follow the developments that have taken place in elucidating the structure of melanosomes and the complex biological roles in which they are implicated. This volume grew from a combination of auspicious factors. In 2009, the 34th Congress of the Federation of European Biochemical Societies (FEBS) was organized in Prague. Naturally, with one of us (J.B.) on the Organizing Committee, one of the scientific sessions was devoted to melanosomes and, in the wake of the discussions at this meeting, it was felt that there was a significant body of new data relating to melanosomes that could usefully be assembled in a volume devoted exclusively to this organelle. It was hoped that such an overview, by integrating the diverse aspects of current knowledge, might help to generate a new understanding of the biological role of melanosomes and stimulate novel research effort in this interesting area of study. We have been fortunate in our publisher, Wiley-VCH, who recognized the timely nature of the proposed volume, and we thank our commissioning editor, Gregor Cicchetti, and his team, especially Anne Chassin du Guerny, for their help and encouragement in bringing the project to fruition. Of course, our main thanks go to our panel of distinguished international contributors who have generously given of their time and expertise in preparing the chapters that we hope form a coherent picture of the up-to-date knowledge in the field. Last, but not least, this book celebrates a long and fruitful collaboration between the Editors involving many visits between Charles University and University College London. It is a pleasure to acknowledge the assistance of the British Council in enabling these exchanges. We had hoped initially to have the opportunity to arrange the order of the chapters in the light of their ultimate content so that overlapping areas were most rationally ordered to enable the volume to be read more or less in sequence while allowing the, perforce abundant, cross-references to act as a secondary web in a cohesive network. However, pressure of time prevented us from completing this task and readers may find it more convenient to skip between the various contributions according to their interests and predilections. In principle, although the topics are inextricably intertwined, we have elected to place the contributions devoted to melanin – its biosynthesis, chemistry, and properties – at the front of the book, and those dealing with melanosomes – their structure, biogenesis, distribution, and properties – in the following chapters. The topic is put into chronological context by a historical Introduction in Chapter 1, in which Jan Borovanský traces the steps in the discovery of the melanosome, illustrated by portraits of the important investigators that took part in these exciting early studies. As this book is directed largely at aspects of human pigmentation, Chapter 2 consists of a detailed overview by Sophie Colombo, Irina Berlin, Véronique Delmas, and Lionel Larue of the specialized cells in vertebrates in which melanin production in melanosomes takes place. In their contribution a distinction is made between “classical” and “nonclassical” melanocytes. Chapter 3, by Patrick Riley,

Preface

Christopher Ramsden, and Edward Land, emphasizes the central role of the generation and reactivity of o-quinones in melanogenesis, and is followed by Chapter 4 in which the biosynthesis of melanins is reviewed by José Carlos Garcia-Borrón and Conchita Olivares Sánchez. Chapter 5, by Alain Taïeb, Muriel Cario-André, Stefania Briganti, and Mauro Picardo, comprises an analysis of inhibitors and enhancers of melanogenesis. The current understanding of the structure of melanins is then reviewed in Chapter 6 by Shosuke Ito, Kasumasa Wakamatsu, Marco d’Ischia, Alessandra Napolitano, and Alessandro Pezzella, and this is followed in Chapter 7 by a description of the properties and functions of ocular melanins and melanosomes by Małgorzata Różanowska. Chapter 8, by Kay Double, Wakako Maruyama, Makako Naoi, Manfred Gerlach, and Peter Riederer, is devoted to the biological role of neuromelanin in the human brain and its importance in Parkinson’s disease. Chapter 9 consists of a detailed review of the biogenesis of melanosomes by Cédric Delevoye, Francesca Giordano, Michael Marks, and Graça Raposo. This is followed in Chapter 10, by Mireille Van Gele and Jo Lambert, by a description of the transport and distribution of melanosomes. The genetics of melanosome structure and function are skillfully summarized in Chapter 11 by Vincent Hearing. Chapter 12, by Jan Borovanský and Patrick Riley, is devoted to the properties and functions of melanosomes, and, in Chapter 13, the abnormalities of melanosomes and melanogenesis in melanoma precursor lesions are discussed by Stan Pavel, Nico Smit, and Karel Pizinger. We firmly believe that this compilation of expertise embodies a significant work of scholarship, and we sincerely hope that the combined wisdom embraced by this volume conveys both the breadth of detailed and exciting knowledge that currently exists about melanin and melanosomes, and also reveals those shadowed areas of doubt and ignorance that await illumination in the future. March 2011

Patrick A. Riley Jan Borovanský

References 1 Eisenhofer, G., Tian, H., Holmes, C.,

Matsunaga, J., Roffler-Tarlov, S., and Hearing, V.J. (2003) Tyrosinase: a developmentally specific major determinant of peripheral dopamine. FASEB J., 17, 1248–1255.

2 Takeda, K., Takahashi, N.-H., and

Shibahara, S. (2007) Neuroendocrine functions of melanocytes: beyond the skin-deep melanin maker. Tohoku J. Exp. Med., 211, 201–221.

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List of Contributors Irina Berlin Institut Curie Developmental Genetics of Melanocytes, INSERM U1021–CNRS UMR3347 Centre Universitaire 91405 Orsay France

Sophie Colombo Institut Curie Developmental Genetics of Melanocytes, INSERM U1021–CNRS UMR3347 Centre Universitaire 91405 Orsay France

Jan Borovanský Charles University First Faculty of Medicine, Institute of Biochemistry and Experimental Oncology U nemocnice 5 128 53 Prague 2 Czech Republic

Marco d’Ischia University of Naples “Federico II” Department of Organic Chemistry and Biochemistry Via Cinthia 4 80126 Naples Italy

Stefania Briganti San Gallicano Dermatological Institute Laboratory of Cutaneous Physiopathology Elio Chianesi 53 00144 Rome Italy Muriel Cario-André Université de Bordeaux INSERM U1035 146 rue Léo Saignat 33076 Bordeaux France

Cédric Delevoye Institut Curie Centre de Recherche Structure and Membrane Compartments 26 Rue d’Ulm 75248 Paris cedex 05 France Véronique Delmas Institut Curie Developmental Genetics of Melanocytes, INSERM U1021–CNRS UMR3347 Centre Universitaire 91405 Orsay France

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List of Contributors

Kay L. Double University of New South Wales Neuroscience Research Australia Barker Street Sydney, NSW 2031 Australia

Jo Lambert Ghent University Hospital Department of Dermatology De Pintelaan 185 9000 Ghent Belgium

José Carlos García-Borrón University of Murcia Department of Biochemistry and Molecular Biology Campus de Espinardo 30100 Murcia Spain

Edward J. Land Keele University School of Physical and Geographical Sciences, Lennard-Jones Laboratories Keele Road Keele ST5 5BG UK

Manfred Gerlach University of Würzburg Clinical Neurobiology, Department of Child and Adolescence Psychiatry, Psychosomatics and Psychotherapy Füchsleinstrasse 15 97080 Würzburg Germany

Lionel Larue Institut Curie Developmental Genetics of Melanocytes, INSERM U1021–CNRS UMR3347 Centre Universitaire, 91405 Orsay France

Francesca Giordano Institut Curie Centre de Recherche Structure and Membrane Compartments 26 Rue d’Ulm 75248 Paris cedex 05 France Vincent J. Hearing National Institutes of Health Laboratory of Cell Biology 37 Convent Drive Bethesda, MD 20892 USA Shosuke Ito Fujita Health University School of Health Sciences Department of Chemistry Toyoake Aichi 470-1192 Japan

Michael S. Marks University of Pennsylvania Departments of Pathology and Laboratory Medicine and Physiology, 513 Stellar-Chance Laboratories 422 Curie Boulevard Philadelphia, PA 19104-6100 USA Wakako Maruyama National Research Center for Geriatrics and Gerontology Department of Cognitive Brain Science Obu Aichi 474-8511 Japan

List of Contributors

Makoko Naoi Gifu International Institute of Biotechnology Department of Neurosciences Kakamigahara Gifu 504-0838 Japan

Mauro Picardo San Gallicano Dermatological Institute Laboratory of Cutaneous Physiopathology Elio Chianesi 53 00144 Rome Italy

Alessandra Napolitano University of Naples “Federico II” Department of Organic Chemistry and Biochemistry Via Cinthia 4 80126 Naples Italy

Karel Pizinger Charles University Department of Dermatology Faculty of Medicine Husova 3 306 05 Pilsen Czech Republic

M. Concepción Olivares Sánchez University of Murcia Department of Biochemistry and Molecular Biology Campus de Espinardo 30100 Murcia Spain

Christopher A. Ramsden Keele University School of Physical and Geographical Sciences, Lennard-Jones Laboratories Keele Road Keele ST5 5BG UK

Stanislav Pavel Leiden University Medical Center Department of Dermatology PO Box 9600 2300 RC Leiden The Netherlands and Charles University Department of Dermatology, Faculty of Medicine Husova 3 306 05 Pilsen Czech Republic

Graça Raposo Institut Curie Centre de Recherche Structure and Membrane Compartments 26 Rue d’Ulm 75248 Paris cedex 05 France

Alessandro Pezzella University of Naples “Federico II” Department of Organic Chemistry and Biochemistry Via Cinthia 4 80126 Naples Italy

Peter Riederer University of Würzburg Clinical Neurochemistry, Department of Psychiatry, Psychosomatics and Psychotherapy, and National Parkinson Foundation Centers of Excellence for Neurodegenerative Diseases Research Füchsleinstrasse 15 97080 Würzburg Germany

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Patrick A. Riley Totteridge Institute for Advanced Studies The Grange Grange Avenue London N20 8AB UK Małgorzata Róz·anowska Cardiff University School of Optometry and Vision Science Maindy Road Cardiff CF24 4LU UK Nico P.M. Smit Leiden University Medical Center Central Laboratory for Clinical Chemistry Albinusdreef 2 2333 AC Leiden The Netherlands

Alain Taïeb Hôpital St André Department of Dermatology and Pediatric Dermatology CHU de Bordeaux 1 rue Jean Burguet 33077 Bordeaux France Mireille Van Gele Ghent University Hospital Department of Dermatology De Pintelaan 185 9000 Ghent Belgium Kazumasa Wakamatsu Fujita Health University School of Health Sciences Department of Chemistry Toyoake Aichi 470-1192 Japan

1

1 History of Melanosome Research Jan Borovanský

1.1 Introduction

Melanosomes were first proposed as specific organelles, unique to pigment cells, in a preliminary publication that appeared on 30 July 1960 [1]. An announcement had been made at the 21st Annual Meeting of the Society for Investigative Dermatology, at Miami Beach, Florida, USA on 13 June 1960 [2] and the news, that the chemical composition and enzyme activities in melanosomes and mitochondria are completely different, was considered to be of such significance that it appeared in a newspaper report (Figure 1.1). Similar data, with an emphasis on terminology, were published in 1963 [3]. This advance was the result of collaborative work between M. Seiji (1926–1982), at that time working at the Department of Dermatology, Harvard Medical School in Boston under the leadership of T.B. Fitzpatrick (1919–2003) (Figure 1.2), and H. Blaschko and M.S.C. Birbeck, with whom Dr Fitzpatrick established scientific cooperation during his tenure of a Commonwealth Fellowship at the Department of Biochemistry, Radcliffe Infirmary in Oxford. The history of melanosome research can be formally divided into three parts: (i) the pre-Seiji era (prior to 1960), (i) the Seiji era (1960–1982), and (iii) the postSeiji era (1983–).

1.2 Melanosome Research in the Pre-Seiji Era

The first description of mammalian pigment cells was published by Gustav Simon in 1841 [4] who observed round and stellate pigment cells in the hair bulbs of pig embryos. It was preceeded in 1838 by Purkynĕ’s description of pigment in the cells of the substantia nigra, which not only drew attention to pigment granules, but also noted the rise in their numbers with age [5]. We have to admire these early reports because their authors, armed only with primitive light microscopes, were able to ascertain that melanin was not diffusely distributed in the cytoplasm Melanins and Melanosomes: Biosynthesis, Biogenesis, Physiological, and Pathological Functions, First Edition. Edited by Jan Borovanský and Patrick A. Riley. © 2011 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2011 by Wiley-VCH Verlag GmbH & Co. KGaA.

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1 History of Melanosome Research

Figure 1.1 Announcement of the independent status of melanosome in Medical News on 5

July 1960.

of pigmented cells, but was present in the form of discrete aggregates [5, 6] (Figures 1.3 and 1.4). Deciphering the old literature is problematical as authors often fail to distinguish between melanin (the pigment itself), melanoprotein (the natural melanin– protein complex), and melanin granules (the subcellular organelle). If the method of separation is not adequately described, it is difficult to be certain what material was studied and any conclusions can be misleading [8]. The lack of electron microscopic identification of isolated material led to many misinterpretations; for example, the “melanopseudoglobulin” studied by Greenstein et al. [9] was later shown to be melanosomes [10] and Bolt’s “melanoprotein” [11], widely used in biophysical studies, turned out to consist of damaged melanosomes [12]. Mason et al. [10] posed the question of whether melanin granules were particles with a specific structure or consisted of random aggregates of precipitated metabolic

1.2 Melanosome Research in the Pre-Seiji Era

Figure 1.2

Professor Makoto Seiji (left) and Professor Thomas B. Fitzpatrick (right) in 1972.

Figure 1.3

“Chromatophore” from donkey conjuctiva [7].

products. The introduction of electron microscopy was able to resolve this matter and Laxer et al. [13] were able to discern an inner ultrastructure in isolated melanosomes. The first clear pictures were obtained only in 1956 [14]. An avalanche of papers in subsequent years brought with it enormous amounts of information on the ultrastructure of melanosomes and its changes during

3

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1 History of Melanosome Research

Figure 1.4 Cells of substantia nigra containing neuromelanin [5].

melanosome development (good examples are [15–17]). Other papers (reviewed in [18]) brought together ultrastructural and biochemical data that, in combination, laid the basis for the nomenclature of melanosomal ontogenesis. By comparison with the morphological data, biochemical investigations of melanosomes were more modest, mainly due to the fact that ultrastructural data were derived from studies of intact cells or tissues, whereas biochemical research used samples prepared by relatively harsh preparative procedures. These samples sometimes consisted of melanins, or altered melanosomes, or their fragments, usually without any check of their nature or homogeneity [18]. The aim of researchers in the nineteenth century was not to prepare subcellular particles or native melanoproteins, but to separate the colored pigment (“Farbstoff” = melanin in the terminology of that time). The presence of protein in the isolated material was considered an unwanted contaminant [19]. Probably the first mild separation protocol was used by J.J. Berzelius [20]. He investigated pigment (melanosomes?) obtained from eye membranes by water extraction, and noticed its insolubility in acids and limited solubility in alkali. Similar mild extraction procedures were used by Landolt [21] and Mörner [22]. The early isolation procedures were reviewed by Waelsch [23]. He studied “natural melanin” from human melanoma metastases and horse choroids, confirmed the presence of protein attached to pigment, and suggested that melanin could be synthesized from the cyclic amino acids present in the protein moiety; this idea has not been

1.3 Melanosome Research in the Seiji Era

abandoned till now. Herrmann and Boss [24] demonstrated dopa oxidase activity in the fraction of melanin granules from ciliary bodies of cattle eyes, but, as their samples were contaminated with mitochondria, they demonstrated the presence of mitochondrial enzyme markers as well. In 1949, du Buy et al. concluded that melanosomes are modified mitochondria typical of pigment cells [25]. It is interesting that du Buy [26] and other authors [27] did not abandon the mitochondrial theory of melanosome origin even in 1963 (i.e., 2 years after the formulation of Seiji’s melanosomal concept) and even published their papers in the same volume in which Seiji et al. published detailed confirmation of their model [28]. It is interesting that history has disregarded the contribution of Stein [29] who, several years before the work of Seiji et al., using a separation procedure of his own, isolated melanin granules from ox choroids and analyzed their content not only of melanin, but also lipids, carbohydrates, RNA, and metals (including the pioneer finding of a high level of zinc), and concluded that the chemical composition of melanin granules is completely different from mitochondria. The ability of melanin in melanin granules, isolated from Harding-Passey melanoma and from the ink sac of Loligo opalescens, to act as a cation exchanger [30], and the demonstration of free radical activity in melanin-containing tissues [31] also rank among the observations of the pre-Seiji era.

1.3 Melanosome Research in the Seiji Era 1.3.1 Terminology of Melanosomes

The demonstration of melanosomes as unique pigment cell organelles possessing developmental stages prompted the introduction of a system of terminology that reflected the characteristics of the various states. Until 1961 the common term for all varieties of these organelles was melanin (or pigment) granule [1, 2]. The first system of nomenclature [2] described three stages in the ontogenesis of melanosomes: i) Premelanosomes: spherical organelles. ii) Melanosomes: organelles with an internal structure and tyrosinase activity. iii) Melanin granules: melanoprotein polymer. A second terminological system was proposed [3, 26] consisting of three developmental stages plus a final product. Thus:

• • • •

Stage I (first stage): biosynthesis of protein. Stage II (intermediate stage): biosynthesis of organelle. Stage III (late phase): biosynthesis of melanin. Final product: melanin granule.

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1 History of Melanosome Research

These nomenclature systems introduced a certain degree of confusion, particularly as the term melanin granule had been used to describe pigment granules at any developmental stage. In an attempt to establish a consensus, Fitzpatrick et al. [32, 33] circulated a postal questionnaire seeking opinions about the adequacy of the terms in common use in pigment cell research and, with the approval of the participants of the Sixth International Pigment Cell Conference in 1965 in Sofia, Bulgaria, recommended the use of two terms:

•

Melanosome: a discrete melanin-containing organelle in which melanization is complete as indicated by its almost uniform density by electron microscopy and the absence of demonstrable tyrosinase activity.

•

Premelanosome: a term applied to all the stages in melanosome biogenesis that precede the fully developed state. Within the restrictions of this general definition, the premelanosomal stage might, at the discretion of the investigator, be subdivided into early, intermediate, and late phases.

The nomenclature in general use today does not adhere to any of the three systems outlined above, but is essentially a system proposed by Toda et al. [34–36] reflecting the earlier descriptions of Birbeck [37, 38] which employs the uniform term “melanosome” with a numerical indication (I–IV) of the degree its ontogenetic development. However, in practice, chaos prevails. While the system of Toda et al. is widely – if somewhat erratically – used, some European authors refer, often incorrectly, to the stages proposed in the second system of nomenclature [3, 26] and some American authors tend to cite nomenclature introduced in their previous papers or those of their friends. 1.3.2 Ultrastructural and Histochemical Studies

The concept of subcellular biosynthesis and localization of melanins and melanoproteins in melanosomes was further confirmed by (i) autoradiographic evidence with [3H]dopa and [2-14C]dopa [39–43]., (ii) incorporation of [2-14C]dopa and monitoring radioactivity in subcellular fractions [44, 45]., and (c) isolation of melanosomes and analysis of their chemical composition [46, 47]. Electron microscopy enabled the definition of the basic morphometric data of isolated melanosomes (i.e., their size, shape, and ultrastructural appearance). The most extensive data were published by Hach et al. [48, 49]. For discussion concerning the ultrastructural appearances of melanosomes, see Section 12.3 in Chapter 12. Various pathological states may be manifested by changes in melanosome morphology. Mishima et al. [50] considered that melanosome polymorphism, such as changes in size, shape, ultrastructural matrix, the manner of melanin deposition, and the degree of melanosome maturation, as a criterion of molecular pathology that could find practical use in the differential diagnosis of various pigmentary disorders.

1.3 Melanosome Research in the Seiji Era

In melanoma cells, various irregularities in the architecture of melanosomes are common. Deposition of melanin can be uneven, leading to a bizarre appearance of melanosomes [51–57]; the presence of melanosomes of all stages of development is typical [51, 52]. Melanoma melanosomes also often exhibit defects of their limiting membranes that may lead to leakage of toxic melanin precursors into the cytosol. The pathological consequences of this failure of containment of melanogenic intermediates are discussed in Section 12.4.2. Extracellular deposition of melanin on fibrils resembling melanosomal matrix fibrils has also been observed in melanoma cells [58, 59]. Early ideas on melanosomal biogenesis were summarized in several studies [52, 60, 61]. Fitzpatrick and Breathnach defined a functional unit in human epidermis named the “epidermal melanin unit.” This was viewed as a symbiotic relationship between melanocytes and keratinocytes in which each melanocyte supplies approximately 36 keratinocytes with melanosomes [62]. The mechanism of melanosome transport and transfer to keratinocytes was outlined in Mottaz and Zelickson [63]. Szabó et al. [64] demonstrated racial differences in the fate of melanosomes in human epidermis. 1.3.3 Biochemical Studies

A prerequisite for classical biochemical studies is the ability to isolate native and pure melanosomes [65]. To this end 17 isolation protocols suggested for the isolation of melanosomes between 1940 and 1973 were critically reproduced [18, 65], and it was concluded that the best samples could be obtained using the procedures described by Stein [29], Doezema [66], and Haberman and Menon [67]. Isolation of melanosomes from keratinous material turned out to be more difficult because the need to release melanosomes from hair required chemical means of tissue disintegration, which always engenders a search for a compromise between sufficient tissue disintegration and minimizing the extent of melanosome modification by the isolation procedure [61, 68]. Ten methods for isolating melanosomes from hair were critically reviewed and half of them, which from the assessment of the isolation conditions seemed to be promising, were reproduced [68]. The best results were obtained with the isolation protocol of Borovanský and Hach [69]. The availability of melanosome samples of adequate quality [18] opened the gate to the subsequent establishment of their basic chemical composition. Melanin and protein moieties were shown to be dominant constituents of melanosomes [46, 47, 61, 70–72]. Isolated melanosomes were reported to contain 5–10% of carbohydrates [29]. Tyrosinase, as a glycoprotein, also brings into melanosomes sialic acids containing N-acetylneuraminic acid [73]. In addition to gangliosides mentioned in Section 12.2, many other lipid constituents in melanosomes have been reported including cholesterol and free fatty acids [74], and phospholipids [3, 75]. The level of total lipids was found to vary between 1–5% [29] and 5–11% [76]. Jimbow et al. [77] made a complete qualitative and quantitative analysis of lipids and their

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1 History of Melanosome Research

Figure 1.5 Professor Kowichi Jimbow, a pioneer in melanosome research, in 1981.

fractions in isolated Harding-Passey and B16 melanoma melanosomes, and found quantitative differences between them (Figure 1.5). The demonstration of the absence of DNA in isolated melanosomes was clear evidence of the difference between melanosomes and mitochondria [78]. Melanosomes are abundant in various metals as described in detail in the Section 12.6. The description of the development of analytical methods including classical chemical techniques such as titration, spectrophotometry, electrochemical and isotope methods, neutron activation analysis, mass spectrometry, and inductively coupled plasma techniques, together with cell biological methods such as histochemistry, autometallography, autoradiography, and microanalytical techniques (using electron, proton, laser, X-ray, and ion beams), and their use in melanosome analyses is comprehensively covered in a review [79]. Nowadays it sounds incredible, but in the 1960s there were doubts as to whether melanosomes merely represented an association of tyrosinase with melanin or whether the organelles contained other proteins as suggested by the electron microscopic appearance. The matter was investigated by electrophoretic studies of isolated melanosomes treated with detergents. Of the many studies summarized in [61], only four used samples of melanosomes that had been checked for purity by electron microscopy [66, 80–82] and these publications unambiguously demonstrated that melanosomes contain several proteins, some of them of brown color and rapid anionic mobility on electrophoresis suggesting their melanoprotein nature [80–82]. The presence of more proteins in melanosomes was later confirmed by comparison of the amino acid composition of tyrosinase with that of melanosome hydrolysates [11]. Matrix proteins were characterized by means of sodium dodecyl sulfate–polyacrylamide gel electrophoresis in melanosomes isolated from Harding-Passey and B16 melanomas and treated with Brij-35 and

1.4 Melanosome Research in the Post-Seiji Era

guanidine hydrochloride. A simultaneous ultrastructural study revealed that treatment of melanosomes with guanidine hydrochloride induced partial degradation detectable by electron microscopy [83]. A strong stream of research represented studies aimed at demonstrating the presence of enzymes in melanosomes. Naturally, special attention was paid to the melanosomal marker enzyme – tyrosinase (EC1.14.18.1) [1–3, 84–89]. Among the common constituents of melanosomes are acid phosphatase (EC 3.1.3.2.) [90–95] and other lysosomal hydrolases, such as β-galactosidase (EC 3.2.1.23) [75], β-glucuronidase (EC 3.2.1.31) [74, 96], β-N-acetyl glucosaminidase (EC 3.2.1.30) [74], cathepsin D (EC 3.4.23.5) [74], and arylsulfatase (EC 3.1.6.1) [97]. The presence of acid phosphatase and other acid hydrolases used to be explained by adhesion of lysosomal enzymes because during isolation melanosomes are contaminated with phagosomes [90, 96] and autophagosomes [94, 97]. However, as removal of superficially bound proteins by detergents [96] or enzymes [74] did not remove the activity of lysosomal enzymes, they seemed to be integral constituents of melanosomes. Tyrosine-2-oxoglutarate amino transferase (EC 2.6.1.5) and tryptophan-2,3dioxygenase (EC1.13.11.11) were demonstrated to be a constant constituent of melanosomes from guinea pig skin [98]. The presence of ATPase (EC 3.6.1.3) is not surprising [45]. γ-Glutamyltransferase (EC 2.3.2.2) was demonstrated in melanosomes and premelanosomes of B16 melanoma cells [99]. γ-Glutamyltransferase is thought to have a role both in melanogenesis and in cellular protection against oxidative stress. Progress in melanosome research was quite rapid. Ten years after the recognition of the melanosome as a unique subcellular particle of pigment cells, the basic biological processes associated with pigmentation were shown to be related to: (i) formation of melanosomes in melanocytes, (ii) melanization of melanosomes in melanocytes, (iii) transfer of melanosomes into keratinocytes, and (iv) transport of melanosomes by keratinocytes, with or without degradation, in lysosome-like organelles [100]. These four processes were partially characterized, and biochemical knowledge of melanosomes reached a level enabling consideration of their function and the possibilities of exploiting these functions in clinical practice [101–105]. A well-balanced review on the melanosome and melanogenesis, describing the situation at the beginning of the twenty-first century, was written by Tolesson [106]. The advent of the techniques of molecular biology has still further accelerated the growth of our knowledge of melanosomes.

1.4 Melanosome Research in the Post-Seiji Era

Professor Makoto Seiji died in 1982. In recognition of his key role and fundamental achievements in melanosome research the Seiji Memorial Lectureship was established in his memory by the International Federation of Pigment Cell Societies to be given every third year at the International Pigment Cell Conferences.

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1 History of Melanosome Research

Symbolically, the first Seiji Memorial Lecture was given by Professor T.B. Fitzpatrick at the International Pigment Cell Conference in Giessen in 1983. In parallel with the “epidermal melanin unit” a “follicular melanin unit” was introduced [107]. Modern analytical techniques of high sensitivity make heavy demands on the purity of the melanosomal fractions studied. Hence, the problem of isolation has resurfaced. For the isolation of melanosomes from keratinized structures enzymatic tissue disintegration has been introduced by Arnaud and Boré [108]. However, they used preliminary treatment of hair either with dimethylsulfoxide at 120 °C or treatment of hair under reflux with an aqueous solution of lithium bromide. Such methods are in absolute contradiction to principles of denaturationfree separation. Isolation methods strongly predetermine the quality of the samples obtained, such as surface area-to-mass ratio as demonstrated by Liu and Simon [109]. In 2000, Prota et al. developed an isolation procedure based only on enzyme digestion [110]. Melanosomes of various stages could be separately isolated by inserting into the protocol a free-flow electrophoresis step [111]. Percoll gradients were also introduced into melanosome isolation [112]. Tyrosine-induced increase of melanin was shown to influence melanosomal size and shape, especially of those originating from the light skin types [113]. The list of lysosomal hydrolases was extended by the detection of cathepsin B (EC 3.4.22.1) and L (EC 3.4.22.15) [114], and α-mannosidase (EC 3.2.1.24) [99]. After the discovery of the acidic pH of melanosomes [115], the presence both of acid hydrolases and lysosome-associated membrane proteins 1, 2, and 3, and evidence of phagocytotic ability, melanosomes were designated as specialized lysosomes [116, 117] and their existence as specific organelles was endangered. The ranking of melanosomes among lysosome-related organelles [118] was the only appropriate solution, which readily explains the common participation of lysosomes and melanosomes in some pigmentary disorders such as Chediak–Higashi syndrome and Heřmanský–Pudlák syndrome. The mechanism of melanosome disintegration and degradation has been studied for a long time [119]. There are many histochemical reports of the presence of acid hydrolases, and particularly acid phosphatase, in melanosome complexes and these have been interpreted as implying their presumptive role in melanosome degradation (reviewed in [119, 120]). However, the reaction specificity of acid phosphatase consists of hydrolyzing phosphate esters and there have been no reports to suggest that phosphates play any part in maintaining the aggregation pattern of melanin or melanosome architecture. Acid hydrolases may have a role the degradation of the protein moiety of the melanosome, but melanin seems to be susceptible mainly to redox reactions [119–122] (see also Sections 12.4.1 and 12.4.2). Immunological techniques have helped a great deal in understanding melanosome structure and biogenesis. Monoclonal antibodies prepared by immunization with melanosomal proteins [123], but especially antibodies prepared by Hearing against synthetic peptides corresponding to the C-termini of melanosomal proteins, have proved to be invaluable tools in melanosome research [124]. In the post-Seiji era the contribution of molecular biological techniques has been enormous and is reflected in Chapters 2, 9, 10, and 11. The group of Professor John

1.5 Other Historical Aspects

Simon has recently introduced new sophisticated biophysical and chemical techniques into melanosome research (e.g., [109, 125]). The combined consensus of the current knowledge of melanins and melanosomes that has emerged from the many investigations briefly alluded to above constitutes the material contained within the chapters written by the leading authorities in the field that illuminate this book.

1.5 Other Historical Aspects

The author has been engaged in pigment research since 1968 and this chapter reflects his subjective preferences for the articles taking into account melanosomes as subcellular organelles. Hundreds of articles (and their authors) dealing with the investigation of processes, control factors, and molecular characteristics of melanocytes, which have no direct relation to melanosomes as functional units, can be found in other reviews. The description of pigment cell research along a time axis was monitored in a unique way by Nordlund et al. [126] and there are also articles with a geographical emphasis on pigment cell research [127, 128]. The history of melanocyte research, mentioning the first description by Sangiovanni in 1819 [129] of a pigment cell as a “chromatophore” in the squid, was summarized by Westerhof [130] and repeated by Falabella [131]. Brief historical remarks can be found in [132, 133]. Melanoproteins were first defined in 1910 [134], and studied again by Serra [135] and reviewed in [6, 136]. Since the formulation of an exact definition of specific melanosomal proteins the general term “melanoprotein” has been fading. However, the terminology reappears on occasion in descriptions of the manner of melanin attachment to proteins such as Pmel-17. Of course, the history of melanin and the development of knowledge in the field is much longer than the time since it was given its name by Berzelius in 1840 [20], and is covered in considerable depth in the books by Nicolaus [137] and Prota [138], and in several reviews (e.g., [139]). In his book, Nicolaus [137] divides the development of melanin chemistry into three periods. (i) The period of frustration, which started with the studies of Dressler and Pribram in 1856 and terminated with Raper’s fundamental work in the 1930s. (ii) The period of uncertainty 1930–?. In 1968, Nicolaus predicted that ever-increasing interest would soon lead to entry into the third period – (iii) the period of elucidation. It is undoubtedly to this era that the articles of this book belong. The ever-increasing interest in the investigation of melanosomes can be illustrated by data from the ISI Web of Knowledge (Table 1.1). Until 1960 the term melanosome on the ISI Web of Knowledge did not exist and a slow increase took place up to the period 1981–1985. The decrease in the subsequent period can be explained by the failure to use the term melanosome among the “key words” as investigators concentrated more on the molecular level. A further complication is that the term melanosome has two meanings: (i) a subcellular particle of pigment cells as described in this book and (ii) a dark region

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1 History of Melanosome Research

Figure 1.6 Melanosome – a dark region present in migmatite rocks – on the staircase of the Institute of Biochemistry and Experimental Oncology, First Faculty of Medicine, Charles University in Prague. Length = 54 mm. Table 1.1 Number of entries under the term “melanosome” in the ISI Web of Knowledge (www.isiknowledge.com).

Period (years)

No. entries

1961–1965 1966–1970 1971–1975 1976–1980 1981–1985 1986–1990 1991–1995 1996–2000 2001–2005 2006–2010

4 13 26 29 49 33 131 167 289 343

present in migmatite rocks [140] (Figure 1.6). The entries in Table 1.1 have been adjusted to exclude the geological citations.

Acknowledgments

Supported by VZ MSTM CR 0021620808 and IGA MZ NT11229-3. Thanks belong to Professor Yasushi Tomita from Nagoya University in Japan, who kindly supplied two photographs (Figures 1.1 and 1.2) from his private archive.

References

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Fitzpatrick, B., and Seiji, M. (1960) Melanin granules and mitochondria. Nature, 187, 392–394. Seiji, M., Fitzpatrick, T.B., and Birbeck, M.S.C. (1961) The melanosome: a distinctive subcellular particle of mammalian melanocytes and the site of melanogenesis. J. Invest. Dermatol., 36, 243–252. Seji, M., Fitzpatrick, T.B., Simpson, R.T., and Birbeck, M.S.C. (1963) Chemical composition and terminology of specialized organelles (melanosomes and melanin granules) in mammalian melanocytes. Nature, 197, 1082–1084. Simon, G. (1841) Zür Entwickelungsgeschichte der Haare. Joh. Muller’s Arch. Anat., 367. Purkynĕ, J.E. (1838) Bericht über die Versammlung deutscher Naturforscher und Aerzte in Prag im September 1837 (eds K. Sternberg and J.V. Krombholz), Prague, pp. 174–180. Sorby, H.C. (1878) On the colouring matters found in human hair. J. Anthropol. Inst., 8, 1–24. Kromayer, E. (1893) Oberhautpigment der Säugethiere. Arch. Mikrosk. Anat., 42, 1–15. Duchoň, J., Fitzpatrick, T.B., and Seiji, M. (1968) Melanin 1968: some definitions and problems, in The 1967–68 Year Book of Dermatology (eds A.W. Kopf and R. Andrade), Year Book Medical, Chicago, IL, pp. 6–33. Greenstein, J.P., Turner, J.C., and Jenrette, W.V. (1940) Chemical studies on the components of normal and neoplastic tissues. IV. The melanincontaining melanopseudoglobulin of the malignant melanoma of mice. J. Nat. Cancer Inst., 1, 377–385. Mason, H.S., Kahler, E., Mac Cardle, R.C., and Dalton, A.J. (1947) Chemistry of melanin. IV. Electron micrography of natural melanins. Proc. Soc. Exp. Biol. Med., 66, 421–431. Bolt, A.G. (1967) Interactions between human melanoprotein and chlorpromazine derivatives. I. Isolation

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and purification of human melanoprotein from hair and melanoma tissue. Life Sci., 6, 1277–1283. Borovanský, J. and Hach, P. (1973) Comments on Bolt’s tumour melanoprotein. Neoplasma, 20, 325–329. Laxer, G., Sikorski, J., Whewell, C.S., and Woods, H.J. (1954) The electron microscopy of melanin granules isolated from pigmented mammalian fibres. Biochim. Biophys. Acta, 15, 174–185. Birbeck, M.S.C., Mercer, E.H., and Barnicot, N.A. (1956) The structure and formation of pigment granules in human hair. Exp. Cell Res., 10, 505–514. Wellings, S.R. and Siegel, J. (1959) Role of Golgi apparatus in the formation of melanin granules in human malignant melanoma. J. Natl. Cancer Inst., 3, 131–140. Drochmans, P. (1960) Electron microscope studies of epidermal melanocytes, and the fine structure of melanin granules. J. Biophys. Biochem. Cytol., 8, 165–180. Drochmans, P. (1960) Study by the electron microscope of the mechanism of melanin pigmentation. Arch. Belg. Dermatol. Syphiligr., 16, 155–163. Borovanský, J. (1975) Isolation of melanosomes and an attempt to quantify melanin content in tissues. PhD thesis, Faculty of General Medicine, Charles University Prague. Sieber, N. (1886) Ueber die Pigmente der Chorioidea und der Haare. Arch. f. Exp. Pathol., 20, 362–367. Berzelius, J.J. (1840) Lehrbuch der Chemie (aus der Schwedischen Handschrift des Verfassers übersetzt von F. Woehler). Dritte ungearbeitete und vermehrte Original Auflage, Dresden & Leipzig: in der Arnoldischen Buchhandlung, vol. 9, 22–24. Landolt, H. (1899) Ueber das Melanin der Augenhäute. Hoppe Seylers Z. Physiol. Chem., 28, 192–211. Mörner, K.A.H. (1887) Zur Kenntnis von der Farbstoffen der melanotischen Geschwülste. Z. Physiol. Chem., 11, 66–141.

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1 History of Melanosome Research 23 Waelsch, H. (1932) Zur Kenntnis der

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natürlichen Melanine. Hoppe Seylers Z. Physiol. Chem., 213, 35–57. Hermann, H. and Boss, M.B. (1945) Dopa oxidase activity in extracts from ciliary body and in isolated pigment granules. J. Cell. Comp. Physiol., 26, 131–138. Du Buy, H.G., Woods, M.W., Burk, D., and Lackey, M.D. (1949) Enzymatic activities of isolated amelanotic and melanotic granules of mouse melanomas and suggested relationship with mitochondria. J. Am. Cancer Inst., 9, 325–336. Du Buy, H.G., Showacre, J.L., and Hesselbach, M.L. (1963) Enzymic and other similarities of melanoma granules and mitochondria. Ann. NY Acad. Sci., 100, 569–583. Woods, M., Burk, D., and Hunter, J. (1963) The ontogenic status of melanin granules. Ann. NY Acad. Sci., 100, 534–539. Seiji, M., Shimao, K., Birbeck, M.S.C., and Fitzpatrick, T.B. (1963) Subcellular localization of melanin biosynthesis. Ann. NY Acad. Sci., 100, 497–533. Stein, W.D. (1955) Chemical composition of the melanin granule and its relation to the mitochondrion. Nature, 175, 256–257. White, L.P. (1956) Melanin: a naturally occurring cation exchange material. Nature, 182, 1427–1428. Commoner, J.B., Townsend, J., and Pake, G.E. (1954) Free radicals in biological materials. Nature, 174, 689–691. Fitzpatrick, T.B., Quevedo, W.C., Jr, Levene, A.L., Mc Govern, V.J., Mishima, Y., and Oettle, A.G. (1966) Terminology of vertebrate melanin-containing cells. Science, 152, 88–89. Fitzpatrick, T.B., Quevedo, W.C., Levene, A.L., Mc Govern, V.J., Mishima, Y., and Oettle, A.G. (1966) Terminology of vertebrate melanin-containing cells, their precursors and related cells. A report of the nomenclature committee of the 6th International Pigment Cell Conference, in Structure and Control of the Melanocyte (eds G. Della Porta and O. Muhlbock), Springer, New York, pp. 1–5.

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(1968) Isolation of the intermediate “vesicles” during ontogeny of melanosomes in embryonic chick retinal pigment epithelium. Fed. Proc., 27, 722. Toda, K. and Fitzpatrick, T.B. (1971) The origin of melanosomes, in Biology of Normal and Abnormal Melanocytes (eds T. Kawamura, T.B. Fitpatrick, and M. Seiji), University Park Press, Baltimore, MD, pp. 265–278. Fitzpatrick, T.B., Hori, Y., Toda, K., Kinebuchi, S., and Szabó, G. (1971) The mechanism of normal human melanin pigmentation and of some pigmentary disorders, in Biology of Normal and Abnormal Melanocytes (eds T. Kawamura, T.B. Fitpatrick, and M. Seiji), University Park Press, Baltimore, MD, pp. 369–401. Birbeck, M.S.C. (1962) Electron microscopy of melanocytes. Br. Med. Bull., 18, 220–222. Birbeck, M.S.C. (1963) Electron microscopy of melanocytes: the fine structure of hair bulb premelanosomes. Ann. NY Acad. Sci., 100, 540–548. Brumbaugh, J.A. and Froiland, T.G. (1973) DOPA and cysteine incorporation into premelanosomes: effects of cycloheximide and gene substitution. J. Invest. Dermatol., 60, 172–178. Hempel, K. and Deimel, M. (1963) Untersuchungen zur gezielten Strahlentherapie des Melanoms und des chromaffinen Systems durch selektive 3 H-Inkorporation nach Gabe von 3H markierten DOPA. Strahlentherapie, 121, 22–45. Hempel, K. (1966) Investigation on the structure of melanin in malignant melanoma with 3H and C14-DOPA labeled at different positions, in Structure and Control of the Melanocyte (eds G. Della Porta and O. Muhlbock), Springer, New York, pp. 162–175. Nakai, T. and Shubik, P. (1964) Electronmicroscopic radioautoraphy: the melanosome as a site of melanogenesis in neoplastic melanocytes. J. Invest. Dermatol., 43, 267–269. Zelickson, A.S., Hirsch, H.M., and Hartmann, J.F. (1964) Melanogenesis – an autoradiographic

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2 Classical and Nonclassical Melanocytes in Vertebrates Sophie Colombo, Irina Berlin, Véronique Delmas, and Lionel Larue

2.1 Definition of Melanogenic Cells

Melanogenic cells produce melanin, a polymer based on tyrosine, in specialized organelles, the melanosomes. This synthesis is catalyzed by an enzyme that converts tyrosine into dopaquinone, tyrosinase (Tyr). In mammals and birds, cutaneous pigment cells are described as “classical melanocytes,” and are involved in skin and hair pigmentation. Melanocytes in other locations, such as the eye, inner ear, meninges, adipose tissue, heart, and, possibly, bone, are described as “nonclassical.” The pigment cell precursor in mammals and birds is called the melanoblast, and the mature pigment cell is called the melanocyte (Figures 2.1 and 2.2). Melanocytes are dendritic cells that produce melanin, the natural pigment of the skin. Melanin is largely responsible for the color of the skin, hair, and eyes, and skin appendages. It plays an important role in protecting skin cells against UV radiation. Melanin is produced in the melanosome, a type of cytoplasmic vesicle related to the lysosome. Some of the quinone intermediate products of melanin synthesis are toxic to cells. The melanosome confines these compounds, thereby protecting the cell against their harmful effects. Two melanin pigments are synthesized during melanogenesis: eumelanin, which is brown/black in color, and pheomelanin, which is yellow/red in color. The overall color of the appendages and skin results from regulation of the balance between these two types of pigment. Only eumelanin can efficiently protect cells against UV. Three enzymes are involved in melanogenesis. The first, Tyrosinase (Tyr), plays an essential role in the early stages of melanin synthesis. It hydroxylates l-tyrosine to generate l-3,4-dihydroxyphenylalanine (l-dopa) and then oxidizes l-dopa to generate l-dopaquinone, which spontaneously polymerizes to generate eumelanin. The other two enzymes, tyrosinase-related protein 1 (Tyrp1) and tyrosinase-related protein 2 (Tyrp2, also called dopachrome tautomerase (Dct)), share some similarity with Tyr and fine-tune the synthesis of eumelanin from l-dopaquinone. Pheomelanin is produced from dopaquinone and cysteine. In many mammals, including mice, the agouti signaling pathway regulates this switching of melanin type within cells.

Melanins and Melanosomes: Biosynthesis, Biogenesis, Physiological, and Pathological Functions, First Edition. Edited by Jan Borovanský and Patrick A. Riley. © 2011 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2011 by Wiley-VCH Verlag GmbH & Co. KGaA.

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2 Classical and Nonclassical Melanocytes in Vertebrates non-classical melanocytes

inner ear

eye

internal organs

RPE

uveal cochlea vestibular meninges harderian others organ gland choroid iris ciliary heart lungs body cartilage, bone gonads, etc classical melanocytes epidermal

dermal

interfollicular follicular niche Figure 2.1 Classical melanocytes are pigmented cells, which (i) are found in the skin (dermis or epidermis), (ii) are involved in skin pigmentation, and (iii) have followed

hair bulb the dorsolateral migratory pathway during development. Melanocytes can be considered as nonclassical if they do not fulfill all of these three criteria.

Melanosomes are elliptical or spheroidal organelles containing melanogenic enzymes and cofactors, including proteins of the Tyr family, passing through four stages of maturation [1]. Stage I melanosomes or premelanosomes probably develop from the endoplasmic reticulum [2]. However, the origin of these organelles remains a matter of debate, as Raposo et al. have suggested that stage I melanosomes are actually a type of multivesicular endosome [3]. Stage I melanosomes have an amorphous matrix and internal vesicles formed by membrane invagination. Stage II eumelanosomes have an organized, structured fibrillar matrix, but no active melanin synthesis, whereas melanin synthesis can occur in stage II pheomelanosomes. Stage II eumelanosomes contain Tyr, despite the absence of active melanogenesis. Melanin is deposited on the fibrillar matrix in stage III eumelanosomes and stage IV eumelanosomes are fully melanized, their internal matrix being masked by melanin deposits (reviewed in [4, 5]). Within the skin, melanosomes are transferred to the surrounding keratinocytes, giving the skin its color. Vertebrate pigment cells can be classified into two types, based on embryonic origin. The cells of one of these types form the retinal pigment epithelium (RPE), which is found in an outer layer of the eye and is derived from an evagination of the developing forebrain, the optic cup. The neural crest-derived pigment cells or melanocytes form the second type of pigment cells. This type of cell includes the pigment cells of the integument and inner ear, the eye and the internal organs. The primary function of these cells is to produce pigment for the coloration of skin, eyes, and appendages (hairs, feathers, scales). Variations in pigment cell

2.1 Definition of Melanogenic Cells a)

b)

c)

(a) Classical skin melanocytes. X-Gal staining of a transverse section of a Dct:LacZ mouse tail showing melanocytes in the basal layer of the epidermis (Ep) and in the hair follicle (HF). Note the dentricity of the melanocytes. (b and c) Nonclassical

Figure 2.2

heart melanocytes in C57BL/6 mice. Ventral view of whole heart (b) and mitral valve (c). LA, left atrium; LV, left ventricle; MV, mitral valve; RA, right atrium; RV, right ventricle. Scale bars: (b) 1 mm and (c) 100 μm.

development and function are readily identifiable and apparent as a particular type of coloration or pattern in vertebrates. Pigment cells are not essential for viability. Mutations affecting their development are, therefore, generally not lethal, but result in obvious changes in the coloring of adult vertebrates. In other vertebrates, the pigment cells are called chromatophores. Chromatophores are pigment-containing, light-reflecting cells found in amphibians, fish,

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reptiles, crustaceans, and cephalopods. They are largely responsible for generating skin and eye color in cold-blooded animals, and are generated from the neural crest during embryonic development. Mature chromatophores are grouped into subclasses based on their color under white light: xanthophores (yellow), erythrophores (red), iridophores (reflective/iridescent), leucophores (white), melanophores (black/brown), and cyanophores (blue). Melanophores, the equivalent of higher vertebrate melanocytes, contain eumelanin, which is packaged in melanosomes and distributed throughout the cell. In some amphibian species, other pigments are packaged with the eumelanin. For example, a deep-red pigment, pterorhodin, a pteridine dimer that accumulates around the eumelanin core, has been identified in the melanophores of some frogs. Melanophores display specialized bidirectional and coordinated translocation of the melanosomes within the cytoplasm. Melanophores develop from the neural crest, and are most abundant in the dermal and epidermal layers of the skin, in which the intracellular distribution of the pigment has a significant effect on the color of the animal. Pigment transport is dependent on an intact cytoskeleton and motor proteins associated with cytoskeletal components. Some species can change color rapidly, through mechanisms involving pigment translocation and the reorientation of reflective plates within chromatophores. Such mechanisms are often used as a type of camouflage. Certain vertebrates, including chameleons, generate this effect by producing cell signaling molecules, such as hormones or neurotransmitters, in response to changes in mood, temperature, stress, or visible changes in local environment. Zebrafish and amphibians have three types of pigment cell (melanophores, xanthophores, and iridophores), and medaka (Japanese killifish) have a fourth type, leucophores. The distribution of the different types of chromatophores is the major determinant of the pattern of coloration in adult fish and amphibians.

2.2 Distribution and Function of Melanogenic Cells

Melanocytes are a relatively recent development on the evolutionary timescale. They appeared after the formation of the fourth germ layer or neural crest. They have since evolved to execute various functions, depending on their location, but they retain common signature functions, to which melanin synthesis makes an essential contribution. The best-known function of melanocytes is the one of classical melanocytes, which, due to their location in the skin, are the most apparent and widely studied. These cells control the pigmentation of the individual, tanning, and protection against UV rays. The melanogenic cells in the eye are also highly apparent. Histological analysis rapidly revealed that the cells of the RPE differ in shape from skin or uveal melanocytes. Melanocytes and/or melanin pigment were subsequently identified within the body, in the inner ear, heart, brain, and adipose tissue. The functions and the origins of these nonclassical melanocytes remain unclear.

2.2 Distribution and Function of Melanogenic Cells

2.2.1 Classical Melanocytes

Classical melanocytes are found in the epidermis and dermis. In the epidermis, they are located either in its basal layer or in the hair follicles. The cells surrounding the epidermal melanocytes are keratinocytes, a type of epithelial cell, whereas dermal melanocytes are surrounded mostly by fibroblasts. Epidermal melanocytes can transfer melanin to the surrounding cells, whereas dermal melanocytes cannot. The distribution of melanocytes may differ with species. In humans, melanocytes are mostly located in the basal layer of the epidermis, whereas, in rodents, they are found mostly in hair follicles. Rodent melanocytes are found in the epidermis of nonhairy skin (tail, ears, nose and footpad) and in the dermis of the pinna of the ears. The skin is the main barrier to the external environment. Melanin synthesis in the skin evolved as a protection against the harmful ionizing radiation of sunlight. The enzymes involved in melanin synthesis are known to modulate the levels of calcium and reactive species [6–8]. Skin melanocytes protect the body from the harmful effects of light and heat, by producing melanin. Skin phototype, corresponding to skin color and ease of tanning, reflects the degree of pigment production in humans and is the best indicator of skin cancer risk in the general population. Individuals with lightly pigmented skin have a much higher risk (15–70 times higher) of skin cancers, including melanomas, than individuals with darker skin [9, 10]. The melanocortin-1 receptor (MC1R) is a major regulator of skin pigmentation phenotype and the gene encoding this protein has thus been identified as a potential susceptibility gene for melanoma [11, 12]. UV radiations are harmful to human skin because they generate several types of cellular damage, including oxidative damage and two major types of DNA damage: cyclobutylpyrimidine dimers and 6,4-photoproducts, which play a major role in UV carcinogenesis [13]. Its seems that DNA damage/repair itself induces skin pigmentation [12, 14, 15]. 2.2.1.1 Melanocytes in the Epidermis Epidermal melanocytes constitute the second largest cell population (about 5%) in the human epidermis after keratinocytes, which account for about 90% of epidermal cells. They synthesize the eumelanins and pheomelanins that give the skin its color and protect it against UV light. They fulfill various roles, including protecting the skin from DNA damage or oxidative stress and immune responses to various stimuli. 2.2.1.1.1 Melanocytes in the Hair Follicle The melanocytes in hair follicles are involved in both hair pigmentation and the elimination of toxic byproducts of melanin synthesis [16]. The bulge region of the hair follicle serves as a reservoir of melanocyte stem cells for the skin and hairs. These stem cells give rise to new melanocytes populating the bulb region of the hair follicle at each hair cycle.

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2.2.1.1.2 Interfollicular Melanocytes Melanin has many roles. It protects against the DNA damage and free radicals induced by UV radiation. It also slows vitamin D production in response to UV-B irradiation and protects against the UV-A-mediated breakdown of folate, which is essential for nucleotide synthesis. It has also been suggested that pigmentation may be involved in adaptation to ambient temperature and sexual selection for certain species. Epidermal melanocytes are surrounded by keratinocytes, which they contact via their dendrites, in a ratio of about 30 keratinocytes per melanocyte. The epidermal melanocyte and its surrounding keratinocytes form the so-called “epidermal melanin unit.” Melanosomes are transferred from the melanocytes to the keratinocytes, in which their distribution is modified by UV radiation such that they form a cap-like structure covering the side of the nucleus exposed to sunlight [17, 18]. They protect epidermal cells from damage by limiting the penetration of UV rays through the epidermal layers and by scavenging reactive oxygen species (ROS) generated in response to UV exposure [19, 20]. Melanocytes express MC1R, which regulates the quality and quantity of melanin production. MC1R is controlled by the agonists melanocyte-stimulating hormone (MSH) and adrenocorticotropic hormone (ACTH) [21], which stimulate the eumelanin synthesis pathway. This receptor is also controlled by the antagonist agouti signaling protein (ASIP), which activates the production of pheomelanin [18, 22]. By responding to paracrine factors, such as α-MSH, epidermal melanocytes can repair DNA more efficiently by nucleotide excision repair [23, 24], and also combat oxidative stress, because α-MSH decreases hydrogen peroxide generation in response to UV irradiation [23]. These consequences are thought to decrease the genotoxic effects of UV irradiation, enabling melanocytes to survive with a stable genome. The maintenance of melanocyte survival in the skin is essential to optimize photoprotection and to prevent photocarcinogenesis [18]. The skin is continually exposed to microorganisms. The epidermis has no vascular circulation, minimizing the likelihood of toxic chemicals, bacteria, or fungi penetrating the stratum corneum and entering the bloodstream. The epidermis protects itself by producing defensins, a group of proteins with antimicrobial properties. It also contains Toll-like receptors, which recognize invading organisms and elicit a host response. The reactive quinone intermediates generated during melanin biosynthesis also have potent antimicrobial/antifungal properties. Consistent with this finding, fungal dermatitis is more prevalent among individuals with fair skin than among those with dark skin [25]. Melanocytes are also phagocytic cells involved in the inflammatory response. Indeed, α-MSH is known to suppress inflammation (particularly by inhibiting the activation of nuclear factor NF-κB). The pigment system responds to almost all inflammatory events in the epidermis, generally by increasing melanin synthesis (postinflammatory hyperpigmentation), or less often, by decreasing melanin synthesis (postinflammatory hypopigmentation). An example of postinflammatory hyperpigmentation is provided by tanning after sun exposure. UV rays damage the epidermis and sunburn is the inflammatory response to this injury. This process is mediated in part by α-MSH, which induces melanin production, result-

2.2 Distribution and Function of Melanogenic Cells

ing in darker skin that is more resistant to subsequent sunburn. Further functions of α-MSH include the stimulation of DNA repair and downregulation of the immune response of the epidermis, possibly preventing the occurrence of autoimmune disorders, such as lupus erythematosus [18]. Melanocytes produce and react to many cytokines and growth factors, thereby acting as sentinels and active players in the immune system of the skin [2]. Melanocytes can present and process antigens, eliciting a T cell proliferative response [26], with interferon-γ inducing the expression of major histocompatibility complex class II (MHC II) antigens [27]. Melanocytes also interact with Langerhans cells, the principal type of antigen-presenting cell in the skin [18, 28]. Melanocytes probably also have an endocrine/sensory function, regulating the skin immune response. The cutaneous pigment system can serve as an important stress response element, through the effects of corticotrophin-releasing hormone (CRH), catecholamines, acetylcholine (ACh), pro-opiomelanocortin (POMC)derived peptides, and steroid hormones [29–31]. CRH has been shown to inhibit NF-κB (a proinflammatory molecule) in melanocytes by producing POMC and may therefore serve as a feedback mechanism for the self-restriction of inflammatory responses in the skin [32]. Lipocalin-type prostaglandin D synthase (L-PGDS) has been identified as a melanocyte marker, due to its specific expression in mouse and human epidermal melanocytes, but not in other skin cells [33]. L-PGDS isomerizes prostaglandin H2 (PGH2) to generate prostaglandin D2 (PGD2) and functions as an intercellular chaperone for lipophilic ligands, such as thyroid hormones, retinoic acid, bilirubin and biliverdin [34]. PGD2 has pleiotropic effects through its specific G-proteincoupled receptors, DP1 and DP2, both of which are expressed in human epidermal melanocytes. Mitf-M regulates L-PGDS gene transcription in melanocytes. L-PGDS and PGD2 regulate sleep and nociception. Melanocytes also produce β-endorphin, an endogenous opioid, from POMC, which, together with MSH and ACTH and opioid receptors, is located in nuclei active in sleep regulation. It has therefore been suggested that melanocytes may be involved in regulating sleep/waking behavior [35]. There is also evidence to suggest that a melanocyte-derived factor, such as L-PGDS, may be involved in regulating the central chemosensor controlling respiratory rhythm, as opioids are respiratory depressants and epidermal melanocytes produce opioids [35]. 2.2.1.2 Melanocytes in the Dermis Dermal melanocytes are considered to be classical melanocytes because they are involved in skin pigmentation. However, they do not transfer melanin to surrounding cells. They do not interact with keratinocytes, instead interacting with fibroblasts and other cell types, which may instruct them. Dermal melanocytes are present in very small numbers in human adult skin and little is known about their function. Their interaction with the surrounding fibroblasts seems to regulate their melanin production. Fibroblasts have been shown to stimulate melanocytes to produce Tyr [36], thereby increasing melanocyte survival and melanin synthesis after the UV irradiation of reconstructed skin [37]. By contrast, Hedley et al. [38]

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showed that fibroblasts decreased pigmentation levels in reconstructed skin models. Yamaguchi et al. [39] showed that Dickkopf1 (DKK1), an inhibitor of Wnt/β-catenin signaling, was produced in larger amounts in palmoplantar dermal fibroblasts than in nonpalmoplantar dermal fibroblasts. DKK1 inhibits melanocyte function and growth, by regulating β-catenin and microphthalmia-associated transcription factor (Mitf) [40]. In addition to modulating melanocyte melanin production, DKK1 also affects keratinocytes, increasing skin thickness and decreasing the transfer of melanosomes from melanocytes to keratinocytes. 2.2.1.3 Melanophores in Lower Vertebrates Frog melanophores of various morphological types are present in both the epidermis, which is populated mostly by keratinocytes, and the dermis, in which fibroblasts predominate. Amphibian and fish melanophores perceive chemical stimuli and play an important role in rapid color changes, through the dispersion or aggregation of melanosomes, and neurogenic changes in pigmentation [41]. These modifications of pigmentation are essential for the survival of these animals in predator-rich environments. Unlike the melanocytes of mammals, amphibian melanophores can cross the basal lamina to reach the dermis, where they transfer melanin to dermal fibroblasts [42]. 2.2.2 Nonclassical Melanocytes

Nonclassical melanocytes can be found in various locations of the body, including the visceral organs. They do not usually contain large amounts of melanin in healthy individuals. However, there are key exceptions to this rule. The abdomen of birds, including quail, chicken, ostrich, and zebra finch, contains pigmented cells, and the spleen and liver of amphibians contain pigmented macrophages. Nonclassical melanocytes are more commonly found in the eye, inner ear, heart, brain, and adipose tissue. 2.2.2.1 Melanocytes of the Eye The eye contains two types of pigment cells: the uveal melanocytes and the RPE cells. RPE cells play a crucial role in retinal development and function, thus controlling visual acuity, whereas uveal melanocytes provide the color of the iris and are responsible for uveal melanoma development. Other melanocytes are found in the Harderian gland of vertebrates with nictitating membranes, which may play a role in photoprotection. Photoreception, in general, and in the eyes, in particular, is commonly associated with dark pigments, such as melanins, because of their ability to absorb light. Such pigments are found in all functional eyes, even the most primitive. By screening out light from one direction only, they protect the photoreceptor cells from excessive exposure to light. The amount of light reaching the photoreceptors is also regulated by the pupil, the diameter of which varies with light intensity. The photoreceptive retina in vertebrates is covered by a layer of choroidal melanocytes and a specialized pigmented monolayer, the RPE. The RPE

2.2 Distribution and Function of Melanogenic Cells

cells and their melanosomes do not seem to be renewed, whereas the uveal melanocytes and their melanosomes are produced continually, throughout the life of the individual. 2.2.2.1.1 RPE The RPE plays a key role in visual acuity, for which melanin production does not appear to be required. In vertebrates, the cells of the RPE are derived from the multipotent optic neuroepithelium. They develop in close proximity to the retina, and are indispensible for eye organogenesis and vision. RPE cells are cuboidal cells that contact the outer segments of the photoreceptors on their apical side and rest against the basal membrane, known as Bruch’s membrane, separating the RPE from the choroid, at their base. During embryogenesis, RPE cells are involved in the formation of the ciliary body and iris, and the control of optic fissure closure. They also influence retinal neurogenesis and ganglion cell projections, and have been implicated in regulation of the choroidal vasculature (for review, see [43, 44]). In adults, RPE cells provide photoreceptors with nutritional support, build up a blood–retina barrier and process retinol and retinoids, controlling ion flow and oxidative damage, and removing the fragments of photoreceptor outer segments shed from the distal end of the photoreceptor by phagocytosis [45]. The melanin in the RPE protects the neural retina against ROS, thereby preventing age-related macular degeneration [18]. RPE melanin is essential for the development of the neural retina. When it is absent or present at low levels, the retina develops abnormally (e.g., in albino mammals) and many temporal retinal axons that should remain uncrossed at the optic chiasm cross inappropriately to innervate the contralateral hemisphere. The central retina is also underdeveloped and there is a rod cell deficit of about 30% (for a review, see [46]). These retinal abnormalities result in significant visual impairment. Albino birds do not display the same retinal abnormalities, probably because, unlike mammals, they have a cone-dominated retina with few rods. The synthetic pathway of melanin is the same in the skin and in the RPE, but unlike epidermal melanocytes, which produce melanosomes continuously, RPE cells are thought to produce melanosomes only during the prenatal period. Both prenatal RPE cells and skin melanocytes present stage I–IV melanosomes. Melanogenesis can occur only if premelanosomes are present, and Tyr and other melanogenic proteins are synthesized. The adult RPE contains Tyr, but not the structural protein Pmel17, which is essential for the formation of premelanosomes in melanocytes. Biesemeier et al. [47] showed that melanogenesis can occur within multivesicular and multilamellar organelles in amelanotic RPE cells in vitro, without the formation of typical premelanosomes. Tyrp1 and Pmel17 are not detectable in these cells and classical stage I–IV melanosomes are also absent. 2.2.2.1.2 Uveal Melanocytes The uvea is the pigmented, highly vascularized middle layer of the eye. It is divided, from anterior to posterior, into the iris, ciliary body, and choroid. The pigment is produced and held in numerous dendritic melanocytes, similar to

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normal dermal melanocytes. It has been suggested that uveal melanin reduces the likelihood of uveal melanoma by protecting melanocytes from oxidative stress. The role of the melanin in uveal melanocytes in protection against UV remains a matter of debate [48, 49]. Choroid The choroid, a layer of highly vascularized tissue surrounding the eye, is essential for normal retinal function. The melanocytes in this structure are localized in the choroidal stroma and suprachoroid. They are pigmented and may be star-shaped, fusiform, round, or oval, with a central nucleus. Their function remains unclear, but the fibrillar structures observed in their cytoplasm may be involved in maintaining capillary tonus. There is no functional contact between the dendrites of the choroidal melanocytes and the other cell types present in uveal tissue. These melanocytes remain quiescent and no longer have the capacity to produce melanin [49]. Choroidal melanin is thought to protect the choroid from oxidative damage due to the stress arising from its location in the highly vascularized posterior segment of the eye. Iris The iris is a small structure consisting of connective tissue and muscle, with a central opening called the pupil. Eye color, reflecting the pigmentation of the iris, depends on the total amount of melanin in the epithelial cells and stromal melanocytes and the relative proportions of the two types of melanin. Brown irises contain abundant melanocytes and melanin in the anterior border layer and stroma, whereas these layers contain very little melanin in blue irises. This pigmentation is essential in the control of pupil aperture (thus controlling the amount of light entering the eye). The pigmentation of the iris appears to be correlated with the incidence of uveal melanoma and age-related macular degeneration, for which it is considered a risk factor [48, 49]. Ciliary Body The ciliary body is the circumferential tissue within the eye consisting of the ciliary muscle and ciliary processes and coated with a double layer, the ciliary epithelium. The outer layer is highly pigmented and continuous with the RPE. These cells constitute the iris dilator muscle, which plays an important role in light accommodation, by controlling pupil aperture. 2.2.2.1.3 Harderian Gland The Harderian gland is found in the orbit of the eye in vertebrates (reptiles, amphibians, birds, and mammals) with a nictitating membrane. Dendritic cells containing melanin granules have been found in the interstitial tissue of the Harderian gland, which is pigmented. Two types of melanocytes, with and without the various developmental stages of melanin granules, have been found in this gland in mice. Cells with developing granules were found to have more dendrites and to contain a larger number of cytoplasmic organelles. The other cells were ellipsoidal or elongated and contained few cytoplasmic organelles and a large number of fully pigmented melanin granules, but no developing granules. The Harderian gland macrophages contain fully pigmented melanin granules of

2.2 Distribution and Function of Melanogenic Cells

various sizes, suggesting that they engulf some of the melanocytes. The numbers of mature melanocytes in the Harderian gland probably varies with retina pigmentation. For example, gerbil strains with pink eyes also have Harderian glands lacking melanocytes. The role of melanocytes in the Harderian gland is unclear, but may be related to photoprotection. The nictitating membrane is also pigmented in many species, with melanocytes present around the duct and its opening [50]. 2.2.2.2 Melanocytes of the Inner Ear Corti was the first to demonstrate the presence of melanocytes in the inner ear, in 1851. The distribution of melanocytes in the inner ear differs between species. These cells are mostly concentrated in the cochlea and the vestibular organ. Melanocytes are abundant in the human inner ear, but are rare in the ears of lower vertebrates. Patients with lacking inner ear melanocytes or with poor or no inner ear melanocyte function, such as those suffering from Waardenburg syndrome or Harada’s disease, display hearing loss and problems with balance. This suggests that melanocytes have important functions in the cochlea and the vestibular labyrinth. Inner ear melanocytes have also been shown to play an important role in animal models, such as viable dominant spotting mice or white spotting rats, which have a mutation in the c-Kit gene (KitW), and in black-eyed white Mitfmi-bw homozygous mice and Mitfmi-vga9 homozygous mice. All these animals have coat color phenotypes due to a lack of melanocytes and are deaf. In addition, mice homozygous for the weak allele, Mitfvit, gradually lose their melanocytes after birth, and thus display hair graying and age-dependent hearing loss, indicating a role for Mitf and melanocytes not only during inner ear development, but also in inner ear function after development has been completed. 2.2.2.2.1 Cochlea The cochlea is the organ responsible for hearing in mammals. Cells containing melanin are present within the stria vascularis and the modiolus. These cells play important roles in the development and function of the cochlea, by regulating endocochlear potential and preventing noise-induced damage (for review, see [51]). The endolymph – the extracellular fluid in the membranous labyrinth of the inner ear – is excreted by the stria vascularis, which consists of three layers of cells: the marginal, intermediate, and basal cell layers. The intermediate layer cells in the stria vascularis of the mammalian cochlea are melanocytes that contain and synthesize melanin. Two types of cochlear melanocytes have been described: light intermediate cells that contain premelanosomes, are positive for the dopa reaction, and capable of melanin synthesis, and dark intermediate cells corresponding to degraded melanocytes incapable of melanin synthesis. In the modiolus, the melanocytes are located in the perivascular and perineural spaces. These cells are closely associated with the spiral modiolus artery. Cochlear melanocytes are required for the normal development and function of the cochlea, as mammals with pigmentation defects display profound cochlear defects. Viable dominant spotting mice (KitWv) and white spotting rats (KitWs) have no cochlear melanocytes and

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display alterations in the development and function of the stria vascularis, including changes in endocochlear potential. However, melanin itself is not essential for normal hearing function. The absence of melanin has no effect on age-related cochlear degeneration [52]. Furthermore, albino mammals, which have melanocytes but no pigment due to a lack of Tyr or related enzyme function, have normal hearing. However, albino cochleas are more susceptible to the ototoxic effects of high-intensity noise than pigmented cochleas [51]. This may be accounted for by the ability of melanin to scavenge the ROS, generated after exposure to noise. Melanin formation in the inner ear seems to be catalyzed by a tyrosine hydroxylase rather than by Tyr [53]. Tyr and Tyrp1 have not been found in cochlear extracts from the gerbil, but these extracts do contain tyrosine hydroxylase with a higher affinity for l-tyrosine, and this enzyme is independent of l-dopa. Strial intermediate cells continually undergo mitosis. Exposure to noncontinuous impulse-type noise or broadband white noise leads to strong hyperpigmentation of the stria vacularis, with some melanosomes being transferred to marginal cells. An increase in prostaglandin levels after noise exposure may lead to an increase in the mitosis rates of intermediate cells [51]. Intermediate cells generate a positive endocochlear potential and secrete K+. The + K channel at the plasma membrane may be involved in the mitosis of intermediate cells. Mice lacking these K+ channels are deaf. Melanocyte-deficient mice display measurable changes in the ionic composition of the endolymph, which bathes the mechanosensory hair cells [54]. Normal endolymph contains high levels of K+ ions, and low levels of Na+ and Ca2+ ions. Cochlear melanocyte deficiencies are associated with a low K+ concentration in the endolymph, affecting the capacity of hair cells to respond to the sound-induced motion of the endolymph [55]. The melanin in cochlear melanocytes may also serve as a biological reservoir for Ca2+ and may be involved in Ca2+ regulation in the inner ear, as it has a high affinity for divalent cations, such as Ca2+ [56, 57]. Variations in endolymph Ca2+ concentration are known to affect the function of hair cells. Albino animals, which have amelanotic melanocytes, also have low concentrations of Ca2+ in the endolymph. However, the molecular mechanisms by which these melanocytes exert their functions are unknown. The glutathione S-transferase (GST) α4 (Gsta4) gene has recently been shown to be expressed specifically in melanocytes of the stria vascularis, but not in other tissues harboring melanocytes [58]. This suggests that cochlear melanocytes play a role independent of their melanogenic function. GSTs carry out important functions in the detoxification processes of many tissues. Other GSTs, including Gsta1/2, Gstm1/2, and Gstp1, are expressed in the lateral wall of the cochlea, which includes the melanocyte-containing stria vascularis. Gstp1 seems to be expressed in the intermediate cells and may therefore colocalize with Gst4. Alpha GSTs play a role in defense against the intracellular lipid peroxidation caused by the reaction of various lipids with ROS. Gsta4-4 (Gsta4 homodimer) is the major enzyme catalyzing the conjugation of glutathione with 4-hydroxynonenal, one of the end products of lipid peroxidation. Gsta4 in melanocytes of the stria vascularis may act as an antioxidant in defenses against the hearing loss induced by loud noises and ototoxic drugs, which have been reported

2.2 Distribution and Function of Melanogenic Cells

to induce oxidative stress through the production of ROS. This stress results in deafness, due to the injury of hair cells in the cochlea. ROS are also inevitably produced during normal aerobic metabolism. Blood vessels in the stria vascularis supply oxygen and may be a source of oxidative stress. The distribution of GSTs may facilitate the detoxification of harmful substances at the boundaries between blood vessels and the tissues of the inner ear. Cochlear melanogenesis is induced by exposure to noise. Pigmented cochleas are less susceptible to noise partly because melanin, as an energy transducer, works as a ROS scavenger. The specific expression of Gsta4 in cochlear pigment cells may therefore be of particular importance, with this molecule acting as an antioxidant and its levels being correlated with cochlear melanogenesis. 2.2.2.2.2 Vestibular Organ Melanocytes are also present in the vestibular labyrinth of the inner ear [59]. In viable dominant spotting mice, which have profound defects of the stria vascularis, the vestibular labyrinth appears to be pigmented in approximately 20% of animals. The melanocytes of the vestibular labyrinth have been shown to display the characteristics of epidermal melanocytes in culture, with the presence of melanosomes and the expression of genes associated with pigmentation, such as the Melan-A, Tyrp1, and Tyr genes. Their function remains unclear. It has been speculated that they could play a role in balance perception, as melanocyte defects have been found to be accompanied by vertigo. The vestibular melanocytes are located in the dark cell areas of the vestibular labyrinth. They form a continuous pigmented layer covered by a secretion epithelium consisting of epithelial dark cells (EDCs) and are presumably in contact with neighboring subepithelial capillaries. These cells have large numbers of cytoplasmic processes and melanosomes at various stages. Gap junctions are found between melanocytes and between melanocytes and EDCs. These junctions may be involved in communication and the endolymph–perilymph transport of molecules. They may also play a role in the regulation of local Ca2+ homeostasis. Changes in the morphology and activity of vestibular inner ear melanocytes (VIEMs) have been observed in pathological changes to the vestibular labyrinth, during experimental hydrops, or in the presence of gentamicin, for example. These changes suggest that VIEM may react to stress conditions in the inner ear. EDCs are involved in endolymphatic K+ circulation. By analogy, melanocytes may therefore be involved in K+ transport or in the constitution of the endolymph in the vestibular labyrinth, in the same way that the melanocytes of the stria vascularis have K+ channels in their membrane and contribute to plasma membrane potential. However, no such K+ channels were found in the dark cell areas and vestibular function remains normal in mice lacking these channels. 2.2.2.3 Melanocytes of the Heart Melanocytes are found in many locations in the heart, including the valves and septa. These melanocytes are dependent on and produce proteins of the classical melanocyte signaling pathway. The level of pigmentation in the heart reflects that

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in the skin. The function of cardiac melanocytes remains unclear, because these cells do not seem to be essential for normal heart function. They may play a role in atrioventricular (AV) valve function, by modulating the viscoelastic properties of these valves. They also contribute to the triggering of arrhythmia, by regulating calcium and ROS levels. It has been known for some time that the neural crest contributes to the formation of the normal heart, particularly in terms of the septation of the outflow tract and the formation of the venous pole. However, melanocytes have only recently been found in the heart [60]. It has been shown in vitro that cardiac neural crest cells (NCCs) form melanocytes. Furthermore, quail-to-chicken transplantation experiments have demonstrated that cranial NCCs form pigmented cells on all the major arteries, including those at the base of the heart. Pigmented cells were also found in the hearts of mutant mice from a line derived from a large-scale N-ethylN-nitrosourea mutation screen. Several groups have investigated the distribution of pigmented cells in the heart [60–63]. l-Dopa-positive melanocytes containing Dct and Tyrp1 are found within the AV mitral (left side) and tricuspid (right side) valve leaflets, and are also present in the chordae tendineae, which extend from the distal edge of the leaflet to the papillary muscle. They are found in the aortic semilunar valve leaflets and in the AV septa. They are present at the apex of the interventricular septum, just below the endocardial layer. They are also present on the dorsal surface of the medial wall of the right atrium, close to the confluence of the coronary sinus and the superior vena cava, and on the outer medial wall of the right atrium. These cells are detected along the right interatrial septum near the foramen ovale and toward the septal leaflet and the aorta. Levin et al. [62] also found melanocytes in the pulmonary veins of mice and humans, although those present in human pulmonary veins were not pigmented, probably because they did not express the Tyrp1 gene. Very small numbers of melanocytes have recently been found in the ductus arteriosus and ligamentum arteriosum of mice [64]. They are never observed in the pulmonary valve leaflets, muscular wall of the interventricular septum or papillary muscle. Melanoblasts are first detected in the heart at embryonic day (E) 12.5, in AV endocardial cushions [61], and prevalvular cushions of the AV region of E13 embryonic hearts are capable of producing pigmented cells in culture [60]. The cardiac melanocytes express markers of the classical melanocyte lineage, such as Dct, Sox10, Mitf, Tyr, Tyrp1, and Pax3. They also differ from cardiomyocytes in terms of their morphology. They have large numbers of caveolae along the plasma membrane and some cells contain mature melanosomes [62]. No melanin appears to be transferred from cardiac melanocytes to the surrounding cells [63]. A cluster analysis of global gene expression suggested that Dct-expressing cells were more closely related to atrial myocytes than to dermal melanocytes. These two cell types have similar calcium-handling proteins (such as phospholamban and ryanodine receptor, type 2) and have some voltage-gated ion channels in common (e.g., the pore-forming subunit of L-type calcium channels and cardiac voltage-dependent sodium channels). However, Dct-positive cells in the heart display no significant expression of some of the signature genes of cardiomyocytes,

2.2 Distribution and Function of Melanogenic Cells

such as those encoding cardiac actin and troponins. Thus, Dct-expressing cells in the heart can be distinguished from atrial myocytes and dermal melanocytes on a molecular basis [62]. The distribution of melanocytes in the heart is similar in mice lacking Dct and wild-type mice. Dct is therefore not required for the migration and survival of melanocytes in the heart. The level of pigmentation in the heart seems to be correlated with coat color in mice. Cardiac melanocytes were not found in the spotting mutants Ednrbsl/sl and KitWv/Wv, which have very few melanoblasts and are almost completely white [61], or in the Mitfvga9/vga9 mutants, which are white due to a lack of melanocytes; bcat* mice also have very few cardiac melanocytes and present hypopigmentation [63]. Conversely, large numbers of cardiac melanocytes are present in Tyr::N-rasQ61K transgenic mice, which produce the oncogenic N-Ras in melanocytes, or in mice that overproduce endothelin 3 (Edn3) or hepatocyte growth factor [61, 63]. In mice overproducing Edn3, ectopic melanocytes have even been found in the pulmonary valve and outflow tract. These results indicate that cardiac melanocytes are dependent on the signaling molecules known to be required for correct skin melanocyte development, suggesting that they may originate from the same precursor population. Cardiac melanocytes have not been found in zebrafish or frog, but are present in quail, suggesting an association between the presence of cardiac melanocytes and four-chambered hearts. The function of cardiac melanocytes remains unclear. These cells are not essential for gross heart morphogenesis and physiology, as mice lacking melanocytes (e.g., KitWv or Mitfvga9 mice) and mice with abundant pigmentation (e.g., Tyr::NrasQ61K mice) in the valves and septa of the heart live well into adulthood, with apparently normal hearts. These cells may therefore play a more subtle role that becomes critical in stress conditions. They may be involved in AV valve development from the endocardial cushions, as they arrive in the heart in these cushions. Melanoblasts have been shown to produce and secrete metalloproteases and may thus participate in valve remodeling. This function may continue into adulthood, during which both melanocytes and interstitial cells may be involved in the maintenance of tissue homeostasis and may affect the mechanical properties of the AV valves. Cardiac melanocytes increase the stiffness and storage of mouse AV valves, thereby affecting their viscoelastic properties, and are important for the correct functioning of these valves in the body [65]. Quasistatic and nanodynamic mechanical analyses of the leaflets of the mouse tricuspid valve have indicated that the mechanical properties of the leaflet vary with the degree of pigmentation. Melanocytes may be stiffer than the endothelial cells overlying the leaflet or the surrounding extracellular matrix. However, it remains unclear whether it is the melanocytes themselves or the presence of melanin that contributes to the mechanical properties of the leaflets. It is also possible that variations in stiffness result from the influence of melanocytes on the surrounding extracellular matrix. As melanocyte precursors reach the heart early in valve development and persist into adulthood, dysfunctions affecting these precursors might lead to valve abnormalities. Indeed, cardiac melanocytes are involved in the triggering of atrial arrhythmia [62]. Atrial fibrillation is the most common form of cardiac arrhythmia. It is often initiated by ectopic beats arising from the pulmonary veins and atrium. The

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dysregulation of intracellular calcium and reactive species levels has been described in patients with atrial fibrillation. Dct is involved in the regulation of these concentrations in melanocytes. Mice with no Dct in their cardiac melanocytes have been shown to be more susceptible than wild-type mice to atrial arrhythmogenesis, in the absence of other cardiac electrophysiological alterations and structural abnormalities. By contrast, mice lacking cardiac melanocytes (KitW/Wv mutants) display no atrial arrhythmia. Cardiac melanocytes express adrenergic and muscarinic receptors and interact with autonomic nerves (sympathetic and cholinergic nerves). They can therefore respond to autonomic efflux, which may contribute to arrhythmia. The treatment of mice lacking melanocytes with muscarinic agonists or of mice with melanocytes with β-adrenergic antagonists reduces atrial arrhythmia. The treatment of mice lacking Dct with antioxidants significantly decreases the frequency of atrial arrhythmia, confirming the antioxidant role of Dct and suggesting a role for ROS in triggering arrhythmia in the absence of Dct. Cardiac melanocytes are excitable and form electrical connections with neighboring cardiomyocytes. In the absence of Dct, cardiac melanocytes display prolonged repolarization, with early after depolarization and frequent calcium oscillations, confirming the role of Dct in calcium handling and that of intracellular calcium dysregulation in arrhythmogenesis. Cells capable of synthesizing melanin may be present in the heart and pulmonary veins to buffer calcium and reactive species. However, during pathologic processes, the capacity of these cells to bind free radicals and calcium may be changed or their binding sites saturated, transforming them into initiators of triggered activity and cardiac arrhythmia. Melanin-synthesizing cells may also be involved in maintaining the normal balance of oxidative species in the myocardium. 2.2.2.4 Melanocytes of the Brain and Neuromelanins Melanin-producing melanocytes are found in the sympathetic cephalic ganglia and leptomeninges, and along cerebral capillaries. In the leptomeninges, these cells principally cover the ventrolateral surfaces of the medulla oblongata. These dendritic cells contain melanosomes, observed as single, membrane-bound granules and displaying all stages of melanization, thus confirming that they are indeed melanocytes and not macrophages. At other locations in the brain, the meninges contain only isolated pigmented cells [66]. Brain melanocytes may have a neuroendocrine and detoxification function. Epidermal melanocytes produce L-PGDS, a potent inducer of sleep, and produce opioids that regulate respiratory rhythm. It is thus possible that brain melanocytes produce such molecules in vivo and regulate these mechanisms. Neuromelanin, a brain-specific pigment, is found primarily in catecholaminergic neurons (which produce norepinephrine and dopamine, but not epinephrine) in the substantia nigra and locus coeruleus [67], and accumulates with age [68]. Neuromelanin has a spherical architecture and is composed of a pheomelanin core covered with eumelanin; it also contains aliphatic compounds and peptides [69]. It is formed from the oxidation products of dopamine and cysteinyldopamine in the substantia nigra, and is derived from norepinephrine metabolism in the

2.3 Embryonic Development of Melanogenic Cells

locus coeruleus. It is abundant in the human brain and present in smaller amounts in some nonhuman primates, but it is not found in the brains of many lower species. Neuromelanins play a critical role in protecting neurons against toxic ROS and metals, such as iron [70–72]. Neuromelanins produced from dopa and cysteinyldopa have recently been found in many other types of neuron, almost everywhere in the brain. They also accumulate with age and may play an essential role in reducing toxicity in these tissues [73]. In Parkinson’s disease, more dopaminergic neurons containing neuromelanin in the substantia nigra than unpigmented neurons are lost [68]. Impairment of the iron-chelating function of neuromelanin plays an essential role in Parkinson’s disease development, in which the amount of toxic iron bound to neuromelanin increases, thus rendering pigmented neurons susceptible to oxidative damage [18, 67, 74]. 2.2.2.5 Melanin in Adipose Tissue Eumelanin has been found to be synthesized in the visceral adipose tissue of morbidly obese patients [75]. However, the presence of pheomelanin was not investigated. The level of melanin biosynthesis in these tissues is three times higher in obese than in nonobese subjects. The expression of melanogenesisrelated genes (Tyr, Tyrp1, Dct, Rab27a, MC1R, and Melan-A) is also stronger in obese than in nonobese subjects. In adipose tissues, melanogenesis occurs in the cytosol rather than in melanosomes. The high levels of the melanogenic peptide α-MSH in the serum of obese subjects may account for the activation of the melanogenic pathway, because human adipocytes express the melanocortin receptor MC1R, which triggers α-MSHinduced melanogenesis in melanocytes. The ectopic synthesis of melanin in obese patients may be a compensatory mechanism conferring benefits due to the antiinflammatory and oxidative damage-absorbing properties of melanin. The excess ROS due to obesity and the higher levels of cellular fat deposition may be neutralized by the melanin produced. According to the definition of melanocytes these cells may be considered nonclassical melanocytes.

2.3 Embryonic Development of Melanogenic Cells

In vertebrates, pigment cells have two embryonic origins: the neural crest and neuroepithelium. The pigment cells populating the retina are derived from the neuroepithelium. All other pigment cells are derived from a transient and pluripotent population of cells – the NCCs – considered to be the fourth germ layer. The neural crest delaminates from the roof of the developing neural tube and overlying ectoderm early in development along the rostrocaudal axis at the cephalic (from mid-diencephalon to rhombomere 8), vagal (from somite 1 to 7), truncal (from somite 8 to 28), and lumbo-sacral (posterior to somite 28) levels, to generate various lineages, such as neurons, glia, endocrine cells, bones, and melanocytes. Unlike other NCCs, melanocyte precursors can differentiate from the neural crest

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irrespective of the region along the rostrocaudal axis in which they are located. The pigment cells derived from neural crest may give rise to classical and nonclassical melanocytes. 2.3.1 Classical Melanocytes 2.3.1.1

Early Determined Melanoblasts: The Dorsolateral Pathway

2.3.1.1.1 General Description Cutaneous melanocytes are derived from NCCs. In the truncal region, NCC precursors are found at the border between the neural ectoderm (or neural plate) and the non-neural ectoderm, in a region constituting the apex of the neural groove. In the trunk, the NCCs emerge when the dorsal end of the neural tube is fused, in the region corresponding to somites 8–28. This emergence follows an epithelialto-mesenchyme transition. The NCCs then enter a region called the migration staging area (MSA) located between the dorsal part of the somite, the lateral part of the neural tube, and the ventral part of the ectoderm [76]. In this region, the NCCs receive signals directing their migration and specification. They take one of two migration pathways: the dorsoventral pathway, between the neural tube and the somites, and the dorsolateral pathway, between the ectoderm and the somites. Most NCCs migrate along the dorsoventral pathway to generate various types of cells, depending on the site of emergence along the rostrocaudal axis: sensory and sympathetic neurons, Schwann cells, endoneural fibroblasts, chromaffin cells of the medullo-surrenal gland, and smooth muscle cells. Cells migrating along the dorsolateral pathway give rise only to melanocytes [77]. The NCCs following the dorsolateral pathway delaminate later than those following the dorsoventral pathway. Some of the NCCs produce Wnt1, Pax3, and Sox10. These cells give rise to the limited number of founder melanoblasts, which develop at about E8.5 [78]. Melanoblasts are nonpigmented cells committed to become melanocytes in the absence of external events preventing this determination. Founder melanoblasts begin to proliferate, generating precursor melanoblasts in the MSA. At E10, these melanoblasts produce Kit and Mitf. At around E10.5, they express a melanogenic enzyme marker, Dct. They proliferate in the MSA for about 1 day and then begin to migrate, colonizing the whole embryo. They migrate between the surface ectoderm and the somites (E10.5–E11), following a temporal rostrocaudal gradient. From E11.5, the migrating melanoblasts start entering the epidermis. They then cross the basal membrane and penetrate into the epidermis, from E12.5 in the mouse [79, 80]. From E15, a subset of melanoblasts migrates toward the matrix of the nascent hair follicles, where these cells express genes encoding proteins acting downstream from Mitf, such as Tyr and Tyrp1. Some follicular melanoblasts concentrate in the niche of the hair follicle, the “bulge.” These cells form the melanocyte stem cells and are responsible for maintaining homeostasis. Others migrate into the bulb of the hair follicle, where they differentiate into mature melanocytes, producing melanin in melanosomes at birth.

2.3 Embryonic Development of Melanogenic Cells

2.3.1.1.2 Genes Involved in Classical Melanocyte Development Many signaling molecules are required at all stages melanocyte development. They instruct the NCC to acquire a melanogenic fate and migrate along the dorsolateral pathway. They are necessary for melanocyte proliferation, differentiation, and survival. The following description is not exhaustive. Wnts and bone morphogenetic proteins (BMPs) are produced early in the development of the dorsal neural tube. BMPs favor the neuronal/glial differentiation of NCCs and inhibit pigment cell specification, whereas Wnts play a dual role in promoting both sensory neurons and melanocytes. Melanoblasts arise from NCCs that delaminate late, at a time at which BMP4 is downregulated and Wnt signaling is maintained. Thus, temporal changes in the signals directing migration trajectories and cell fates in the dorsal neural tube may be involved in specification of the melanocyte lineage [81]. Wnt/β-catenin signaling plays a major role in the determination of NCC cells to differentiate into melanocytes. Wnt1 and Wnt3a are expressed in the dorsal portion of the neural tube. The culture of mouse or avian NCCs with Wnt results in an increase in the number of pigment cells at the expense of other cell types [81]. In zebrafish, the overproduction of β-catenin in migrating NCCs promotes the formation of pigment cells at the expense of neurons and glia [82]. Wnt signaling promotes pigment cell fate through direct activation of the melanocyte transcription factor Mitf-M by β-catenin (reviewed in [83]). Pax3, which is also produced early in the dorsal neural tube and in NCCs, is important for expansion of the melanoblast population [84] and for subsequent differentiation, as it transactivates Tyrp1 and acts in synergy with Sox10 to activate Mitf [85]. The FoxD3 transcription factor plays a critical role in determining the balance of NCC migration between the dorsolateral and dorsoventral migratory pathways. This factor inhibits melanoblast development. It is produced in the dorsal neural tube and in NCCs migrating early in the dorsoventral pathway. It continues to be produced in immature NC-derived cells, including Schwann cell precursors (SCPs), in which it may prevent differentiation. Its depletion from avian neural tubes promotes pigment cell development, whereas its overproduction prevents migration along the dorsolateral pathway [86]. Thomas and Erickson suggested that FoxD3 regulates the assignment of cells to a particular lineage by repressing Mitf gene expression in the early stages of neural crest development [87]. The two major signaling pathways stimulating the differentiation of melanocytes from the neural crest are the stem cell factor (SCF/Kitl)/Kit and Edn3/Ednrb pathways. Both Ednrb and Kit are involved in multiple steps in pigment cell development. Kit signaling is important at several timepoints in melanocyte development, having independent effects on both migration and survival [88–90]. Its role has been widely studied in the many Kit mutant mice generated to date, all of which present pigmentation defects. Kit is produced, together with Mitf, in both premigratory and migrating melanocytes, throughout their development [91], and its ligand, Kitl, is produced in a complementary manner in the dermamyotome, dermis, and hair follicles. Kit also seems to promote melanocyte

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differentiation through post-translational modifications of Mitf [92]. Edn3 signaling is also important during many steps of melanocyte development, for migration, proliferation, survival, and differentiation [93]. In avian embryos, premigratory NCCs produce Ednrb, whereas Ednrb2 is activated in migrating and differentiated melanocytes [94]. In mice, Ednrb is required between E10 and E12.5, when melanoblasts migrate and proliferate [95]. Edn3/Ednrb also promotes Tyr gene expression [96]. Edn3 has a tissue distribution complementary to that of Ednrb, particularly in the epidermis [97, 98]. Edn3 is required earlier than Kit for the early migration of melanoblasts in the dermis [99]. These two signals are thought to influence each other. The gene encoding Mitf-M is considered to be the pigment cell master regulatory gene. Mitf-M directly regulates the expression of the melanogenic genes encoding Pmel17, Melan-A, and Trpm1, and the melanin synthesis enzyme genes encoding Tyr, Tyrp1, and Dct. Many Mitf mutant mice have been generated and all present pigmentation defects [100]. Mitf homologs are found in zebrafish and Xenopus, and are also required for pigment cell development. In addition to regulating melanocyte differentiation, Mitf also regulates the survival of these cells, by controlling Bcl2 levels [101], and proliferation, by adjusting Ink4a and p21 levels [102, 103]. Many genes regulate Mitf expression and, thus, melanocyte development, either directly or indirectly. These genes include members of the Snail family (Snail and Slug), the Sox9, Sox10, and Ap2a genes. Sox10 transactivates Mitf [104], with which it acts in synergy to activate Dct [105]. A first wave of differentiation of melanocyte occurs at birth. Other waves of differentiation occur periodically during life, melanocyte production is required to repopulate the adult hair follicles at each hair cycle. These melanocytes are produced from Dct-positive melanocyte stem cells present in the bulge area of the hair follicle [106]. Cell division and differentiation must be tightly regulated, to maintain the stem cell population and to guide their differentiation into melanocytes at each hair cycle. Stem cell maintenance is regulated by Mitf, Pax3, and Wnt signaling, all of which are also required for melanocyte development. Defects in the maintenance of this stem cell population may be responsible for hair graying [100, 107]. 2.3.1.2 Late Determined Melanoblasts: A Common Origin with SCPs and the Dorsoventral Migratory Pathway There seems to be a second pathway for the generation of melanocytes in the skin [108]. Growing nerves projecting throughout the body serve as a niche for stem/ progenitor cells, containing SCPs, which give rise to large numbers of skin melanocytes. These melanocytes develop after those following the dorsolateral pathway. They colonize the dorsal and lateral body walls, and seem to be the major melanocytes present in the limbs, as blocking the dorsolateral pathway has no effect on the number of melanoblasts in the limb bud. NCCs that delaminate early follow the ventral pathway of migration, with temporal distinctions between the various cells types generated in a ventral to dorsal direction, with sympathetic

2.3 Embryonic Development of Melanogenic Cells

neurons produced first, followed by sensory neurons, while SCPs are produced throughout this period. SCPs are defined as Sox10+ NCC-derived cells that are tightly associated with neuronal projections early in embryonic development and able to migrate long distances along the nerves. Sox10 is also expressed by multipotent NCCs and melanocytes. Melanocytes produced from the nerves innervating the skin originate from Sox10+/Krox20− SCPs of the nerve and produce Mitf and Sox10. They appear in the distal ventral ramus of the spinal nerve and then along the dorsal ramus nerves, which innervate the skin and axial muscles of the dorsal and lateral trunk. SCPs are generated due to a lack of neuronal specification by Hmx1 gene function. This homeobox transcription factor may control a critical transcriptional switch between neuronal and glia melanocyte fates in the ventral NCC pathway. It is produced only in cells committed to a neuronal fate and is required for neurogenesis. Its absence leads to a shift in the balance between glial cells and neurons, increasing the numbers of SCPs and, thus, of melanoblasts. The determination of cell fate (SCP versus melanocyte) depends on contact with the nerve. For cells to be maintained as SCPs, they must be in contact with nerves. In the absence of signals provided by the nerve, some SCPs instead acquire a melanocyte fate. Promyelinating and myelinating Krox20+ Schwann cells do not normally differentiate into melanocytes, but they retain the competence to do so if nerve contact is lost. Krox20 expression defines a restriction of cell fate that normally prevents SCPs from differentiating into melanocytes. Cell fate determination also depends on interactions with neuregulin-1, which is expressed in neuron axonal membranes, and the ErbB2/ErbB3 complex receptor expressed by Schwann cells. Neuregulin signaling promotes the survival and proliferation of SCPs, and determines whether they have a glial or melanocyte fate. In addition, insulin-like growth factor-1 and platelet-derived growth factor, soluble factors produced in Schwann cells, have opposite effects to neuregulin-1, promoting melanocyte differentiation, survival and expansion from SCPs. Thus, Schwann cells are the source of both glia and melanocytes. This may help to account for the combination of changes in skin pigmentation and neurological disorders observed in patients with neurofibromatosis type 1. 2.3.2 Nonclassical Melanocytes

With the exception of the RPE cells, which are derived from the neuroepithelium, nonclassical melanocytes, like classical melanocytes, are derived from the neural crest. These melanocytes may follow other migration routes, such as the dorsoventral pathway between the neural tube and the somites in the vagal region. They also differ from classical melanocytes in terms of their signaling pathway dependence. In general, decreases in Kit receptor tyrosine kinase signaling affect melanocyte development. However, the melanocytes in the eye, ear, and Harderian gland seem

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to be less sensitive to Kit signaling than cutaneous melanocytes, as their precursors produce very small amounts of Kit and they are stimulated more effectively by Edn3 or hepatocyte growth factor (HGF) signals. Inhibition of these pathways specifically prevents the growth and differentiation of these noncutaneous melanocytes. Conversely, the induction of Edn3 or HGF expression in mouse skin and epithelial tissues promotes the survival and differentiation of noncutaneous and dermal melanocytes, with no similar effect on epidermal melanocytes, regardless of Kit signaling. Dermal melanocytes behave more like those of noncutaneous tissues than like epidermal melanocytes. There seem to be two major melanocyte populations: Kit-sensitive cutaneous melanocytes in the epidermis, and weakly Kit-sensitive noncutaneous and dermal melanocytes. These molecular differences between noncutaneous and dermal melanocytes, on the one hand, and epidermal melanocytes, on the other, may be important in the pathogenesis of melanocyterelated diseases and melanomas [109]. Little is known about the embryonic development of nonclassical melanocytes in mice and humans. A little information is available concerning the origin of the melanocytes found in the eye and the heart, but almost nothing is known about the origin of the melanocytes found in the brain or inner ear. Even less is known about the origin of nonclassical melanocytes in other species. 2.3.2.1 Melanocytes of the Murine Eye Melanocytes found in the eye (RPE and uveal melanocytes) have two different origins: neuroepithelium and neural crest. 2.3.2.1.1 Melanocytes of the RPE RPE cells are generated directly from the optic neuroepithelium and remain embedded in their local epithelial environment. An evagination of the neuroepithelium in the ventral forebrain is at the origin of the vertebrate eye. The optic neuroepithelium will be partitioned into three distinct territories, soon after the formation of the evagination: the distal territory will give rise to the future retina, the proximal territory to the optic stalk, and the dorsal territory to the RPE. The transition zone between the future retina and the RPE, the ciliary margin zone (CMZ), is at the origin of the ciliary body and the iris [110]. Eye development in the mouse starts at around E7.5. The neuroepithelium evaginates laterally in a dorsal-distal direction and extends toward the surface ectoderm, generating the optic stalk (which will become the optic nerve) proximally and the optic vesicle (which will become neuroretina, RPE, ciliary body, and iris) distally. The distal-most part of the vesicle forms the optic cup once it comes into contact with the surface ectoderm. By E13.5, the outer wall of the cup consists entirely of RPE. At E9.5, Mitf, a key pigmentation gene essential for skin melanocyte development, is expressed throughout the budding optic vesicle, together with Pax2 and Pax6. However, as early as E10.5, its expression is downregulated in the future retina, but becomes stronger in the future RPE. By contrast, Pax2 and Pax6 are downregulated in the future RPE, with Pax2 becoming concentrated in the optic stalk and Pax6 in the future retina [111].

2.3 Embryonic Development of Melanogenic Cells

Genes Involved in RPE Development The patterning of the optic neuroepithelium results from exposure to various extracellular ligands produced either by the neuroepithelium itself or by the surrounding tissues. Indeed, the extraocular mesenchyme promotes RPE development by expressing activins [112]. Hedgehog (HH) proteins have been found in the RPE of zebrafish, Xenopus, and mouse, and their receptors and signal transduction proteins are expressed in the corresponding areas [113]. HH signaling promotes RPE formation [114, 115]. Fibroblast growth factors (FGFs) are negative regulators of RPE development. They are expressed in the surface ectoderm overlying the eye primordium and the corresponding receptors are found in the future neuroretina. In addition, the future retina itself produces FGFs. Despite this retinal expression, however, removal of the surface ectoderm and, thus, of a major source of FGFs, leads to the conversion of the presumptive neuroretina into pigmented RPE-like cells in both chicken embryos and optic vesicles harvested from mouse embryos. Conversely, exposure of the presumptive RPE to FGF leads to its development into neuroretina [116]. Nevertheless, FGFs are highly redundant in the eye. As a result, genetic studies have shown that no single ligand/receptor pair seems to be critical. The optic vesicle initially displays a high degree of developmental plasticity, indicating that each cell in the neuroepithelium can, in principle, respond to each of these ligands. The cells are, thus, initially equipotent. This raises questions about how the RPE is separated from the neuroretina distally and from the optic stalk proximally. Mitf is a critical transcription factor in the eye, playing a key role in separating the neuroretina from the RPE. It is produced in large amounts in the RPE [117, 118] and mutations rendering this molecule nonfunctional result in a hyperproliferating, unpigmented RPE, the dorsal part of which assumes a neuroretinal fate. These RPE abnormalities lead to small eyes. For more details on Mitf regulation in the RPE, see [110]. Vax proteins are responsible for separating the optic stalk from the RPE. Vax1 is confined to the optic stalk, whereas Vax2 is restricted to the ventral neuroretina [119, 120]. The expression patterns of the Vax genes are therefore complementary to that of the Mitf gene, which is retained in the RPE, with a sharp boundary toward the optic stalk. Mitf and Vax are involved in mutual repression, leading to the formation of sharp boundaries between the optic stalk and RPE. The Kit and Edn3 signaling pathways, which are essential for melanocyte development, do not seem to be important in the RPE. In the RPE, cell proliferation is compatible with pigmentation and, thus, with differentiation. Mitf, which is essential for RPE proliferation, is also involved in the regulation of pigment gene expression and pigmentation in the RPE [118]. 2.3.2.1.2 Uveal Melanocytes Uveal melanocytes are neural crest-derived cells that migrate towards the eye during development. In humans, uveal melanogenesis begins in the 20th week of embryonic development and finishes some weeks after delivery. This is the reason for which definitive eye color – iris color – cannot be determined until the age of 6 months.

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Choroid The choroid accounts for the vast majority of uveal melanocytes. The RPE is essential in the development of the choroid. Molecular interactions between the RPE and periocular mesenchyme are essential for melanocyte differentiation and vascular development in the choroid. The melanocytes of the choroid are derived from the cranial NCCs. In the human embryo, the pigmentation of choroidal melanocytes occurs late in gestation and is complete at birth. In the mouse, the pigmentation of choroidal melanocytes begins soon after birth and is complete by two weeks of age. In the absence of the RPE – if FGF9 is expressed in the presumptive RPE, resulting in a switch to neural retina, for example – the choroid fails to develop, demonstrating the requirement for the RPE as a source of inductive signals for choroid development [49]. Iris The iris consists of five cell layers, two of which are pigmented: the stroma and the posterior pigment epithelium [48]. The iris pigment epithelium consists of a double layer of cuboidal pigmented cells that are tightly fused. These cells are of neuroectodermal origin, and are derived from the CMZ at the anterior end of the optic cup. By contrast, the stromal melanocytes are derived from NCCs and migrate through the uveal tract during development. Ciliary Body Like the iris, the ciliary body – a structure linking the iris to the choroid and forming part of the uveal tract – has a pigmented epithelium derived from the CMZ and stromal melanocytes derived from the neural crest. 2.3.2.2 Melanocytes of the Murine Heart The melanocytes of the heart are derived from cardiac NCCs, which leave the neural tube between the postotic area and the first four somites (a domain that overlaps the cranial and vagal neural crest). Dct-positive melanoblasts have been found in the postotic area extending through the second and third branchial arches, and in a more posterior position, in the region of somites 2 and 3. They have also been seen migrating close to the sixth branchial arch arteries. At E11.5, they are found close to the endocardial cushions and appear to enter the heart via the septum primum. These cardiac melanoblasts seem to emigrate from the neural tube earlier than those migrating dorsolaterally to colonize the skin. At E12.5, the cardiac melanoblasts are found in the AV endocardial cushions, and in the areas surrounding the aortic sac and common cardinal vein. They are also found on the dorsal side of the right atrium, apparently traveling along the anterior cardinal vein and the sixth branchial arch artery [61]. By E13.5, these cells are found in the pulmonary veins and septal leaflets of the AV valves. Between E14.5 and E16.5, they colonize the posterior atrium and atrial-pulmonary venous anastomoses and, by late gestation, they are found in regions of the compact AV node and sinoatrial node [62]. This pattern of expression is subsequently maintained throughout adulthood. Cardiac melanoblasts originate from the same axial levels as cardiac NCCs, with which they share migratory pathways, but they constitute a separate population in terms of their level of commitment, the timing of their arrival in the heart, and their dependence on signaling. They seem to be derived

2.4 Transfer of Melanin from Classical and Nonclassical Melanocytes

from a subpopulation of cardiac neural crest precursors that do not express Wnt1 and Pax3 at early timepoints [61, 62]. 2.3.2.3 Other Nonclassical Murine Melanocytes In the brain, the melanocytes in the sympathetic cephalic ganglia and leptomeninges are derived from cephalic NCCs. In the inner ear, the melanocytes of the stria vascularis are derived from cephalic NCCs. 2.3.2.4 Other Organisms Chromatophores are just one of a number of cell types generated by the neural crest during embryonic development in lower vertebrates. They migrate long distances to populate many organs of the body, including the skin, eye, ear, and brain. In fish and Xenopus, pigment is produced in the chromatophores before migration is completed. Chromatophores leave the neural crest in waves and follow either a dorsolateral route through the dermis, entering the ectoderm through small holes in the basal lamina, or a ventromedial route between the somites and the neural tube. In fish, the melanophores appear laterally at about 24 h of development and are subsequently found along the trunk. The xanthophores are the next to appear, followed by the iridophores [121]. The melanophores of the RPE of the eye constitute an exception. They are not derived from the neural crest and originate instead from the optic cup generated from the neuroepithelium, as in higher vertebrates. The development of chromatoblasts – the precursor cells of chromatophores – seems to be dependent on the same signaling pathways as that of higher vertebrate melanocytes, because Kit, Sox10, and Mitf play an important role in controlling chromatophore differentiation in fish [121, 122]. Defects in these proteins may result in chromatophores being entirely absent or absent from particular regions, resulting in a leukistic disorder.

2.4 Transfer of Melanin from Classical and Nonclassical Melanocytes

Melanin is actively transferred principally from epidermal melanocytes. We will not focus here on the biogenesis of melanosomes and the transport of the melanin, which are dealt with in other chapters of the book (see Chapters 9 and 10). Instead, we describe here the conditions and proteins essential for the correct transfer of melanin from the donor cells to the recipient cells. The presence or absence of crucial players may account for the lack of melanin transfer from nonclassical melanocytes. The epidermal melanin unit is a functional unit consisting of one melanocyte and several neighboring keratinocytes. The melanocytes synthesize the melanin and donate it to the recipient cells, the epidermal keratinocytes, which acquire and retain most of the melanin in the skin. The melanosome is a unique membrane-bound organelle in which melanin biosynthesis takes place. Melanin is a complex pigment that protects the skin

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against the potentially harmful effects of light, by absorbing and scattering light and reducing the production of ROS [123]. For this to be achieved, melanosomes must be transported to the tips of melanocyte dendrites and transferred into the surrounding keratinocytes [124]. This transfer is essential for photoprotection of the skin and the maintenance of normal skin color. Melanosomes are lysosomerelated organelles [125] and they display four stages of maturation, based on their appearance under an electron microscope and their melanization status [1]. Stage I melanosomes (or premelanosomes) are round vesicles found in the perinuclear zone, where they probably develop from the endoplasmic reticulum [2], or from endosomes [3]. The transition of premelanosomes to stage II of maturation involves an elongation of the vesicles and the organization of a distinct fibrillar matrix, but no melanogenesis activity is detected, despite the presence of Tyr. Melanin is first synthesized in stage III, when it is deposited on the fibrillar matrix. In stage IV, deposition continues until the internal structure is no longer visible [4, 5]. 2.4.1 Melanosome Transport

Skin pigmentation is dependent on the transport of melanosomes within melanocytes, from the area of synthesis close to the center of the cell to the peripheral dendrites, before transfer to keratinocytes [126–129]. The intracellular transport of melanosomes is directed by two major polarized cytoskeletal macromolecular polymers – actin filaments and microtubules – composed of actin monomers and dimers of α- and β-tubulin, respectively. Melanosomes are shuttled along the microtubules by certain members of two major families of motor proteins: dyneins and kinesins [130]. Transport along actin filaments is mediated by myosins [131]. Dynein and kinesin act as short cross-bridge structures connecting the organelle to the microtubules [130, 132]. The net transport of melanosomes depends on the balance between the forces directed toward the center and those directed toward the periphery in melanosomes. Myosin Va is an important motor in the actin filament-based transport of organelles [133]. It captures melanosomes delivered to the periphery via the centrifugal component of microtubule-dependent movement, by mediating their interaction with actin filaments forming a cortical shell running the length of the dendrites right to their tips [134]. The long-range, bidirectional, microtubule-dependent movements of melanosomes, coupled with the actomyosin Va-dependent capture of melanosomes in the periphery, is the principal mechanism underlying the centrifugal transport and peripheral accumulation of melanosomes in melanocytes. 2.4.2 Melanosome Transfer

In mammals, skin and coat colors are regulated by melanosome transfer from melanocytes to neighboring keratinocytes and/or to the hair bulb cells. In humans,

2.4 Transfer of Melanin from Classical and Nonclassical Melanocytes

pigment is transferred principally to protect against the damaging effects of UV radiation from the sun [135]. Melanocytes close to the basal lamina in the skin export melanin to the surrounding keratinocytes in the epidermis, resulting in a tanning effect. The melanized keratinocytes are constantly shed and renewed, thus creating a need to produce new melanin continually. The number of melanocytes is similar in humans with different skin types, the differences between ethnic groups resulting mostly from differences in the melanogenic activity of each melanocyte and the size and maturation of the melanosomes [135, 136]. Most studies on pigment transfer have been carried out in mammals – principally mice and humans – but some investigations have focused on amphibians [41, 137]. Several models of melanosome transfer are based on the assumption that melanosomes can be correctly retained close to the cell membrane. This maintenance of melanosomes at the cell membrane is achieved by the association of melanosomes with actin filaments, which are abundant at the tips of the dendrites. 2.4.2.1

Melanosome Transfer from Classical Melanocytes

2.4.2.1.1 Mechanisms of Melanin Transfer Melanocytes have many dendrites, making it possible for each melanocyte to maintain contact with several keratinocytes simultaneously. This allows for the phagocytosis of dendrite tips by the keratinocytes, but also makes it possible for large numbers of melanosomes to be maintained close to the cell membrane, facilitating other modes of transfer. The mechanism underlying melanosome transfer is less well understood than the mechanism of melanosome transport from the perinuclear area to the tips of the dendrites. Several models of the transfer of mature melanosomes to neighboring keratinocytes have been proposed, based on different model systems and observation methods: exocytosis, cytophagocytosis, fusion of plasma membranes, and transfer by membrane vesicles. The large number of models proposed reflects the possibility of several of these mechanisms operating simultaneously in the same organism or of different mechanisms operating in animals of different species or ages or in different tissues. The mechanisms of melanosome transfer are described in Chapter 10. 2.4.2.1.2 Molecular Players in Melanin Transfer The molecular and cellular mechanisms involved in melanosome transfer have been only partly elucidated, but membrane fusion between the melanosome and the plasma membrane of the melanocyte dendrite or the keratinocyte membrane seems likely to be involved in the transfer process. Melanogenic paracrine and autocrine cytokines have been discovered in vitro, mediating interactions between melanocytes and other types of skin cells. α-MSH, ACTH, basic FGF, nerve growth factor (NGF), endothelins, granulocyte-macrophage colony-stimulating factor (GM-CSF), SCF, leukemia inhibitory factor (LIF), and HGF have also been reported to be produced by keratinocytes and involved in regulating the proliferation and/ or differentiation of mammalian epidermal melanocytes [138]. Indeed, keratinocytes may produce and release many factors involved in regulating the proliferation

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and differentiation of mammalian epidermal melanocytes through receptormediated signaling pathways. Foxn1 For many years, little was known about the role of epithelial cells in the development of pigmentary interactions. Until recently, it remained unclear whether epithelial cells were “passive,” acquiring pigment only if spontaneously provided by a melanocyte, or “active,” recruiting melanocytes and inducing the transfer of pigment. The group of Janice Brissette has reconsidered this issue of the passive/active role of keratinocytes in melanin transfer from melanocytes. They have shown that Foxn1, a transcription factor, is responsible for identifying pigment recipient cells and recruiting pigment donors to generate pigmentary units. Foxn1 stimulates keratinocytes to emit signals, one of which is FGF2. Melanocytes make use of these signals to recognize the Foxn1-positive cells as targets, to which they connect, via dendrites, and transfer pigment [139]. Foxn1 is the first gene to be implicated in the uptake of pigment, but it is not the only one. Foxn1 is not universally expressed in the pigment recipient cells, being limited to only some of the keratinocytes. Other mechanisms must therefore be involved in the reception of pigment. PAR-2 It has been reported that keratinocytes express a proteinase-activated receptor 2 (PAR-2) [140, 141], from the large family of seven-transmembraneregion receptors that bind guanosine nucleotide-binding proteins. PAR-2 is widespread throughout the body, being found in the gastrointestinal tract, pancreas, kidneys, liver, lung, cardiovascular system, ovary, eye and brain [142–144]. The physiological activation of PAR-2 is induced by serine protease cleavage of its extracellular region, resulting in a new N-terminus that acts as a tethered ligand, free to interact with another region of the receptor, leading to activation. PAR-2 stimulation has been shown to induce a rearrangement of cytoskeletal proteins [145] and an increase in the phagocytic activity of keratinocytes. This increases melanin transfer, thereby increasing the overall pigmentation of the epidermis [145–147]. Intimate contact between melanocytes and keratinocytes is required for PAR-2-mediated pigmentation effects. PAR-2 is expressed in melanocytes only during interactions with keratinocytes [147]. Consistent with the role of PAR-2 in the phagocytosis of melanosomes by keratinocytes and with the stimulation of melanosome transfer by UV irradiation, UV upregulates PAR-2 production and induces its activity, both in vitro and in vivo. Proteases with PAR-2 cleavage activity are also induced in UV-treated keratinocytes [148]. The effect of UV on PAR-2 synthesis and activity is more significant in coloredskinned individuals (skin phototype II and III) than in those with fair skin (skin phototype I) [148]. In vitro experiments have shown that PAR-2 activation stimulates serine protease secretion by keratinocytes, thus creating a positive feedback loop [148]. Analyses of the cellular signaling pathways regulating the effects mediated by PAR-2 have shown that PAR-2-mediated phagocytosis is Rho-dependent, Rho being a downstream mediator of PAR-2. PAR-2 activation results in a rapid and

2.4 Transfer of Melanin from Classical and Nonclassical Melanocytes

concentration-dependent increase in cAMP levels, but PAR-2-mediated Rho activation is not linked to the protein kinase A signaling pathway [149]. In addition to inducing phagocytosis, PAR-2 mediates skin pigmentation by stimulating melanocyte dendrite development through the release of prostaglandins – principally prostaglandin E2 (PGE2) and PGE2α [150], which are released by keratinocytes in response to UV stimulation [151, 152]. KGF The inhibition of serine protease activity does not completely prevent melanosome transfer [146], suggesting that PAR-2 is just one of a number of participants in the melanin transfer process. Indeed, keratinocyte growth factor (KGF/FGF7) has been implicated in melanosome transfer, in which it exerts its effects principally by acting on the recipient keratinocytes [153]. KGF is secreted by dermal fibroblasts [154, 155] and acts by binding to the KGF receptor (KGFR), a splicing variant of fibroblast growth factor receptor 2. KGF binding, together with UV exposure, triggers the activation and internalization of KGFR, thereby inducing phagocytosis and melanosome transfer in vitro [153]. The phagocytosis promoted by KGF involves actin reorganization and the activation of both Rho- and Cdc42/Rac-mediated mechanisms [153]. Furthermore, KGFR is detected in perinuclear phagosomes, suggesting a possible role in the intracellular translocation of melanosomes from the periphery to the central area of the keratinocytes, to protect the nucleus [156]. KGFR activity and signaling are observed in keratinocytes from both light and dark skins, but expression levels are higher in light than in dark skins. This accounts for the weaker lower level of responsiveness of dark keratinocytes to KGF treatment and further demonstrates that the receptor is responsible for differences in melanosome uptake, but that its presence on the membrane is not sufficient in itself [157]. The stronger effect of KGF on melanosome transfer to keratinocytes from light skin than on that to keratinocytes from dark skin suggests that other pathways, in addition to the well known PAR-2-activated pathway [148], must be activated in light skin, thereby enhancing protection against UV-induced damage. Cadherins Cadherins are calcium-dependent transmembrane glycoproteins involved in promoting cell–cell adhesion via homophilic binding and acting as the transmembrane components of cell–cell junctions [158]. Human keratinocytes [159] and melanocytes have been shown to express both E-cadherin and P-cadherin, and these proteins have been shown to play a major role in the establishment of melanocyte–keratinocyte adhesion. P-cadherin seems to be less important than E-cadherin, which has been shown to play a major role in the adhesion of melanocytes to keratinocytes [160]. The role of E-cadherin in melanosome transfer is highlighted by the dissociation of melanocytes and keratinocytes in the acantholytic lesions of Darier’s disease [160, 161], in which there is a loss of keratinocyte– melanocyte adhesion that results in a loss of pigment transfer. Lectins and Glycoproteins In addition to cadherins, the melanocyte–keratinocyte adhesion binds lectins and glycoproteins, via sugar-binding adhesion receptors

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on the surface of the delimiting membranes. Plasma membrane lectins and neoglycoproteins have been shown to be involved in melanocyte–keratinocyte recognition and to facilitate melanin transfer [162]. Some of these membrane receptors have been shown to be upregulated in melanoma cells in response to UV irradiation [163], which is known to increase melanosome transfer [164]. The membrane lectins binding fucose neo glycoproteins are strongly expressed on melanoma cells and only weakly expressed on keratinocytes, consistent with the expression of fucose receptors on melanocytes and the attachment of fucose residues to proteins on the keratinocytes [163]. The addition of specific lectins and neoglycoproteins to cocultures of melanocytes and keratinocytes may disrupt melanosome transfer. These ligands seem to bind their specific plasma membrane receptors and interfere with the melanocyte and keratinocyte intercellular contacts essential for melanosome transfer [162]. This inhibition is reversible and may be enhanced by adding niacinamide [165] SNAREs and Rab Membrane fusion events and exocytosis are often mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and Rab GTPases [166]. These proteins are present in melanocytes, suggesting that they may regulate the targeting of melanosomes to the plasma membrane and the transfer of melanosomes to keratinocytes [148]. SNAREs are mostly membrane-anchored proteins that serve as membrane receptors. On the basis of their distribution, SNARE proteins are classified as either vesicle SNAREs (VAMP/ synaptobrevin) or target SNAREs (syntaxins and SNAP proteins). VAMP proteins can bind to syntaxins and SNAP proteins, forming core complexes that regulate membrane fusion [167]. Melanocytes have been shown to produce both vesicle SNARE (VAMP-2) and target SNARE (SNAP-25, SNAP-23, and syntaxin-4) proteins [148, 168]. The treatment of melanocytes with α-MSH leads to an increase in the production of several SNARE proteins, consistent with a role for these proteins in melanosome transfer [169]. VAMP-2, on melanosomes, and SNAP-23, on the plasma membrane, associate in melanocytes to facilitate membrane fusion. Despite its presence on the plasma membrane of melanocytes, syntaxin-4 does not associate with VAMP-2 and SNAP-23, suggesting that another, as yet unidentified, syntaxin may be involved. Following fusion, melanosomes may be released into the extracellular space, to be taken up by keratinocytes through phagocytosis or a membrane fusion event. Rab GTPases constitute another family of proteins involved in membrane fusion, particularly in the tethering and attachment of melanosomes to the plasma membrane, before fusion actually occurs. Rab3a inhibits regulated exocytosis [170–172]. Its expression is downregulated by UV irradiation, inhibiting melanosome transport and transfer [148, 173]. Rab GTPases involved in melanosome transport include Rab27a, which mediates myosin Va binding to melanosomes through another linker protein, melanophilin. Rab27a keeps the melanosomes attached to the plasma membrane, which is particularly important for exocytosis.

2.4 Transfer of Melanin from Classical and Nonclassical Melanocytes

2.4.2.1.3 Melanosome Positioning within Keratinocytes Following melanosome uptake by keratinocytes, there may be one melanosome per phagosome or several melanosomes within a single large phagosome [174]. Melanosome sorting seems to differ between skin types, as dark-skinned individuals store their large melanosomes individually in single vesicles, whereas fairskinned individuals store smaller melanosomes in larger vesicles within keratinocytes [136, 175]. Within the keratinocytes, melanosomes aggregates towards the apical pole of the nucleus, forming a supranuclear cap protecting the cell from damage due to UV irradiation. They are degraded when the keratinocytes undergo terminal differentiation and desquamation. 2.4.2.2 Transfer of Melanin from Nonclassical Melanocytes Melanin and melanocytes are not restricted to the skin and hair (classical melanocytes) as previously mentioned. They are also found in the eyes (uveal tract, choroid, ciliary body and iris), inner ear (stria vascularis and modulus of the cochlea, dark cells of the vestibular organ), leptomeninges of the entire brain, heart, and adipose tissue (nonclassical melanocytes). Melanocytes are known, as described above, to produce melanin, which they transfer to keratinocytes to protect these cells from the harmful effects of sun exposure [176]. The presence of melanocytes in organs that are not exposed to the sun suggests that these cells must have other functions [35]. Indeed, whereas melanin is continually produced and secreted in the skin and hair, the melanosomes of nonclassical melanocytes are not transferred to the surrounding cells. They are instead retained in the melanocytes, accumulating in the cytoplasm of these cells within the various stromas [63, 67]. The absence of melanin transfer from nonclassical melanocytes to the neighboring cells may be accounted for by the lack of keratinocytes continually producing specific factors modulating melanocyte–keratinocyte contact and controlling the mechanism of melanin transfer to keratinocytes. For example, the cells surrounding melanocytes in the heart are not pigmented, so melanin does not seem to be transferred to or to accumulate in the neighboring cells in this organ, although electron microscopy experiments would be required to confirm this. It has been shown that the skin cells surrounding melanocytes produce specific proteins, such as Foxn1 [139]. Reverse transcription polymerase chain reaction experiments failed to detect Foxn1 mRNA in the heart [63], potentially accounting for the lack of melanin transfer from melanocytes to adjacent cells in the heart. Furthermore, the PAR-2 gene, which is widely expressed in the human body, is only very weakly expressed in the heart and its expression has never been detected in the brain. PAR-2 expression in melanocytes has also been reported to be induced only on interaction with keratinocytes [147]. In conclusion, melanocytes alone, in the absence of keratinocytes, have limited pigmentation power. They require the effective modulators of melanocyte– keratinocyte interactions and melanin transfer produced by the neighboring keratinocytes.

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keratinocytes: analysis of prostaglandin receptors on human melanocytes and effects of PGE2 and PGF2alpha on melanocyte dendricity. J. Invest. Dermatol., 122, 1214–1224. Hanson, D. and DeLeo, V. (1990) Long-wave ultraviolet light induces phospholipase activation in cultured human epidermal keratinocytes. J. Invest. Dermatol., 95, 158–163. Pentland, A.P., Mahoney, M., Jacobs, S.C., and Holtzman, M.J. (1990) Enhanced prostaglandin synthesis after ultraviolet injury is mediated by endogenous histamine stimulation. A mechanism for irradiation erythema. J. Clin. Invest., 86, 566–574. Cardinali, G., Ceccarelli, S., Kovacs, D., Aspite, N., Lotti, L.V., Torrisi, M.R., and Picardo, M. (2005) Keratinocyte growth factor promotes melanosome transfer to keratinocytes. J. Invest. Dermatol., 125, 1190–1199. Marchese, C., Maresca, V., Cardinali, G., Belleudi, F., Ceccarelli, S., Bellocci, M., Frati, L., Torrisi, M.R., and Picardo, M. (2003) UVB-induced activation and internalization of keratinocyte growth factor receptor. Oncogene, 22, 2422–2431. Belleudi, F., Leone, L., Aimati, L., Stirparo, M.G., Cardinali, G., Marchese, C., Frati, L., Picardo, M., and Torrisi, M.R. (2006) Endocytic pathways and biological effects induced by UVBdependent or ligand-dependent activation of the keratinocyte growth factor receptor. FASEB J., 20, 395–397. Boissy, R.E. (2003) Melanosome transfer to and translocation in the keratinocyte. Exp. Dermatol., 12 (Suppl. 2), 5–12. Cardinali, G., Bolasco, G., Aspite, N., Lucania, G., Lotti, L.V., Torrisi, M.R., and Picardo, M. (2008) Melanosome transfer promoted by keratinocyte growth factor in light and dark skin-derived keratinocytes. J. Invest. Dermatol., 128, 558–567. Takeichi, M. (1991) Cadherin cell adhesion receptors as a morphogenetic regulator. Science, 251, 1451–1455. Hirai, Y., Nose, A., Kobayashi, S., and Takeichi, M. (1989) Expression and role of E- and P-cadherin adhesion molecules

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in embryonic histogenesis. II. Skin morphogenesis. Development, 105, 271–277. Tang, A., Eller, M.S., Hara, M., Yaar, M., Hirohashi, S., and Gilchrest, B.A. (1994) E-cadherin is the major mediator of human melanocyte adhesion to keratinocytes in vitro. J. Cell Sci., 107, 983–992. Hakuno, M., Shimizu, H., Akiyama, M., Amagai, M., Wahl, J.K., Wheelock, M.J., and Nishikawa, T. (2000) Dissociation of intra- and extracellular domains of desmosomal cadherins and E-cadherin in Hailey–Hailey disease and Darier’s disease. Br. J. Dermatol., 142, 702–711. Minwalla, L., Zhao, Y., Cornelius, J., Babcock, G.F., Wickett, R.R., Le Poole, I.C., and Boissy, R.E. (2001) Inhibition of melanosome transfer from melanocytes to keratinocytes by lectins and neoglycoproteins in an in vitro model system. Pigment Cell Res., 14, 185–194. Condaminet, B., Redziniak, G., Monsigny, M., and Kieda, C. (1997) Ultraviolet rays induced expression of lectins on the surface of a squamous carcinoma keratinocyte cell line. Exp. Cell Res., 232, 216–224. Jimbow, K., Pathak, M.A., and Fitzpatrick, T.B. (1973) Effect of ultraviolet on the distribution pattern of microfilaments and microtubules and on the nucleus in human melanocytes. Yale J. Biol. Med., 46, 411–426. Greatens, A., Hakozaki, T., Koshoffer, A., Epstein, H., Schwemberger, S., Babcock, G., Bissett, D., Takiwaki, H., Arase, S., Wickett, R.R., and Boissy, R.E. (2005) Effective inhibition of melanosome transfer to keratinocytes by lectins and niacinamide is reversible. Exp. Dermatol., 14, 498–508. Jahn, R. and Sudhof, T.C. (1999) Membrane fusion and exocytosis. Annu. Rev. Biochem., 68, 863–911. Osen-Sand, A., Catsicas, M., Staple, J.K., Jones, K.A., Ayala, G., Knowles, J., Grenningloh, G., and Catsicas, S. (1993) Inhibition of axonal growth by SNAP-25 antisense oligonucleotides in vitro and in vivo. Nature, 364, 445–448.

References 168 Araki, S., Tamori, Y., Kawanishi, M.,

Shinoda, H., Masugi, J., Mori, H., Niki, T., Okazawa, H., Kubota, T., and Kasuga, M. (1997) Inhibition of the binding of SNAP-23 to syntaxin 4 by Munc18c. Biochem. Biophys. Res. Commun., 234, 257–262. 169 Virador, V.M., Muller, J., Wu, X., Abdel-Malek, Z.A., Yu, Z.X., Ferrans, V.J., Kobayashi, N., Wakamatsu, K., Ito, S., Hammer, J.A., and Hearing, V.J. (2002) Influence of alpha-melanocytestimulating hormone and ultraviolet radiation on the transfer of melanosomes to keratinocytes. FASEB J., 16, 105–107. 170 Johannes, L., Lledo, P.M., Roa, M., Vincent, J.D., Henry, J.P., and Darchen, F. (1994) The GTPase Rab3a negatively controls calcium-dependent exocytosis in neuroendocrine cells. EMBO J., 13, 2029–2037. 171 Annaert, W.G., Partoens, P., Slembrouck, D., Bakker, A., Jacob, W., and De Potter, W.P. (1997) Rab3 dissociation and clathrin-mediated endocytosis, two key steps in the exo-endocytotic pathway of large dense-cored vesicles in primary cultures

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of superior cervical ganglia. Eur. J. Cell Biol., 74, 217–229. Geppert, M., Goda, Y., Stevens, C.F., and Sudhof, T.C. (1997) The small GTP-binding protein Rab3A regulates a late step in synaptic vesicle fusion. Nature, 387, 810–814. Araki, K., Horikawa, T., Chakraborty, A.K., Nakagawa, K., Itoh, H., Oka, M., Funasaka, Y., Pawelek, J., and Ichihashi, M. (2000) Small Gtpase rab3A is associated with melanosomes in melanoma cells. Pigment Cell Res., 13, 332–336. Yamamoto, O. and Bhawan, J. (1994) Three modes of melanosome transfers in Caucasian facial skin: hypothesis based on an ultrastructural study. Pigment Cell Res., 7, 158–169. Minwalla, L., Zhao, Y., Le Poole, I.C., Wickett, R.R., and Boissy, R.E. (2001) Keratinocytes play a role in regulating distribution patterns of recipient melanosomes in vitro. J. Invest. Dermatol., 117, 341–347. Lin, J.Y. and Fisher, D.E. (2007) Melanocyte biology and skin pigmentation. Nature, 445, 843–850.

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3 Biological Chemistry of o-Quinones Patrick A. Riley, Christopher A. Ramsden, and Edward J. Land

3.1 General Biological Significance of o-Quinones

An evolutionary approach to the topic of melanins prompts some interesting considerations, for it is clear that, if we adopt an inclusive definition of melanins (i.e., pigments derived from quinone precursors), these compounds arose early in evolution, and are very widespread both in the animal and plant kingdoms. On the other hand, the specialized organelles in which melanin is synthesized and distributed in vertebrates (the melanosomes) are of much more recent evolutionary origin. While this chronological discrepancy poses many biologically significant questions we take the view that the purposes fulfilled by melanosomes remain closely bound to the physicochemical properties resulting from the structure of quinone-derived pigments. Quinones are of central importance in biochemical systems, particularly because of their ability to undergo facile one-electron redox reactions, and p-quinones, by virtue of their relative stability, feature among the electron transporters that permit electron movements across lipid membranes (e.g., ubiquinone). By contrast, oquinones are relatively less stable on account of their susceptibility to nucleophilic addition reactions and this property makes them valuable as cross-linking agents. As a result of their reactivity o-quinones possess many biologically significant functions, as outlined in the following sections. 3.1.1 Antibiosis

The ability of o-quinones to undergo facile reactions with nucleophiles such as thiol and amino groups (see Section 3.2.2) has been utilized in plants both as a means of antibiosis by damaging invasive organisms (“cytotoxic” action), and as a way of modifying and hardening the protective exterior layers such as are found in seed envelopes (“cross-linking” action). The participation of quinone formation in the “stress responses” of plants is exemplified by the browning reactions in

Melanins and Melanosomes: Biosynthesis, Biogenesis, Physiological, and Pathological Functions, First Edition. Edited by Jan Borovanský and Patrick A. Riley. © 2011 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2011 by Wiley-VCH Verlag GmbH & Co. KGaA.

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potatoes and bananas that takes place following any surface damage. The browning of fruits appears to be a reaction to invasion by parasitic organisms as well as a generalized response to surface trauma permitting the access of melanogenic substrates (such as dopamine) to catechol oxidases that are normally separately stored in segregated intracellular compartments. As well as exerting an antibiotic action by attacking surface structures the localized production of quinones may have an action in cross-linking structural molecules and rendering them resistant to degradation. This structural reinforcement appears to be the major function of quinonoid polymers in the cell walls of spores. 3.1.2 Defensive Secretions

In insects the cytotoxic potential of o-quinone generation is the basis of their immune system and quinones form an important component of defensive secretions. Quinones have been shown to be a significant component of defensive secretions of many small insects such as millipedes and Bombardier beetles. The red-legged millipede (Metiche tanganyiense) is able to prevent predation by the dwarf mongoose with a hot quinone-containing spray. The irritant properties of quinones have been recognized for millennia and they have been employed as laxatives for over 4000 years, and concoctions of madder as a protection against witchcraft have a history extending back to the time of the Essenes [1]. It is likely that the ink of cephalopods exerts its action not so much by obscuring vision (which is of limited significance in guiding predators in deep waters) as by the deterrent action on sensitive chemoreceptor organelles of reactive o-quinones generated in the ink, which contains a mixture of tyrosinase and substrate molecules. 3.1.3 Balanid Adhesion

An interesting example of the secretion of tyrosinase utilizing the action of the enzyme on extracellular substrates, in particular on proteins or peptides containing tyrosine residues, is the involvement of quinone cross-linking in adhesion of barnacles. The cements involved in adhesion of several species of barnacle include tyrosine-rich peptides: the protein glues of the blue mussel have been characterized and are members of a 3,4-dihydroxyphenylalanine (dopa)-rich family of polypeptides that cross-link by an oxidative tanning process. Mussels produce protein “byssus” threads secreted by the mussel foot that anchors the mussel to solid substrata in the inter-tidal region. These threads consist of collagen fibers embedded in a polyphenolic protein matrix in which there is a high level of posttranslational hydroxylation of tyrosine and proline residues to give dopa and hydroxyproline, respectively. On oxidation to the corresponding o-quinone of the dopa residues, these proteins form cross-links that stabilize the adhesion. Similar substrate peptides have been isolated in several species of balanids and the crosslinking reactions are tyrosinase-catalyzed [2].

3.1 General Biological Significance of o-Quinones

3.1.4 Cuticular Hardening in Insects

The cross-linking action of o-quinones is involved in insect cuticular hardening. The process has been studied quite extensively and relies on the ability of certain side-chain substituted substrate molecules to be successively oxidized so as to form cross-links between adjacent proteins [3]. 3.1.5 Pigmentation

The previous examples are what may be termed initial benefits of melanogenesis in that they derive from the primary oxidation that leads to the production of pigment. The phases of the evolution of melanin pigmentation have been summarized [4] as involving the following steps: i)

Secretion of an o-quinone-generating enzyme with defensive and crosslinking potential.

ii) Intracellular retention of the enzyme with accumulation of a polymerized product of o-quinones and their derivatives, permitting photoreceptor screening. iii)

Limitation of pigment production to specific cells leading to organismal patterning with roles in camouflage and display.

iv)

Transference of pigment to acceptor cells enabling regional effects on durability, photoprotection, and excretion.

Some of these actions are dependent on the structure and properties of melanin. For example, the increased durability of pigmented tissues, demonstrated by the greater resistance to mechanical damage and greater strength of black compared to white feathers, derives from the polymeric structure of melanin [5]. The detailed structure of melanin is unknown, but there is overwhelming evidence that the basic structure entails a backbone of indole moieties that contains both quinone and hydroquinone residues [6]. A high degree of conjugation exists in the polymer which imbues it with strong photon absorption in the visible and UV spectrum. An interesting feature of melanins is the modification of optical absorbance characteristics on exposure to light due to oxidation of catecholic groups to quinones. This has the effect of increasing the degree of conjugation of the polymer and the electron delocalization reduces the energy gap between bonding and antibonding π-orbitals. The effect of this is that, as the degree of conjugation increases, lower and lower quantal energies are required for absorption. This bathochromic characteristic of melanin results in significant photon absorption even in the IR portion of the spectrum, and this may permit it to act as a means of thermal absorption in poikilotherms and animals in cold climates where solar radiation constitutes a significant factor in energy conservation.

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3 Biological Chemistry of o-Quinones

66

Photoprotection is widely considered to be the most significant action of melanin in humans and there is good evidence that increased epidermal melanization is associated with a significantly decreased risk of skin cancer related to sun exposure. The mechanism may involve the absorption of energetic photons that might damage important cellular structures such as DNA and, therefore, constitutes an aspect of screening. Another mechanism of protection by melanin may involve scavenging of free radicals. However, an alternative aspect of the protective action of melanin is to consider its functions to include the elimination of cells that may have undergone deleterious mutation. Thus, a genoprotective mechanism exhibited by melanin may involve the generation of free radicals that are toxic to cells that have been exposed to potentially genotoxic doses of light [7]. Owing to the conjugated structure of melanin there is an equilibrium between the quinone and hydroquinone moieties in the polymer that renders the pigment a facile electron exchanger. It can, thus, act as a free radical generator or scavenger [8]. In addition, the relatively high density of negative surface charge on melanin renders it a cation trap and it has been suggested that this constitutes one of the biological benefits conferred by surface pigment since by desquamation in keratocytes melanin provides an excretory pathway for metals and other cationic materials [9]. Finally, as the most prominent surface pigment of vertebrates and some other classes of animals and plants, melanins are important components of external display and camouflage.

3.2 o-Quinone Reactivity 3.2.1 Structure and Reactivity

Although o-quinones (1,2-benzoquinones) can often be isolated as crystalline compounds, they are inherently unstable. This is primarily because the six-membered ring is not aromatic (i.e., it does not contain six π electrons in cyclic conjugation in accord with Hückel’s 4n + 2 rule). The driving force for many of the reactions of o-quinones is the formation of an “aromatic sextet,” which is associated with thermodynamic stability. This is illustrated by the facile reduction of o-quinones 1 by ascorbic acid 2 (and many other reducing agents) to give aromatic catechols 3 (Equation 3.1).

R

O

HO +

O

O HO

1

H CH OH 2

H

1

OH

H HO

R

2

O

+ HO

O

O 3

H

O

1

CH2OH OH

O

(3.1)

3.2 o-Quinone Reactivity

Owing to their instability, little structural information is available for simple monocyclic o-quinones. Stability can be increased by substituent effects and the molecular structure of the sterically hindered 3,5-di-tert-butyl derivative 4 has been determined using X-ray diffraction [10]. In contrast to catechol 5, in which all the C–C bond lengths (average 1.385 Å) are characteristic of an aromatic ring (benzene 1.39 Å)[11], the C–C bonds of the o-quinone 4 have lengths corresponding to either double bonds (average 1.342 Å) or single bonds (range 1.439 to 1.554 Å). Magnetic criteria such as nucleus-independent chemical shifts (o-quinone NICS(1) 1.3; benzene NICS(1) −12.8) and proton chemical shifts also indicate that o-quinones are non-aromatic [12].

O

But 1.340

1.399

HO

1.378 O

HO

But 1.344 4

1.391 5

In addition to reduction to a catechol (e.g., Equation 3.1), there are other mechanisms by which o-quinones react to achieve aromaticity. A common mode of reaction is addition of a nucleophile (Equation 3.2), which also leads to aromatic catechol derivatives (e.g., 6).

O

R1

H2N–R2

O

R1 NH2 R2

–

O

HO

R1

HO

N R2 H

+

O H

6

(3.2)

It is beyond the scope of this chapter to survey all the modes of reactions of oquinones. Only reactions of known biological significance are covered in the following sections. However, cycloaddition is a common mode of reaction and the following examples are worthy of brief mention. [4 + 1] Cycloaddition readily occurs with phosphorus derivatives to give the cycloadducts 7 (Equation 3.3), which also achieve aromatic stability. The stable adducts 7 can be useful for characterizing o-quinones [13]. O O

R1

PR3

R1

O R3P

(3.3)

O 7

Another common type of cycloaddition is the [4 + 2] Diels–Alder reaction with alkenes that can form the cycloadducts 8 and/or the cycloadducts 9 (Equation 3.4).

67

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3 Biological Chemistry of o-Quinones

In some cases an o-quinone will react with itself to give a dimeric cycloadduct. Other types of cycloaddition reaction of o-quinones have been reviewed [13, 14]. R2

R1

O

R2

R1

O

R2 and/or

+ O

R2

R2

R2

O 8

CO CO

R1

9 (3.4)

It should also be noted that because of their inherent reactivity, some o-quinones rapidly react via variations of the above modes of reaction to give unexpected products. Thus, the o-quinone 10, formed from pyrogallol, rapidly dimerizes with elimination of carbon monoxide to give purpurogallin 11 (Equation 3.5) [15]. OH O

OH O

–CO

2

OH

HO

(3.5)

HO

O 10

11

3.2.2 Reduction

Reduction is the process of adding electrons. o-Quinones readily accept one electron to form a semiquinone radical anion 12 followed by a second electron to form a catechol dianion 15 (Scheme 3.1). Depending upon the conditions these species R1

O

e–

• –

O

R1

O

O

1

H+

13 e–

R1

HO –

Scheme 3.1

H+

–

14

R1

O

–

O

3

R1

O

12

R1

HO

•

HO

H•

HO

H+

O 15

3.2 o-Quinone Reactivity Table 3.1

Redox potentials (Eo) of selected quinones.

Entry

Quinone

Eo (V)

Reference

1 2 3 4 5 6 7 8

1,2-benzoquinone 1,4-benzoquinone 2,3,4,5-tetrachloro-1,2-benzoquinone 3,5-di-tert-butyl-1,2-benzoquinone 4-hydroxy-1,2-benzoquinone 1,2-naphthoquinone 1,2-anthraquinone 9,10-phenanthraquinone

0.795 0.711 0.830 0.580 0.594 0.576 0.490 0.460

[16] [17] [16] [18] [19] [20] [20] [20]

may be protonated (13 and 14). In some cases the second reductive step may occur via hydrogen atom transfer from the partially oxidized reducing agent (XHᠨ). This → 3) accounts for many of the biologically signifireversible reductive process (1 ← cant reactions of o-quinones. The ease of reduction of o-quinones to catechols (1 → 3) is indicated by the redox potential (Eo), which can be measured potentiometrically. Substituents, including fused rings, can significantly modify the redox potential. As the redox potential increases the quinone becomes a more powerful oxidizing agent. Selected redox potentials are shown in Table 3.1. Electron-withdrawing groups increase the redox potential (e.g., Entry 3) and hence the oxidizing power of the o-quinones, that is, they are more easily reduced. Conversely, electron-donating substituents (e.g., Entries 4 and 5) and fused rings (e.g., Entries 6–8) reduce the oxidizing power. A difference in redox potentials of o-quinones leads to a process known as redox exchange, which plays a significant role in the early stages of melanin biosynthesis. This process is summarized in Equation 3.6 in which an o-quinone 17 oxidizes a catechol 16 to give a new o-quinone 18 and is itself reduced to the catechol 19. Thus, an o-quinone with a high redox potential such as 2,3,4,5-tetrachloro-1,2benzoquinone (Entry 3, Table 3.1) will oxidize a wide variety of catechols to the corresponding o-quinones. This process occurs via intermediate semiquinone radicals, as shown in Scheme 3.1, with the catechol acting as the reducing agent. In fact, if semiquinone radicals (e.g., 12 → ← 13) are generated in solution by, for example, pulse radiolysis, they very rapidly disproportionate to a mixture of the corresponding catechol and o-quinone.

R1

OH

R2

OH 16

+

O

R3

O

R4 17

R1

O O

R2 18

+

HO

R3

HO

R4 19 (3.6)

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3 Biological Chemistry of o-Quinones

Scheme 3.2 summarizes the role of redox exchange in the early stages of melanin biosynthesis [21, 22]. Since dopachrome 23 is a quinone with a strongly electron-donating substituent (NH), it has a smaller redox potential than dopaquinone 21. This, therefore, favors redox exchange between cyclodopa 20 and dopaquinone 21 to give dopa 24 by a nonenzymatic mechanism. In a similar way, cysteinyldopa 22 is oxidized to cysteinyldopaquinone 25. For further discussion of the balance between these alternative redox exchange pathways, and their influence on tyrosinase kinetics, see Section 3.2.3.

HO

CO2–

O

–

CO2 HO

NH2

O

N H

NH2

HO Cys

20 cyclodopa

21 dopaquinone

22 cysteinyldopa redox exchange

redox exchange

O CO2 O

CO2–

HO

Cys

CO2–

HO

–

NH2

HO

N H

CO2–

O

NH2

O Cys

23 dopachrome

24 dopa

25 cysteinyldopaquinone

eumelanin pathway

pheomelanin pathway

Scheme 3.2

o-Quinones with a tertiary amine side-chain 26 cyclize to the betaines 27 that in aqueous solution are in equilibrium with the catechols 28 [23]. As the species 27 and 28 are associated with a strongly electron-withdrawing substituent (R2N+), they would be oxidized to a quinone derivative 29 with a much higher redox potential than the quinone precursor 26. Consequently, in contrast to the cyclodopa– dopaquinone system (Scheme 3.2), redox exchange does not take place between 26 and 27/28 (Scheme 3.3) and this has been used to study the mechanism of activation of tyrosinase in vitro (see Section 3.3) [24, 25]. –

O NR2

O

O

H+ +

HO

N R2 27

26 Scheme 3.3

O

HO +

HO

N R2 28

X

+

N R2

O

29

3.2 o-Quinone Reactivity

3.2.3 Addition Reactions: Intermolecular addition

o-Quinones are electron-deficient molecules and react with nucleophiles such as amines to give addition products (e.g., 6 in Equation 3.2). The example shown in Equation 3.2 is formally a 1,4-addition to a conjugated carbonyl, commonly referred to as Michael addition. Since the nucleophile can attack more than one position of the ring, including the carbonyl carbons, nucleophilic attack can lead to a variety of addition reactions that are largely beyond the scope of this chapter. One important case of intermolecular addition that merits mention is the addition of cysteine to dopaquinone 21 to give cysteinyldopa 22 (Scheme 3.2). This is formally a 1,6-addition to a conjugated carbonyl. Although 5-S-cysteinyldopa 30 is the major product (74%), there is a smaller amount of the alternative 1,6-adduct (2-S-cysteinyldopa 31) (14%) and a tiny amount (1%) of the 1,4-adduct (6-S-cysteinyldopa 32) (Scheme 3.4) [21, 26, 27]. The small amount of the 1,4-adduct 32 is surprising since Michael addition is favorable for amine nucleophiles. The product distribution for cysteine addition has not been rationalized. Amines are hard nucleophiles (charge controlled), whereas thiols are soft nucleophiles (orbital controlled) and it is possible that the nature of the o-quinone frontier molecular orbitals controls the regioselectivity. Alternatively, thiols are reducing agents and addition may take place via a radical mechanism, possibly involving a semiquinone radical.

CO2–

O

NH2

O

21 dopaquinone Cys

CO2–

HO

NH2

HO

+

Cys HO HO

CO2– NH2

+

CO2–

HO HO

Cys

NH2

Cys 30 5-cysteinyldopa 74%

31 2-cysteinyldopa 14%

32 6-cysteinyldopa 1%

Scheme 3.4

3.2.4 Polymerization

A special case of intermolecular addition is the formation of melanin polymer from indole building blocks. Structural studies suggest that each step in the process is an addition to an indolequinone intermediate. In the eumelanin pathway decarboxylation of dopachrome 23 and subsequent tautomerization gives

71

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3 Biological Chemistry of o-Quinones

5,6-dihydroxyindole (DHI) 33 that is partially oxidized to highly reactive 5,6-indolequinone (IQ) 34 [22, 28]. Product analysis of oligomers suggests that polymerization occurs by nucleophilic attack of DHI 33 on the IQ 34 to give a mixture of intermolecular addition products. The mode of addition seems to favor 2,2′-, 2,4′-, and 2,7′-linkages (i.e., 35–37). This is surprising since indoles usually react with electrophiles at the 3-position. The dihydroxyindole dimers 35–37 then react further in a similar manner giving polymeric material. The dimeric structures in Scheme 3.5 are only representative since DHI-2-carboxylic acid (DHICA) is also known to form polymeric material. 4

HO

dopachrome 23

3

O

2 HO

7

N H

O

N H

33

34

NH HO

2 4′

HO

N H HO

HN HO + HO

OH

35

HO

2 7′

2 2′

+ HO

N H HO

H N

OH OH

N H

OH

36

37

Scheme 3.5

3.2.5 Intramolecular Addition (Cyclization)

In contrast to intermolecular addition, a simpler situation arises when a nucleophile is attached to a side-chain on an o-quinone ring, as is the case for dopaquinone 21. In this case the side-chain limits the rapid nucleophilic attack by the amine to either position 3 or position 5. In practice attack at position 3 is kinetically unfavorable [29] and only the product 20 is formed (Scheme 3.6). The alternative intramolecular cyclization product 38 is not detected. CO2– HO HO

CO2– 5 N H 20 cyclodopa

Scheme 3.6

O O

3

4 5

CO2– NH2

21 dopaquinone

HO X

HN 3

HO

38

3.2 o-Quinone Reactivity

73

It is noteworthy that when the side-chain is lengthened by one carbon atom (i.e., propylamines 40) nucleophilic attack at position 4 to give a spiro product 39 is the fastest reaction (Scheme 3.7). However, this spiro-cyclization is reversible, since the kinetic product 39 is unstable (not aromatic) and the system rapidly equilibrates giving the thermodynamic product 41 [23, 30, 31]. Dopaquinone 21, and related ethylamines, do not undergo spiro-cyclization because four-membered ring formation is unfavorable. +

–

O

3

R2N 4

O

4 5

O

O

–

39

O

HO

NR2

40

+

5 N R2 41

Scheme 3.7

3.2.6 Addition–Elimination (Substitution) Reactions

Addition of a nucleophile to an o-quinone is sometimes followed by elimination of a small molecule, which is often water. The overall reaction is commonly referred to as substitution. An example is the spontaneous cyclization of 5-Scysteinyldopaquinone 42 to the quinonimine 43 with elimination of water (Scheme 3.8). Subsequent tautomerization gives the 1,4-benzothiazine ring 44 characteristic of pheomelanin pigment [21].

CO2–

O

NH2

O H2N

S

CO2–

O

NH2

N H2O

–

O2C

–

O2C

42

CO2–

HO

NH2

N

S

–

O2C

43

S 44

Scheme 3.8

Another example is the cyclization of 6-fluorodopaquinone 45 with elimination of hydrogen fluoride (Equation 3.7), which gives dopachrome 23 directly without requiring oxidation (cf. Scheme 3.2) [32]. CO2–

O O

O

F NH2

O HF

45

CO2– N H

23 dopachrome

(3.7)

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3 Biological Chemistry of o-Quinones

3.3 Role of o-Quinones in Melanogenesis 3.3.1 Nonenzymatic Formation of Melanogenic Intermediates 3.3.1.1 Contributions from Pulse Radiolysis to the Chemistry of Eumelanogenesis and Pheomelanogenesis Up to the mid-1980s, much of what was known about the initial chemical steps of melanogenesis had been discovered by enzymic oxidation studies in which slow enzyme-catalyzed steps are rate determining, precluding the build-up of high enough concentrations of the reactive intermediates to allow their kinetics to be investigated. This pathway, which shows the principal steps of melanin formation, is summarized in Scheme 3.9, essentially an updated version of the classic Raper– Mason scheme for melanogenesis [33–35]. The conversion of tyrosine 46 to melanin was proposed to involve a series of oxidation steps, the initial being the tyrosinase-catalyzed oxidation of tyrosine to the highly reactive o-quinone dopaquinone, the subsequent reactions leading eventually to the eumelanic and pheomelanic polymers proceeding spontaneously. From the mid-1980s, the application of the technique of pulse radiolysis to this field enabled the reaction rate constants associated with a number of the steps shown in Scheme 3.9 to be measured, at the same time providing very strong direct evidence of the accuracy of the commonly accepted scheme of melanin formation. This technique is, for example, able to generate “instantaneously” (i.e., within a few milliseconds) relatively high concentrations (e.g., up to around 10−4 M) of dopaquinone 21. This can be achieved in simple aqueous solution by the careful choice of various solutes including, for example, dopa, KBr, and phosphate buffer, at specific relative concentrations, all under an atmosphere of nitrous oxide. The technique of pulse radiolysis consists of delivering short pulses of ionizing radiation, typically of the order of up to a few microseconds in duration, from an electron linear accelerator to samples contained in high-purity quartz optical cells. Light, often from a xenon arc lamp, is passed continuously through the sample cell, usually at right angles to the path of the electron beam. The short pulse of radiation results in the formation within the sample of transient species that have characteristic absorption spectra. The remainder of the pulse radiolysis equipment is, in essence, a very fast response spectrophotometer, able to follow changing absorption spectra over timescales ranging from microseconds, or less, to minutes. Most of the relevant melanin precursor studies employed the pulse radiolysis apparatus developed from the 1960s by Keene [36] at the Paterson Laboratories, now the Paterson Institute for Cancer Research, Christie Hospital and Holt Radium Institute, Manchester, UK. In 2000, the detection equipment was transferred to the Synchrotron Radiation Laboratory, Daresbury, Warrington, UK, where it continued to be used with a different linear accelerator until 2008 (Figure 3.1).

3.3 Role of o-Quinones in Melanogenesis

HO

COOH NH2 46

O

COOH

r1

HO

COOH HO

N 20 H

HO

cysteine

NH2

O

r3

COOH NH2

HO

21

S

r2

H2N

r4

COOH

O

O

COOH HO

NH2

HO

23

N 47

dopa quinone

HO

r8

25

S

r5

COOH

H2N

COOH HO

NH2

O

24 HO

HO

COOH

COOH

N

HO

22

COOH

O

N 33

N r9

S

HOOC

43

r6

dopa O

O

COOH

HO

COOH N

O

N

O

48

NH2

34

NH2

N (HOOC)

S

44 r7

Eumelanins

Pheomelanins

Scheme 3.9 Outline of reactions involved in melanogenesis showing the reactions for which rate constants have been determined (see Table 3.2).

When ionizing radiation is incident upon a dilute aqueous solution, the radiation, unlike light, is almost completely absorbed by the solvent (water) generating within it mainly two types of very reactive free radicals, namely hydrated electrons (e−aq) and HOᠨ radicals. If the solution is saturated with N2O, all the e−aq are converted into further HOᠨ radicals. These hydroxyl radicals are highly oxidizing and have a tendency to add to aromatic solutes, as well as causing one-electron oxidations. In the presence of excess KBr, these HOᠨ radicals, from both sources, generate Br2ᠨ− radicals which then cause exclusive one-electron oxidation, in the

75

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3 Biological Chemistry of o-Quinones

Figure 3.1 The Manchester Pulse Radiolysis apparatus reinstalled at the Daresbury Synchrotron Radiation Laboratory. The reaction chamber is being set up by Professor E.J. Land, Dr Ruth Edge, and Dr Suppiah Navaratnam.

case of dopa yielding dopasemiquinone radicals. The unstable dopasemiquinones then disproportionate within a few milliseconds into dopaquinone and reformed dopa. From measurements made at 380 nm (the wavelength maximum of dopaquinone 21) in the absence of further additives, the dopaquinone decays over hundreds of milliseconds, with the subsequent formation of dopachrome 23, a precursor of eumelanin, with a wavelength maximum of 480 nm. However, the kinetics of formation at 480 nm does not match exactly the decay at 380 nm, there clearly being an intermediate between dopaquinone and dopachrome. This is interpreted in terms of dopaquinone cyclizing over hundreds of milliseconds (k = 3.8 s−1) [37], forming cyclodopa 20 which rapidly cross-reacts with the remaining dopaquinone 21 to yield reformed dopa 24 and dopachrome 23. In order to measure directly the rate constant of redox exchange between dopaquinone and cyclodopa, the moderately stable cyclodopa was synthesized chemically, and the kinetics of formation of dopachrome upon addition of cyclodopa to pulseradiolytically generated dopaquinone followed. The addition of cyclodopa 20 did indeed result in an increase in the rate of formation of dopachrome 23 absorption at 480 nm, leading to a rate constant for redox exchange between dopaquinone 21 and cyclodopa 20 of 5.3 × 106 M−1 s−1. This rate constant, together with the rate constant for the unimolecular decay of dopaquinone, led to a satisfactory simulation of the optical density versus time curves at 380 and 480 nm obtained from the pulse radiolysis of dopa alone [37].

3.3 Role of o-Quinones in Melanogenesis

In the presence of cysteine, dopaquinone 21 undergoes reductive addition to give cysteinyldopa 22, mainly the 5-S isomer 30, thus initiating the pheomelanic pathway. From studies of the enhanced rate of decay of dopaquinone absorption at 380 nm, a rate constant of 3 × 107 M−1 s−1 was obtained for the reaction of cysteine with dopaquinone [38]. Dopaquinone 21 can also be shown by pulse radiolysis to undergo spontaneous redox exchange with 5-S-cysteinyldopa 30, leading to 5-Scysteinyldopaquinone 25. Although the latter, like dopaquinone, has an absorption maximum at 380 nm, its extinction coefficient is over 3 times greater. This enabled the transition from dopaquinone to cysteinyldopaquinone to be followed easily, and the resultant rate constant of 8.8 × 105 M−1 s−1 was obtained [39]. 5-S-Cysteinyldopaquinone 25 was shown to cyclize (k = 10 s−1) spontaneously to a quinonimine 43, wavelength maximum 540 nm, which, in turn, decayed by tautomerization to a benzothiazine 44 (k = 6.0 s−1). The latter was found to decay subsequently with a rate constant of 0.5 s−1 [40]. Later isolatable intermediates in the series of chemical reactions leading to eumelanin are DHICA (47), a rearrangement product of dopachrome, and its decarboxylation product DHI (33). In an analogous way in which the above redox exchanges between dopaquinone and cyclodopa or 5-S-cysteinyldopa were studied, pulse radiolysis investigations were carried out seeking redox exchange between dopaquinone and DHI or DHICA [41]. Evidence for a redox exchange occurring between dopaquinone 21 and DHI 33 with a rate constant of 1.4 × 106 M−1 s−1 was gained, although the identity of the actual DHI quinoid tautomer 34 awaits application of a more structurally informative detection technique, such as time-resolved resonance Raman spectroscopy. The results obtained with mixtures of dopaquinone 21 and DHICA 47 were interpreted as demonstrating that, in this case, redox exchange is around 10 times slower than for dopaquinone to DHI and also goes less to completion [41]. A summary of the rate constants, obtained by pulse radiolysis for the early chemical reactions of eumelanogenesis and pheomelanogenesis, is provided in Table 3.2 (see also [31]).

Table 3.2

Reaction r1 r2 r3 r4 r5 r6 r7 r8 r9

Rate constants for the reactions shown in Scheme 3.9. First order (s−1)

Second order (M−1 s−1)

3.8 5.3 × 106 3.0 × 107 8.8 × 105 10.0 6.0 0.5 1.6 × 105 1.4 × 106

Reference [37] [37] [38] [39] [40] [40] [40] [41] [41]

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3 Biological Chemistry of o-Quinones

3.3.2 Balance between Eumelanogenesis and Pheomelanogenesis

Both eumelanin and pheomelanin are derived from dopaquinone, and the divergent pathways of biogenesis may be described by the balance between the rate constants of the nonenzymatic reactions involved in the initial steps. Thus, taking dopachrome and cysteinyldopaquinone as representative of the divergent pathways, an index of divergence (D), defined as the ratio of the products of the rate constants of the reactions involved, has been proposed [37]: D=

r3 ⋅ r4 ⋅ [cysteine ] r1 ⋅ r2

where r1, r2, and r3 are the rate constants determined by pulse radiolysis for, respectively, dopaquinone cyclization, redox exchange with cyclodopa, and cysteine addition, and r4 is the rate constant for dopaquinone redox exchange with 5-Scysteinyldopa. This leads to a “cross-over value” (i.e., for D = 1) for switching between predominance of eumelanogenesis to predominant pheomelanogenesis, when the cysteine concentration at the site of melanogenesis is 7.6 × 10−7 M [37]. The factors regulating the intramelanosomal cysteine concentration are discussed elsewhere in this volume (see Chapter 6). 3.3.3 Control of Melanogenesis: Phase I Melanogenesis

The initial reactions in the melanogenic pathway (often referred to as phase I melanogenesis) are of signal importance because of their influence on the activity of tyrosinase and their part in the control of the nature of the final pigmentary product (see Scheme 3.10). An important feature of the spontaneous reactions outlined above is their significance in generating the catecholic intermediate, dopa. Dopa is central to the control of melanogenesis because of its two actions in influencing the activity of tyrosinase both as an activator of mettyrosinase by acting as a reducing substrate and as a suicide inactivator of tyrosinase. 3.3.4 Tyrosinase Activation

Tyrosinase is unusual in that it exhibits two catalytic functions: (i) oxygenase activity (“cresolase” action, shown as “t’ase I” in Scheme 3.10) in which monohydric phenols have oxygen substituted at the 2-position giving an o-quinone [42, 43], and (ii) oxidase activity (“catecholase” action, shown as “t’ase II” in Scheme 3.10) [44] in which a catechol is oxidized to the corresponding o-quinone. One of the characteristics of in vitro oxidation of tyrosine 46 is a “lag period” [45]. Native tyrosinase occurs mainly as met-tyrosinase 49, which cannot oxidize phenols (e.g., tyrosine) and needs to be reduced to deoxy-tyrosinase 50 by a catechol before phenol oxida-

3.3 Role of o-Quinones in Melanogenesis tyrosine

t’ase I cysteine

dopaquinone

cyclodopa

cysteinyldopa

t’ase II

dopachrome

dopa

cysteinyldopaquinone

tyrosinase inactivation Eumelanin

Pheomelanin

pheo/eumelanin balance Scheme 3.10

Phase I reactions and the control of melanogenesis.

tion can begin [46]. In vitro this activating catecholic substrate is generated indirectly by fast redox exchange of dopaquinone formed slowly by the small amount of oxy-tyrosinase 51 present in native tyrosinase. The lag period ends when all the enzyme has been activated by this indirect and relatively slow nonenzymatic formation of dopa (Scheme 3.11) [24, 25]. 3.3.5 Tyrosinase Inactivation

Another unusual feature of tyrosinase is that the oxidase pathway exhibits inactivation kinetics [47–53]. It was shown in 1982 by Dietler and Lerch [54] that this inactivation is linearly correlated with the loss of 50% of the copper atoms from the active site, but the mechanism to account for the loss of copper remained elusive. Dietler and Lerch suggested that it might be the result of oxidative modification of the histidine residues that coordinate the copper atoms in the active site of the enzyme [54], but radical scavengers were without effect on the inactivation process [51, 54] and it is probable that the histidine oxidation they observed was secondary to the inactivation process.

79

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3 Biological Chemistry of o-Quinones

oxygenase pathway

N

– N O 2+ N Cu O N –

51 oxy-tyrosinase

R HO O

R

oxidase pathway

Cu2+ N N

HO HO

R

R

O X

O2

O

O +

+

N N

N

N N Cu1+ N

1+

Cu

50 deoxy-tyrosinase

HO HO

R N N

N

H

O 2+ –

Cu

N N Cu2+ N

49 met-tyrosinase

N = histidine ligand Scheme 3.11

Recently a mechanism was proposed to account for the copper loss during tyrosinase inactivation [55]. Briefly stated, this “Quintox mechanism” postulates that oxy-tyrosinase 51 sometimes binds catechol substrates in the oxygenase mode (i.e., as if they were phenols). Scheme 3.12(a) shows the intermediate 52 formed by binding of a phenol to oxy-tyrosinase followed by elimination of an o-quinone 54 and generation of deoxy-tyrosinase 50. If a catechol were to bind to the active site of the enzyme in a similar way (53, Scheme 3.12b), the extra hydroxy substituent could deprotonate leading a 3-hydroxy-o-quinone 55, reductive elimination of Cu(0), and an irreversibly inactivated form of tyrosinase 56. This processing of catechols as phenols in the oxygenase cycle accounts for the observed suicide inactivation and is consistent with a number of experimental studies [56, 57]. A caveat is that the majority of the recent experimental data bearing on the question of suicide inactivation are derived from work with tyrosinase from mushrooms (Agaricus bisporus) and this limits the extent to which the conclusions drawn from them can be generalized. However, the strongly conserved active center of tyrosinases obtained from different sources [58] makes it probable that very similar, if not identical, mechanisms apply to this class of enzyme in general. While there are no studies unequivocally demonstrating the formation of zerovalent copper during the suicide inactivation of tyrosinase, this mechanism seems to be the most plausible explanation for the loss of enzymatic activity and a variation of the reductive–elimination mechanism has also been proposed [59]. The current state of the evidence has been the topic of a recent review [60] and may be summarized as follows.

3.3 Role of o-Quinones in Melanogenesis a)

b)

H O –

N

N

N Cu2+ – N– O O

Cu2+

N

N N

N

O

O

N N

R

53

O

Cu2+

O

O

52

O

N

– Cu2+ –

–

H

R

H O

N

OH

O R

R

54

N N

+ H2O N

N Cu

55

1+

Cu1+

+ N

N

N

N

50 deoxy-tyrosinase

H2 O N N Cu2+ Cu N N

56 inactivated tyrosinase

Scheme 3.12

A strong prediction arising from the mechanistic hypothesis proposed by the Quintox group [55] is that catechol oxidases (EC 1.10.3.1), enzymes closely related to tyrosinase but which lack oxygenase activity due to subtle differences in the active site [61–63], will fail to exhibit suicide-inactivation kinetics since the copper reduction requires oxygenase-catalyzed generation of a hydroxylated intermediate (Scheme 3.12b). This is not predicted by the alternative mechanism recently advanced by Muñoz-Muñoz et al. [59] since their proposal depends on a protonation of the peroxy oxygen in a bidentate catechol complex. The catechol oxidase extracted from bananas (Musa cavendishii) shows no activity towards monohydric phenols and, in contrast to tyrosinase, it oxidizes a number of catecholic substrates without exhibiting suicide-inactivation kinetics [56]. Further indirect evidence of the involvement of an oxygenase presentation in the inactivation mechanism is the inhibitory effect of addition of a monohydric phenol to the catechol oxidation mixture. The relative inactivation rate (the ratio of the inactivation rate to the initial rate of oxidase activity) of 4-methylcatechol oxidation by tyrosinase is diminished with increasing concentrations of 4-methylphenol [55]. According to the Quintox mechanism the inactivation comes about by a proportion of the substrate binding to the active site in an alternative orientation. The catechol may bind either in the usual bidentate oxidase mode to the active-site copper atoms 57 or like a phenol in the oxygenase mode 58. The orientation of

81

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3 Biological Chemistry of o-Quinones

these binding modes to oxy-tyrosinase differs in respect of the plane defined by the copper atoms and the coordinated molecular oxygen [64–67]. Both modes (57 and 58) have well-defined and inflexible locations of the catechol unit and its associated substituents. It might be anticipated, therefore, that structural constraints associated with different binding modes would be reflected by a differential influence of substituents on the rate of oxidation (k1) and the rate of inactivation (k2). Such differences have been observed by quantitative analysis of a series 4-substituted catechols with ring substituents chosen to give a wide variation and minimal correlation of substituent properties [68].

N N

H O

N

N

– Cu2+ –

2+

Cu

O O H O–

–

H N O N 2+ – Cu – N– O O

N

N H

O

N Cu2+

R

N N

N N

H O

N

– Cu2+ –

N Cu2+

O O H O–

–

F

N N

F

R 57

58

59

There is evidence in the literature of the 5-hydroxylation of dopa that confirms the ability of tyrosinase from different sources to process catechols by the oxygenase pathway [69–72] and a strong prediction of the Quintox mechanism is the generation of a hydroxylation product 55 of the catecholic substrate during the inactivation reaction (Scheme 3.12b). As the inactivation reaction is relatively rare and involves small quantities of enzyme, in the experimental system used the o-hydroxylated quinone product 55 is a minor component of the reaction mixture. Nevertheless, the product 55 has been identified by high-performance liquid chromatography/mass spectrometry in the case of 4-methylcatechol oxidation by tyrosinase and shown to be correlated with the loss of enzyme activity [56]. Since the Quintox suicide-inactivation mechanism requires o-hydroxylation to take place, the inactivation kinetics of tyrosinase during catechol oxidation should be prevented if an unsubstituted ring carbon adjacent to the catecholic function is not available. Since this is not a requirement of the mechanism suggested by Muñoz-Muñoz et al. [59] this provides a critical experimental test of the mechanisms. As predicted by the Quintox mechanism, 3,6-difluorocatechol 59, which cannot act as an oxygenase substrate, acts as an oxidase substrate for tyrosinase, but does not exhibit suicide-inactivation kinetics [57]. The oxygen utilization during the oxidation of 3,6-difluorocatechol 59 by tyrosinase shows first-order kinetics and contrasts with the kinetics of inactivating substrates. All the experimental tests so far carried out have been consistent with the predicted outcome of the Quintox inactivation mechanism of tyrosinase.

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Systems, vol. 13 (ed. H. Sigel), Decker, New York, pp. 143–186. Nelson, J.M. and Dawson, C.R. (1944) Tyrosinase, in Advances in Enzymology, vol. 4 (eds F.F. Nord and C.H. Werkman), Interscience, New York, pp. 99–152. Asimov, I. and Dawson, C.R. (1950) On the reaction inactivation of tyrosinase during aerobic oxidation of catechol. J. Am. Chem. Soc., 72, 820–828. Ingraham, L.L., Corse, J., and Makower, B. (1952) Enzymatic browning of fruits. III. Kinetics of the reaction inactivation of polyphenoloxidase. J. Am. Chem. Soc., 74, 2623–2626. Seiji, M., Sasaki, M., and Tomita, Y. (1978) Nature of tyrosinase inactivation in melanosomes. Tohoku J. Exp. Med., 125, 233–245. Tomita, Y., Hariu, A., Mizuno, C., and Seiji, M. (1980) Inactivation of tyrosinase by dopa. J. Invest. Dermatol., 75, 379–382. García-Cánovas, F., Tudela, J., MartínezMadrid, C., Varón, R., Garcia-Carmona, F., and Lozano, J.A. (1987) Kinetic study on the suicide inactivation of tyrosinase induced by catechol. Biochim. Biophys. Acta, 912, 417–423. Tudela, J., Garcia-Cánovas, F., Varón, R., Jiménez, M., Garcia-Carmona, F., and Lozano, J.A. (1988) Kinetic study in the transient phase of the suicide inactivation of frog epidermis tyrosinase. Biophys. Chem., 30, 303–310. Dietler, C. and Lerch, K. (1982) Reaction inactivation of tyrosinase, in Oxidases and Related Redox Systems (eds T.E. King, H.S. Mason, and M. Morrison), Pergamon, New York, pp. 305–317. Land, E.J., Ramsden, C.A., and Riley, P.A. (2007) The mechanism of suicideinactivation of tyrosinase: a substrate structure investigation. Tohoku J. Exp. Med., 212, 341–348. Land, E.J., Ramsden, C.A., Riley, P.A., and Stratford, M.R.L. (2008) Evidence consistent with the requirement of cresolase activity for suicide inactivation of tyrosinase. Tohoku J. Exp. Med., 216, 231–238. Ramsden, C.A., Stratford, M.R.L., and Riley, P.A. (2009) The influence of catechol structure on the suicide

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inactivation of tyrosinase. Org. Biomol. Chem., 7, 3388–3390. Spritz, R.A., Ho, L., Furumara, M., and Hearing, V.J. (1997) Mutational analysis of copper binding by human tyrosinase. J. Invest. Dermatol., 109, 207–212. Muñoz-Muñoz, J.L., Garcia-Molina, F., Garcia-Ruiz, P.A., Molina-Alcaron, M., Tudela, J., Garcia-Cánovas, F., and Rodriguez-Lopez, J.N. (2008) Phenolic substrates and suicide inactivation of tyrosinase: kinetics and mechanism. Biochem. J., 416, 431–440. Ramsden, C.A. and Riley, P.A. (2010) Mechanistic studies of tyrosinase suicide inactivation. Arkivoc, 2010 (i), 260–274. Eicken, C., Zippel, F., BüldtKarentzopoulos, K., and Krebs, B. (1998) Biochemical and spectroscopic characterization of catechol oxidase from sweet potatoes (Ipomoea batatas) containing a type-3 dicopper center. FEBS Lett., 436, 293–299. Gerdemann, C., Eicken, C., and Krebs, B. (2002) The crystal structure of catechol oxidase: new insight into the function of type-3 copper proteins. Acc. Chem. Res., 35, 183–191. Klabunde, T., Eicken, C., Sacchettini, J.C., and Krebs, B. (1998) Crystal structure of a plant catechol oxidase containing a dicopper center. Nat. Struct. Biol., 5, 1084–1090. Matoba, Y., Kumagai, T., Yamamoto, A., Yoshitsu, H., and Sugiyama, M. (2006) Crystallographic evidence that dinuclear copper center of tyrosinase is flexible during catalysis. J. Biol. Chem., 281, 8981–8990. Decker, H., Schweikardt, T., and Tuczek, F. (2006) The first crystal structure of tyrosinase: all questions answered? Angew. Chem. Int. Ed., 45, 4546–4550. Van Gastel, M., Bubacco, L., Groenen, E.J.J., Vijgenboom, E., and Canters, G.W. (2000) EPR study of the dinuclear active copper site of tyrosinase from Streptomyces antibioticus. FEBS Lett., 474, 228–232. Bubacco, L., van Gastel, M., Groenen, E.J.J., Vijgenboom, E., and Canters, G.W. (2003) Spectroscopic characterization of the electronic changes in the active site of Streptomyces antibioticus tyrosinase upon

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3 Biological Chemistry of o-Quinones binding of transition state analogue inhibitors. J. Biol. Chem., 278, 7381–7389. 68 Ramsden, C.A. and Riley, P.A. (2010) Studies of the competing rates of catechol oxidation and suicide inactivation of tyrosinase. Arkivoc, 2010 (x), 248–255. 69 Hansson, C., Rorsman, H., and Rosengren, E. (1980) 5-Hydroxydopa, a new compound in the Raper–Mason scheme of melanogenesis. Acta Derm. Venereol., 60, 281–186. 70 Agrup, G., Rorsman, H., and Rosengren, E. (1982) 5-OH-dopa, product of and

substrate for tyrosinase. Acta Derm. Venereol., 62, 371–376. 71 Carlberg, M., Jergil, B., Lindbladh, C., and Rosengren, E. (1984) Enzymatic 5-hydroxylation of l-dopa by a tyrosinase isolated from the sea anemone Metrium senile. Gen. Pharmacol., 15, 301–307. 72 Burzio, L.A. and Waite, J.H. (2002) The other Topa: formation of 3,4,5-trihydroxyphenylalanine in peptides. Anal. Biochem., 306, 108–114.

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4 Biosynthesis of Melanins José Carlos García-Borrón and M. Concepción Olivares Sánchez

4.1 Introduction

Melanogenesis, the biochemical pathway responsible for the biosynthesis of the chemo- and photoprotective melanin pigments mostly from phenolic precursors, is a widespread process occurring in all types of organisms throughout the phylogenetic scale. In prokaryotes, melanogenesis consists of a rather simple series of reactions involving just one melanogenic enzyme, while in mammals it is a tightly regulated pathway coordinated by the combined action of a family of melanocytespecific gene products, some of which associate to form a multienzymatic complex. The mammalian melanogenic enzymes are three related and highly similar metalloproteins: tyrosinase, tyrosinase-related protein 1 (Tyrp1 or gp75) and tyrosinaserelated protein 2 (Tyrp2, also called dopachrome tautomerase (Dct), and designated here Dct/Tyrp2). These proteins share numerous structural similarities, and follow quite similar biosynthetic, processing, and trafficking pathways [1]. All three enzymes are type 1 membrane-bound melanosomal glycoproteins. They are synthesized by ribosomes and transported through the rough endoplasmic reticulum (ER) and the Golgi apparatus, where they undergo post-translational processing and glycosylation. They are then delivered to the early melanosomes and the synthesis of melanin ensues. The present chapter describes the currently accepted biochemical pathway for melanin synthesis, which is in fact a modified version of the Raper–Mason route outlined in the first half of the twentieth century. We summarize the current knowledge of the structural features and catalytic mechanism of the melanogenic enzymes, focusing on the mammalian proteins (mainly tyrosinase and Dct/Tyrp2, which are better characterized). We do not address specific aspects of the melanogenic pathway in prokaryotes or in plants, and the reader interested in these organisms is referred to one of various recent and authoritative reviews [2–5]. We also outline several aspects of the regulation of the melanogenic pathway in order to give an integrated view of mammalian melanogenesis. We also refer to other chapters of this book for further details on related topics such as the genetics of the melanogenic proteins, the biogenesis of the melanosome, and the chemical Melanins and Melanosomes: Biosynthesis, Biogenesis, Physiological, and Pathological Functions, First Edition. Edited by Jan Borovanský and Patrick A. Riley. © 2011 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2011 by Wiley-VCH Verlag GmbH & Co. KGaA.

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structure of the melanin pigment, as they are deeply related to those covered here, but beyond the scope of the present chapter.

4.2 Raper–Mason Pathway

In vertebrates, melanins are formed from the amino acid l-tyrosine by means of a unique series of enzymatic and chemical reactions known as the Raper–Mason pathway. Based on the observation that melanins could be formed from phenolic precursors in the presence of purified tyrosinase, the melanogenic pathway was originally delineated by Raper [6] and Mason [7] taking into account the involvement of a single enzyme, tyrosinase, as well as a series of spontaneous chemical reactions. However, comparison of the structure of natural melanins and melanins synthesized in vitro by the action of tyrosinase on several melanogenic precursors, as well as the study of the action of melanosomal extracts on melanin biosynthesis intermediates, led to the discovery of several other enzymatic melanogenic activities in addition to tyrosinase. These enzymatic activities have been incorporated to the currently accepted version of the Raper–Mason pathway. In addition, genetic evidence arising from the study of mutant mice with various coat color phenotypes demonstrates that certain nonenzymatic melanosomal proteins such as Pmel-17, the product of the silver locus [8], act to support a correct melanogenic function. For simplicity, the Raper–Mason pathway can be divided into two phases. A proximal phase contains the rate-limiting tyrosinase-catalyzed reactions and leads to the formation of a key intermediate, l-dopachrome, from the amino acid ltyrosine. In the subsequent distal phase, l-dopachrome evolves spontaneously and/or enzymatically to reactive intermediates and the chemical polymerization steps that ultimately yield the melanogenic pigments take place. 4.2.1 Phase I Melanogenesis: The Proximal Raper–Mason Pathway – From L-tyrosine to L-dopachrome

The key regulatory and rate-limiting melanogenic enzyme is tyrosinase, a coppercontaining, membrane-bound glycoprotein located in the melanosomes. This enzyme is able to catalyze the first two steps of the proximal phase: the hydroxylation of l-tyrosine and the subsequent oxidation of the intermediate o-diphenol (l-3,4-dihydroxyphenylalanine (l-dopa)) to yield l-dopaquinone (monophenolase or cresolase activity, EC 1.14.18.1, and diphenolase or catechol oxidase activity, EC 1.10.3.1, respectively) (Figure 4.1). Hydroxylation of l-tyrosine is the rate-limiting step in melanin synthesis. Although tyrosine hydroxylase and dopa oxidase activities occur in the same active site [10], the diphenolase activity is approximately 7 times faster than monophenolase activity. The mechanism of these reactions has been controversial and will be discussed below (Section 4.3.2). In any case, there is no doubt that the final product of tyrosinase action on l-tyrosine, l-dopaquinone,

4.2 Raper–Mason Pathway

The mammalian melanogenic pathway. Tyrosinase is the only enzyme in the proximal phase of melanogenesis. The role of the Tyrps in the distal phase is critical to determine the type and amount of eumelanins synthesized: l-dopachrome can undergo a spontaneous decarboxylative

Figure 4.1

reaction to form DHI or a nondecarboxylative rearrangement catalyzed by Dct/Tyrp2 and subsequent oxidation to IQCA catalyzed by Tyrp1 (at least in mouse melanocytes). The pathway for pheomelanin synthesis is less well understood. (Adapted from [9].)

is the first branch point of two connected biosynthetic routes in melanogenesis leading to the two main types of melanin pigments: dark (brown/black) eumelanin or light (yellow/red) pheomelanin [1]. During eumelanin biosynthesis (Figure 4.1, left), l-dopaquinone can undergo two types of spontaneous chemical reactions: an intramolecular 1,4-addition to the benzene ring and/or a water addition reaction. In the first case, the amino group of the l-dopaquinone side-chain engages in an intramolecular Michael addition with cyclization to yield l-cyclodopa (also termed leucodopachrome). This intermediate is quickly and spontaneously oxidized to l-dopachrome by another molecule of l-dopaquinone, which is reduced back to l-dopa [11, 12]. Both reactions are fast and proceed without enzymatic control, although cyclization of ldopaquinone is favored over conjugation with thiols and other nucleophilic chemicals due to its intramolecular nature and to the presumably low concentrations of these compounds inside the eumelanosome. Note that half the l-dopa oxidized by tyrosinase to l-dopaquinone is reduced back to l-dopa. This is most likely a relevant feature of the pathway owing to the regulatory role of l-dopa on the tyrosine hydroxylase activity of the enzyme (see below). On the other hand, the formation of l-cyclodopa is favored as the pH increases, because the amino group should be

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nonprotonated to allow for the nucleophilic attack on position 6. Therefore, at acidic intramelanosomal pH values cyclization should be rather slow and water addition to the benzene ring may become possible, thus leading to the formation of a highly reactive three-hydroxylated phenol, topa (3,4,5-trihydroxyphenylalanine). Presumably, topa would also evolve to l-dopachrome through a series of slow spontaneous chemical reactions ([13]; not shown in Figure 4.1). 4.2.2 Distal Melanogenic Steps: From L-Dopachrome to Eumelanins

l-Dopachrome is considered the final product of the proximal phase in the Raper– Mason pathway and is in fact a second branch point in melanogenesis leading to slightly different types of eumelanins, with varying proportions of carboxylated or decarboxylated monomeric units. The distal melanogenic reactions begin with the spontaneous, nonenzymatic decarboxylative rearrangement of l-dopachrome to 5,6-dihydroxyindole (DHI) and CO2. DHI is then oxidized to 5,6-indolequinone (IQ). Under aerobic conditions, this oxidation occurs spontaneously, but tyrosinase significantly accelerates the rate of the reaction. Alternatively, orange red l-dopachrome may be enzymatically transformed into colorless DHI-2-carboxylic acid (DHICA) [14]. The corresponding catalytic activity was originally designated dopachrome conversion factor, then dopachrome isomerase, and is now named Dct (EC 5.3.3.12), also known as Tyrp2 [15]. It is worth noting that traces of divalent transition metal cations, such as Cu2+ or Ni2+, also bring about this isomeric rearrangement of l-dopachrome, and can yield mixtures of DHI and DHICA [16]. Although the enzymatic and chemical conversions of l-dopachrome are not mutually exclusive, the enzymatically catalyzed reaction is highly stereo-specific for l-dopachrome and, inside the melanosome, the level of Dct activity controls the DHICA/DHI ratio [17]. DHICA is relatively stable at physiological pH and temperature, and does not lose its carboxyl group easily to form DHI. Further oxidation of DHICA yields indole-2-carboxylic acid-5,6-quinone (IQCA). Spontaneous oxidation is much slower for DHICA than for DHI because of the electron-withdrawing effect of the carboxyl group at position 2, so that DHICA oxidation in vivo is most likely an essentially enzymatic reaction. However, the details of the enzymology of DHICA oxidation are complex and have not yet been completely worked out. Indeed, it has been shown that whereas mouse tyrosinase is unable to recognize DHICA as a diphenolic substrate to promote its oxidation, mouse Tyrp1 behaves as a low-specific-activity DHICA oxidase [18, 19]. Conversely, human tyrosinase shows DHICA oxidase activity [20]. Therefore, it is possible that the oxidation of DHICA in vivo is carried out by different enzymes, Tyrp1 or tyrosinase, depending on the particular mammalian species considered. Accordingly, the physiological role of Tyrp1 in some species, especially in human melanocytes, has been and still is a matter of discussion (see below). In any case, it is firmly established that l-dopachrome formed in the initial phase of the melanogenic pathway is transformed into mixtures of DHICA and DHI,

4.2 Raper–Mason Pathway

and that the relative proportions of these two diphenols are dictated by a complex set of intramelanosomal conditions with a major role for Dct/Tyrp2 activity. The next steps in eumelanogenesis are less well characterized. DHI, DHICA, and their corresponding o-quinones undergo relatively unordered and intermixed spontaneous polymerization to form black, insoluble eumelanin (DHI-melanin) and golden-brown, poorly soluble eumelanin (DHICA-melanin). The size, chelating, and absorption properties of the final eumelanin depend on the initial DHICA/ DHI ratio [14, 17]. DHICA-melanin exerts a stronger antioxidant effect than DHImelanin, and this behavior may be due to the different structure and solubility of the two melanins. Furthermore, the DHICA branch of eumelanogenesis is less cytotoxic than the DHI branch. In fact, the oxidative metabolism of DHI, and to a lesser extent DHICA, is a source of cytotoxic species, including highly reactive quinone intermediates and oxygen species such as hydrogen peroxide [21]. Tyrp1 and Tyrp2 are thought to be critical to protect melanocytes from the inherent toxicity of melanogenic reactions. Indeed, in the presence of these proteins the synthesis of DHI is kept to a minimum, whereas the production and oxidative metabolism of DHICA is favored, so that melanogenesis follows primarily the less-toxic DHICA pathway. Given our incomplete knowledge of the distal portion of melanogenesis, one open question is the possible involvement of other melanosomal proteins different from tyrosinase and the Tyrps in the control of the distal steps of the pathway. In this respect, the contribution of the silver protein as an accelerating factor for the polymerization of monomeric intermediates has been proposed [22, 23], but a precise catalytic activity has never been definitively proven for this protein (discussed below). In summary, the type of melanin produced depends not only on the availability of substrates [24], but also on the relative activities of at least three melanogenic enzymes, which leads to a refined control of melanosynthesis in animals compared with lower organisms [17] and accounts for the occurrence of a wide spectrum of natural melanin pigments with subtly different physicochemical properties. 4.2.3 Biosynthesis of Pheomelanins

The switch from eumelanin biosynthesis to production of pheomelanins depends upon the availability of low-molecular-weight sulfydryl compounds such as the amino acid l-Cys or reduced glutathione. In the presence of these compounds, the rapid conjugation between l-dopaquinone and the thiol group yields a family of sulfur-containing intermediates related to pheomelanins [25, 26]. However, the pheomelanogenic intermediates and the regulatory events that control the availability of thiolic substrates within melanosomes are poorly known. All the reactions in the pheomelanin-specific pathway are thought to involve highly unstable intermediates and, accordingly, they might occur spontaneously. In fact, efforts to identify melanosomal enzymes responsible for the catalysis or regulation of these

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reactions have been largely unsuccessful. A simple model of pheomelanogenesis (Figure 4.1, right) assumes that the nucleophilic attack of the thiol group of l-Cys to l-dopaquinone yields mainly 5-cysteinyldopa [27]. However, in addition to carbon 5 other free positions in the l-dopaquinone ring such as 2 and 6 are also reactive, so that a complex mixture of isomers is obtained. 5-cysteinyldopa would undergo several structural rearrangements and dehydration to form an alanylhydroxy-benzothiazine monomer, the proposed monomeric subunit for pheomelanin. However, pheomelanin biosynthesis could proceed through a more complex pathway involving glutathione rather than Cys, since this tripeptide is much more abundant in the intracellular medium than the free amino acid. Accordingly, it has been proposed that the first major thiol-conjugated species formed inside melanocytes is 5-glutathionyldopa. A dipeptidase would then catalyze the release of 5-cysteinyldopa and the pathway would progress as described above ([28]; not shown in Figure 4.1 for simplicity). This glutathione-dependent branch may still be an oversimplification of the in vivo situation, where still uncharacterized enzymatic activities might be involved.

4.3 Structural and Functional Properties of the Melanogenic Enzymes

As discussed above, tyrosinase is the key melanogenic regulatory enzyme in vertebrates. In plants, the highly similar catechol oxidases catalyze the oxidation of o-diphenols, but they are unable to carry out the hydroxylation of monophenolic substrates. The active sites of the two types of enzymes (termed phenoloxidases) are very similar in terms of their sequence and physicochemical properties. Phenoloxidases and the oxygen carrier proteins hemocyanins belong to the type 3 copper protein family. Collectively, they are responsible for pigmentation of skin and hair, browning of fruits, and wound healing in plants and arthropods. Since melanin pigments are found in all types of organisms, tyrosinases are ubiquitously distributed throughout the phylogenetic scale, but the following discussion is focused on the mammalian enzymes. 4.3.1 Structure of Tyrosinase and Related Proteins

The overall structure and the active site of tyrosinases are highly conserved among different vertebrate species. Moreover, mammalian tyrosinases show high homology with Tyrp1 and Dct/Tyrp2 (up to 40% amino acid identity and approximately 70% amino acid homology [29]). There are several residues strictly conserved in all tyrosinases, the more important being the copper-binding ligands and other amino acids establishing interactions critical to maintain the globular folding of the protein. In addition, tyrosinases from higher organisms display some other conserved structural features, such as the C-terminal motifs for trafficking and targeting, the Cys clusters, several N-glycosylation sites, and a putative transmem-

4.3 Structural and Functional Properties of the Melanogenic Enzymes a)

b)

(a) Schematic representation of the tyrosinase family proteins. The numbering, precise positions of domains, and amino acid sequence of metal binding sites correspond to the mouse proteins. SP, signal peptide; Cys, cysteine-rich segments; Cu or Me, copper- or metal-binding domains (sequence comprised between the first and third His detailed below); TM, transmembrane fragment. (b) Proposed consensus for tyrosinases [30]. Bold: the six His directly bound to Cu. Subscript indicates the first (A)

Figure 4.2

or second (B) binding site and the order of the His in the primary sequence. Φ means aromatic residue (F, Y, or W). B means hydrophobic residue. The residues are labeled with a subscript indicating their position in relation to the closest H. Residues in italics mean that the residue changes from tyrosinase to Tyrps. Underlined: conserved residues important to maintain the three-dimensional structure of tyrosinase and its family.

brane domain near its C-terminus that ensures their association with the melanosomal membrane in a similar orientation relative to the melanosomal lumen [30] (Figure 4.2). Most of these elements are also conserved in mammalian Tyrps. However, in spite of the remarkable sequence similarity of their active site, the three proteins of the tyrosinase family have distinct enzyme activities and function at different steps of melanin biosynthesis [1]. This is most likely a consequence of the specific binding of different metal cofactors in the two conserved metal-binding motifs that are critical to their enzymatic functions: copper in tyrosinase [31] and zinc in Dct/Tyrp2 [32]. The precise metal cofactor in Tyrp1 is still unknown [33]. So far, no mammalian tyrosinase has been crystallized, probably because it is a somewhat heterogeneous transmembrane glycosylated protein. Nevertheless, a variety of data based on mutagenesis studies [34, 35], the available crystallographic data on a plant catechol oxidase (Ipomoea batatas) [36], and the first published crystal structure of a prokaryotic tyrosinase (Streptomyces castaneoglobisporus) [37]

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allow a model for the mammalian tyrosinase active site and reaction mechanism to be defined. In this model, the two copper-binding sites in tyrosinase, named CuA and CuB, are peptidic regions containing three perfectly conserved His residues. CuA displays a H-xn-H-x8-H motif and CuB displays a H-x3-H-xn-H motif, where “n” is a variable number of residues [30]. The six ion-binding His residues are adjacent due to the folding of the protein at the active site, consisting of a hydrophobic pocket inside a helix bundle with four densely packed helices. The overall structure is maintained by electrostatic and cation-π interactions. Oxygen binds as a side-on (μ–η2:η2) peroxide bridge [38] and, in the resting form of the enzyme, the pair of antiferromagnetically coupled copper ions are pentacoordinated in a distorted square pyramidal geometry [39]. Although it is generally accepted that each copper ion is bound only by the three His residues in either CuA and CuB, some other alternative modes of cofactor quelation have been proposed, with four putative ligands (one Cys and three His) [40]. Apart from the invariant His residues in the copper-binding sites, all tyrosinases throughout nature display several other strictly conserved residues most likely involved either in the binding and docking of the substrates or in the maintenance of the structural integrity of the active site. Accordingly, we proposed a broader consensus for the copper-binding regions with a new notation to distinguish residues essential for tyrosinase active-site folding [30] (Figure 4.2). This allows for the correlation of the available crystallographic data and of studies on the functional effects of naturally occurring or artificially created mutations, with the contribution of these residues to the active-site structure or their potential catalytic role [35, 41, 42]. Notably, some of these residues are also present in the Tyrps. The structure of all mammalian tyrosinases characterized to date also comprises the following common features [30] (Figure 4.2). The N-terminal signal peptide targets the nascent polypeptides to the ER for its folding, processing, and sorting. The signal peptide must be cleaved for correct folding due to its hydrophobicity. It is generally assumed that cleavage takes place between G18 and H19 in human and mouse tyrosinase. Within the carboxyl tail of the protein, mammalian tyrosinases contain a single transmembrane segment followed by a short extension. There is no doubt that the bulk of the protein containing the active site faces the lumen of the melanosome, whereas the short C-terminal extension is located in the cytosol. The transmembrane fragment anchors the protein to the melanosomal membrane. The C-terminal cytosolic peptide contains essential signals for targeting the enzyme to the melanosome [43, 44]. Some of these signals are a dileucine motif (LL, with some variants) and the tyrosine-based motif (YXXB, where B is any bulky hydrophobic residue) [45]. Mammalian tyrosinase family proteins also display conserved Cys-rich domains. The number and relevance of Cys residues in tyrosinase is variable. The Streptomyces enzyme is completely devoid of Cys, while mammalian tyrosinases have 17 Cys residues clustered in three regions. Notably, the second cluster has been denominated the epidermal growth factor (EGF)-like region [46] as it partially matches the two EGF-like motifs and an EGFrelated domain, the laminin-LE. Plant catechol oxidases contain only the first cluster. Even though almost all Cys seem to be important for a correct protein

4.3 Structural and Functional Properties of the Melanogenic Enzymes

folding and copper acquisition [47, 48], the function of the clusters and the location of disulfide bridges in mammalian tyrosinases and Tyrps remain mostly unknown. In higher organisms, the tyrosinase family proteins are glycoproteins containing several N-glycosylation sites. The role of N-glycans in sorting, stability, and activity of mammalian tyrosinase has been extensively studied. Post-translational processing of tyrosinase and its traffic through the secretory pathway are critical for the acquisition of an active form of the enzyme. Some N-glycosylation sequons are strongly conserved, such as the one present within the CuB site. This sequon appears particularly important for the correct folding of the enzyme [49]. When initially synthesized, the nonglycosytaled protein backbone of tyrosinase has an apparent molecular weight of 55 kDa; after terminal glycosylation, the electrophoretic pattern of tyrosinase is indicative of some heterogeneity and the molecular weight of the mature protein shifts to about 65–75 kDa [50]. Tyrosinase folding and maturation is assisted by ER-resident chaperones, with the interaction of calnexin and the carbohydrate chains in tyrosinase playing an important role [49, 51]. Although it is still unclear exactly where or how copper ions are transferred into apo-tyrosinase, it is accepted that terminal glycosylation and copper acquisition of mammalian tyrosinase occurs as the enzyme is processed through the Golgi complex and delivered to stage II melanosomes via early endosomal intermediates [44, 52], where enzyme activity is expressed. In fact, it has been proposed that late copper binding could prevent abnormal and undesirable melanogenesis in proximal compartments of the biosynthetic-secretory pathway [53]. Moreover, the involvement of the Menkes copper transporter (MNK), located in the trans-Golgi network, has been demonstrated [54]. Interestingly, it has been shown that proper tyrosinase folding and post-translational glycosylation can occur in the absence of copper loading [49]. This suggests that copper is transferred to a well-structured active site able to bind the metal ion with high affinity, thus avoiding leakage of the cofactor. Since tyrosinase is the key and rate-limiting melanogenic enzyme, loss-offunction mutations on the tyrosinase gene result in reduction or absence of melanin of melanocytes in the skin, hair follicles, and eyes, causing oculocutaneous albinism (OCA) type 1 (tyrosinase-negative, OMIM 203100) [55]. As discussed below, OCA1 is often associated with retention of defective tyrosinase in the ER and subsequent degradation [56]. At present, more than 200 mutations in the tyrosinase gene have been associated with albinism (reviewed by Oetting [57]). Tyrosinase and the Tyrps exist as a high-molecular-weight complex with relatively stable interactions putatively involving the highly conserved Cys-rich EGF domains. The strong intermolecular association of Tyrp1 and tyrosinase enhances the intracellular stability of tyrosinase [58, 59] and, as discussed below, has a significant effect on its maturation and intracellular transport [60]. 4.3.2 Catalytic Cycle of Tyrosinase

The current view of the mechanism of tyrosinase catalysis considers several forms of the enzyme and two connected cycles for the cresolase (tyrosine hydroxylase)

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and catecholase (dopa oxidase) activities, occurring in a single active site. Depending on the absence/presence of oxygen and the oxidation state of the copper ions [Cu(II)/Cu(I)], the enzyme can exist in three interconvertible forms, called met, oxy, and deoxy. The mechanism is well supported by structural data from the first published crystal structure of a tyrosinase (from S. castaneoglobisporus) [37]. This structure provides a good model to interpret the function of residues in the active site of the mammalian tyrosinases due to the high degree of conservation within this region in all known tyrosinases. Indeed, homology modeling of mouse tyrosinase based on this structure already provided a framework to successfully explain the effects on enzyme kinetics of artificial and naturally occurring albino mutations [61]. Both the tyrosine hydroxylase or dopa oxidase cycles are initiated when the enzyme is in the oxy state and the appropriate substrates enter the active site (Figure 4.3). However, there are important differences between the two types of reactions. For instance, the tyrosine hydroxylase activity, but not the dopa oxidase activity, shows a characteristic lag period before the reaction reaches its maximal rate. This lag phase can be shortened by catalytic amounts of l-dopa [63]. Therefore, l-dopa is not only the product of the monophenolase activity of tyrosinase acting on l-tyrosine as substrate and a substrate for the catecholase activity, but also a cofactor for the tyrosine hydroxylase cycle. Moreover, the affinity of mammalian tyrosinases for l-dopa acting as cofactor for tyrosine hydroxylase activity is about two orders of magnitude higher than those for the compound acting as a catecholic substrate [64]. The mechanism that follows accounts for these kinetic features as well as for some other differences in the catalytic requirements of tyrosine hydroxylase and dopa oxidase activities. 4.3.2.1 Cresolase (Tyrosine Hydroxylase) Reaction Cycle In the oxy form, molecular oxygen is coordinated between the two active-site copper ions, each of which is bound to the protein matrix by three His residues held in place by a pair of antiparallel α-helices. The only exception is one His in the CuA site (His54 in the S. castaneoglobisporus enzyme and His202 in mouse tyrosinase), located in a flexible loop. This residue could be responsible for a proton shift from the ortho position of l-tyrosine during the catalytic cycle of the Streptomyces enzyme [65]. The tyrosine hydroxylase cycle is initiated by the binding of l-tyrosine to the active site (Figure 4.3). The structures mentioned before provide information on the possible orientation of monophenols at the active site, although this aspect may not be universal. At least in mammalian tyrosinases, the most likely orientation of l-tyrosine would be forced by the π–π interaction between the aromatic ring in the substrate and the second His in the CuB site. One copper of the cluster forms a tyrosine hydroxylate complex placing carbon 3 of the substrate in position to accept the terminal oxygen of the peroxide, which is bound to the second copper, to form l-dopa and then l-dopaquinone. The o-quinone then leaves the reduced bicuprous site, allowing for the entrance of a new oxygen molecule. Therefore, even in the tyrosine hydroxylase cycle the released substrate should be l-dopaquinone [66] rather than l-dopa, as previously suggested. Oxygen

4.3 Structural and Functional Properties of the Melanogenic Enzymes

Mechanism of action for tyrosinase. The two catalytic cycles of tyrosinase are depicted (the His numbering corresponds to the mouse enzyme). Note that met-tyrosinase is involved in the dopa

Figure 4.3

oxidase (DO) cycle, but not in the tyrosine hydroxylase (TH) cycle: when l-tyrosine binds to this form of the enzyme, a dead-end complex is formed. (Adapted from [62].)

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binding then induces an oxidative change in the valence of the copper ions: the two oxygen atoms bind to the Cu(I)–Cu(I) center of deoxy-tyrosinase to yield a peroxide bound Cu(II)–Cu(II) center in oxy-tyrosinase. The unusual enzyme kinetics observed in this reaction are due to the nonenzymatic indirect formation of l-dopa by the chemical reactions accompanying dopaquinone cyclization [67] (Figure 4.1). On the other hand, plant and fungal catechol oxidases lack tyrosine hydroxylase activity despite the high similarity with animal tyrosinases. This could be explained by the presence in all catechol oxidases sequenced this far of a bulky aromatic residue shielding the CuA site [36], which is not found in animal tyrosinases. In keeping with this possibility, it has been proposed that monophenols would dock to CuA whereas o-diphenols would dock to CuB in the active site of mammalian tyrosinases [35]. 4.3.2.2 Catecholase (Dopa Oxidase) Reaction Cycle In the dopa oxidase cycle, the o-diphenolic substrate would bind to the oxy form of the enzyme. Binding of the catechol to CuB would also labilize the oxygen, resulting in the oxidation of the substrate and release of l-dopaquinone. Biochemical and kinetic evidences suggest that the His residue adjacent to the third copper ligand in the CuB site is involved in stereospecific binding of diphenols, but not monophenols [35], and that binding involves only one of the two available hydroxyl groups in the substrate. The met bicupric state of the enzyme accounts for the kinetic characteristics of the lag phase in the tyrosine hydroxylase cycle previously mentioned. This enzymatic form can bind either l-tyrosine or l-dopa. l-Dopa would dock to the two copper ions through both catecholic hydroxyl groups, hence with higher affinity than when the binding proceeds only through CuB (oxy form). This differential mode of binding explains the 100-fold higher affinity of l-dopa acting as a cofactor than as a substrate [64]. Moreover, this orientation accelerates the oxidation of the substrate and the reduced deoxy-tyrosinase is reoxidized upon oxygen binding. Docking of l-tyrosine to met-tyrosinase at the catalytic center would lead to a dead-end complex (Figure 4.3). The inhibition by high concentrations of l-tyrosine reported for tyrosinase [68] could be related to the formation of this dead-end complex. Finally, tyrosinase is characterized by an irreversible inactivation that occurs during the oxidation of catechols. A recently proposed mechanism accounting for this suicide inactivation is based on the ortho-hydroxylation of catecholic substrates, where Cu(II) is reduced to Cu(0), which no longer binds to the enzyme and is eliminated (reductive elimination) [69]. This process is dependent on the cresolase activity of tyrosinase and, accordingly, it is not exhibited by enzymes lacking tyrosine hydroxylase activity [70]. 4.3.3 Dct/Tyrp2

Dct/Tyrp2 is encoded at the slaty locus [71], whose mutation in mice is responsible for the slaty phenotype, characterized by the production of DHICA-poor eumela-

4.3 Structural and Functional Properties of the Melanogenic Enzymes

nin. Dct/Tyrp2 shows remarkable homology to tyrosinase and the protein displays most of the salient conserved structural elements discussed above. However, although Dct/Tyrp2 is also associated to the melanosomal membrane by a single membrane-spanning helix, the sorting and trafficking signals in its short cytoplasmic C-terminal extension are different from those in tyrosinase, suggesting a differential intracellular processing [72]. Dct/Tyrp2 displays several glycosylation sequons, whose occupancy is important for stability and enzymatic function [73]. The sequences of the metal ion binding sites in Dct/Tyrp2 (MeA and MeB) share with tyrosinase the position and number of the His residues likely involved in chelation of the metal cofactor. However, the nature of the metal cofactor is different. Purified Dct/Tyrp2 contains bound zinc ions and enzymatic activity can be reconstituted from apoenzymatic preparations exclusively by addition of zinc, but not by copper or iron ions [32, 74]. Therefore, it is now widely accepted that Dct/ Tyrp2 is a zinc protein. This is consistent with the nature of the reaction catalyzed by Dct: Zn(II) is devoid of redox properties and therefore useless for oxidative reactions, but is highly efficient as a tautomerization catalyst. Accordingly, the presence of zinc ions instead of copper in the metal-binding site is considered the most important factor determining the catalytic events at the active site of Dct/ Tyrp2. Each Zn(II) is probably bound to the protein moiety of the enzyme by the three His residues conserved in the MeA and MeB sites, most likely forming a distorted tetrahedron, the typical geometry of this ion. l-dopachrome would bind to the Zn(II) ions through the semiquinonic face of the molecule, displacing a water molecule bound to the fourth position of the tetrahedron, so that each ion would remain linked to the polypeptidic chain by the three His residues (Figure 4.4). This enzyme–substrate complex initiates an electronic rearrangement in the indolic ring with subsequent hydrogen migration from position 3 to position 2, leading to the formation of DHICA as the final product of the reaction. Although the mouse and human enzymes act on l-dopachrome stereospecifically to release DHICA as the unique product, there are some reports on Dctrelated enzymes in mammals and lower organisms able to act on d-dopachrome or dopaminochrome to give DHI [75, 76]. The specificity of murine and human Dct/Tyrp2 for the l-enantiomer could be explained by the stereospecific interaction of the carboxyl group of l-dopachrome with some residue different from the conserved histidines. This amino acid would anchor the substrate at the active site with the appropriate orientation, similarly to tyrosinase with l-dopa [35]. Unfortunately, attempts to identify this residue have been unsuccessful [77]. In addition to its role in the metabolism of carboxylated melanogenic intermediates, Dct/Tyrp2 might also fulfill still uncharacterized functions. In keeping with this possibility, Dct/Tyrp2 is expressed earlier in the developing mouse than either tyrosinase or Tyrp1 and hence is already present in melanoblasts at developmental stages where no l-dopachrome is expected to be formed [78]. Moreover, a recent report revealed an intriguing link between Dct/Tyrp2 and hypoxia-inducible factor (HIF), a transcription factor that regulates cellular responses to changes in oxygen concentration and has an antiapoptotic effect against DNA-damage-induced apoptosis in certain model systems. This antiapoptotic activity of HIF-1 was shown to

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Figure 4.4 Mechanism of dopachrome tautomerization catalyzed by Dct/Tyrp2. In this catalytic cycle the enzyme presents only the met form and no redox reactions or oxygen binding take place at the active site.

l-Dopachrome binds to zinc ions through the semiquinonic part of the molecule, and subsequent electronic and proton rearrangements lead to DHICA. (Adapted from [62].)

depend on the transcriptional upregulation of Dct/Tyrp2 in a defined population of sensory neurons in Caenorhabditis elegans. The TYR-2 homolog is secreted by ASJ sensory neurons to antagonize CEP-1-dependent germline apoptosis (CEP-1 is the homolog of the tumor suppressor p53). Importantly, the antiapoptotic effect of Dct/Tyrp2 might be a general feature of the enzyme and an evolutionarily conserved function, since knock-down of the protein in human melanoma cells increased apoptosis [79]. 4.3.4 Tyrp1

Tyrp1 is the most abundant glycoprotein expressed specifically in melanocytes. Although the Tyrp1 gene mapping at the brown locus was the first member of the tyrosinase gene family to be cloned, its catalytic function (which results in the production of black rather than brown melanin) remains controversial and several lines of evidence suggest that the protein might even serve noncatalytic roles. Despite extensive homology with tyrosinase, Tyrp1 from mouse melanocytic cells binds indolic substrates, but displays an affinity for phenols much lower than tyrosinase. This switch in substrate specificity could be a consequence of subtle changes within the hydrophobic residues in the respective active sites that have

4.3 Structural and Functional Properties of the Melanogenic Enzymes

been extensively discussed elsewhere [30]. In keeping with these binding properties, the protein purified from mouse melanocytes behaves as a low-specificactivity tyrosinase isoenzyme with both tyrosine hydroxylase and dopa oxidase activities [80, 81]. Moreover, mouse Tyrp1 shows DHICA oxidase activity in vitro and mouse melanocytes mutated at the brown locus do not exhibit detectable DHICA oxidase activity, whereas this activity can be readily detected in wild-type melanocytes [18, 19]. However, a role for Tyrp1 as a DHICA oxidase may not be general, as some crucial differences regarding substrate specificities between the mouse and human melanogenic enzymes have been reported. Indeed, human Tyrp1 does not display DHICA oxidase activity [82], while human tyrosinase does [20]. This suggests that DHICA metabolism could follow alternative enzymatic pathways in mouse and human melanocytes. Apart from its catalytic potential, Tyrp1 participates with tyrosinase in the formation of multimeric complexes [59], which may be important for the regulation of melanogenesis or for the assembly of a melanogenic apparatus. Tyrp1 assists the correct trafficking of tyrosinase to the melanosomes [60] and mediates a significant stabilization of the enzyme [58]. It has been proposed that Tyrp1 would be mostly devoid of enzymatic activity and that its strong stabilizing effect on tyrosinase could be the main role for Tyrp1 in melanogenesis. In agreement with this view, mutation of Tyrp1 causes OCA3, which is associated with moderate hypopigmentation of the skin, hair, and eyes. In OCA3, mutated Tyrp1 is retained within the ER and appropriate processing of normal tyrosinase is also aborted, thus accounting for the observed decrease in the production of melanin pigments [60]. 4.3.5 Other Melanosomal Proteins

In addition to the tyrosinase family proteins, genetic evidence shows that other melanosomal proteins are needed for normal melanogenesis, although they may lack a defined enzymatic activity and may serve structural or regulatory roles. The relevance of these proteins for melanogenesis is highlighted by the pigmentation phenotypes of mice mutated at the corresponding loci. We will briefly mention three of them, silver/gp100/gp87/Pmel-17, P (pink-eyed dilution), and MATP (membrane-associated transporter protein)/AIM-1/underwhite/SCL45A2, because these proteins appear to modulate either tyrosinase activity or the rate of distal steps in melanogenesis. The Pmel-17 gene maps at the silver locus whose mutation causes a pigmentation phenotype in mice characterized by premature graying [83]. The silver protein undergoes proteolytic processing within the eumelanosome that provokes its aggregation and contributes to the formation of the lamellar network of the organelle [84], which may trigger the deposit of melanin [85]. Moreover, the possible involvement of the silver protein in facilitating the polymerization of DHICA to melanin has been proposed [22, 23], but so far no precise enzymatic activity has been demonstrated for this protein.

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Mutations of the P and MATP/AIM-1/underwhite/SCL45A2 proteins lead to OCA2 and OCA4 oculocutaneous albinism, respectively. Both proteins display the 12 transmembrane fragments structural arrangement typical of membrane transporters, but their substrate specificity has not yet been definitively demonstrated. It has been postulated that the P protein might be an anionic transporter involved in the control of the melanosomal pH, which is critical for tyrosinase activity and processing, and therefore melanogenesis [86]. Regarding MATP, although its substrate for transport is still unknown, in OCA4 melanocytes tyrosinase processing and intracellular trafficking to the melanosome are also disrupted and, consequently, the normal maturation of these organelles is impaired [87].

4.4 Regulation of the Melanogenic Pathway

Mammalian melanogenesis is a highly regulated pathway, as shown by the large number of genes that impinge on the process [88]. Regulatory events may take place in virtually any aspect of melanocyte biology, including melanocyte development and differentiation, the expression of melanosomal components, organelle biogenesis and/or transport, control of pigment-type switching, and others [89]. We discuss here only mechanisms that act directly on the melanogenic pathway to regulate either the type or the amount of pigment formed. 4.4.1 Eumelanogenesis versus Pheomelanogenesis: Regulation of the Type of Melanin Pigments

A wealth of genetic, biochemical and pharmacological evidence has established that the main factor governing the type of pigment formed in melanocytes is signaling from the melanocortin 1 receptor (MC1R). MC1R belongs to a fivemember subgroup of class A G-protein-coupled receptors designated MC1R to MC5R. Their specific ligands, called melanocortins, are peptide hormones formed by proteolytic cleavage of proopiomelanocortin. MC1R is expressed preferentially but not exclusively in epidermal melanocytes [90, 91] and its specific ligands are α-melanocyte-stimulating hormone (α-MSH) and adrenocorticotrophin (ACTH). Importantly, these ligands are synthesized not only in the pituitary gland but also by skin keratinocytes, thus allowing for a local paracrine regulation [92] that may be critically involved in physiological adaptations of the skin such as the tanning response [93]. Following activation by α-MSH or ACTH, MC1R stimulates cAMP synthesis. cAMP, the principal intracellular mediator of α-MSH [94], promotes the melanogenic response to the hormones with an increase in tyrosinase activity and a switch from production of light-colored and poorly photoprotective pheomelanins to synthesis of darker and more photoprotective eumelanins [24]. Accordingly, high MC1R signaling in mice melanocytes due to high levels of agonists [95] or gain-of-function mutations [96] leads to eumelanogenesis and dark coats.

4.4 Regulation of the Melanogenic Pathway

Conversely, absent or poor MC1R signaling due to loss-of-function mutations or expression of the endogenous inhibitor agouti signaling protein (ASIP) leads to pheomelanogenesis [97], with the MC1R null phenotype corresponding to a yellow fur color. In humans, a pigment type switch similar to the one in mice is not observed, but there is no doubt that MC1R signaling is also a major determinant of the type of pigment produced. Human MC1R is an extremely polymorphic gene, with more than 100 natural nonsynonymous variants reported to date [98]. Several hypomorphic MC1R alleles are strongly associated with a phenotype known as RHC (for red hair color), consisting of pheomelanin-rich red hair, abundant freckles, inefficient tanning upon sun exposure, high sensitivity to UV radiationinduced skin damage, and increased skin cancer risk. Moreover, a carrier of two MC1R mutant alleles likely corresponding to complete loss-of-function forms of the protein, and hence considered a MC1R null individual, showed a typical RHC phenotype, thus suggesting that red hair and fair skin is the null human MC1R phenotype [99]. Although no chemical characterization of the pigment produced by this likely MC1R-null individual was reported, there is little doubt as to a preferentially pheomelanic pattern. Whereas the positive relationship between MC1R signaling and eumelanogenesis as opposed to pheomelanogenesis is well established, the molecular events responsible for the stimulation of eumelanogenesis following activation of the MC1R by its peptide ligands are only partially understood. Given that pheomelanogenesis depends upon the conjugation of l-dopaquinone and the thiol group of sulfur-containing compounds such as Cys, the simplest possible scenario would posit that under conditions of low tyrosinase activity, the supply of thiol compounds would continuously exceed the low rate of l-dopaquinone formation. Accordingly, essentially all the l-dopaquinone formed within melanosomes would be trapped by conjugation with low molecular weight thiolic compounds, thus directing the melanogenic pathway towards pheomelanogenesis. Conversely, MC1R activation following binding of melanocortin peptides would stimulate tyrosinase. Eventually, a threshold of enzymatic activity would be reached where the rate of production of l-dopaquinone would exceed the supply of thiolic compounds. In these circumstances, this excess of l-dopaquinone would be allowed to evolve to dopachrome and would eventually be incorporated into a growing eumelanin polymer. In support of this simple model, stimulation of mouse melanocytes with α-MSH first increases pheomelanin levels, prior to the synthesis of eumelanins, whereas ASIP decreased the production of both types of pigment, probably as a result of downregulation of tyrosinase [100]. Although it is clear that the melanocortin-dependent stimulation of eumelanogenesis in rodent melanocytes and melanoma cells occurs in large part via the increase of tyrosinase activity, the relative contributions of transcriptional and post-transcriptional mechanisms remain uncertain. Transcriptional stimulation is mainly achieved through the cAMP dependent activation of the expression of Microphthalmia (MITF), a master regulator of melanocyte biology that activates several melanogenic genes [101]. MITF binds to specific target sequences in the

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promoter of the tyrosinase gene, resulting in increased levels of tyrosinase mRNA with a subsequent increase in tyrosinase abundance. However, in human melanocytes stimulated with α-MSH the increases in tyrosinase abundance are modest and it has been shown that intracellular cAMP elevating agents such as forskolin increase MITF protein levels without a concomitant increase in tyrosinase mRNA, thus suggesting the occurrence of post-transcriptional regulatory mechanisms [102]. Moreover, careful studies have established that even in mouse melanocytes the observed increase in tyrosinase mRNA abundance and protein levels is usually lower that the stimulation of tyrosinase specific activity, thus showing that in addition to transcriptional effects, α-MSH triggers a post-translational stimulation of tyrosinase [103]. The possible molecular basis of these post-translational events will be briefly discussed below. Changes in the levels and/or activity of other proteins in addition to tyrosinase are thought to contribute to the switch of pigment type production in response to α-MSH. Interestingly, in addition to tyrosinase, Dct/Tyrp2 gene expression is upregulated by α-MSH and downregulated by ASIP [104, 105], thus suggesting a role for Dct/Tyrp2 as a positive regulator of eumelanogenesis. In keeping with this possibility, Dct activity and the relative levels of eumelanins and pheomelanins are significantly decreased in mouse melanocytes carrying the slaty or slaty light mutations in the Dct locus [106]. Moreover, other factors such as the availability of thiol compounds might be modulated by α-MSH and may contribute to the regulation of the pigment type produced in melanocytes. 4.4.2 Regulation of the Amount of Pigment

Tyrosinase-catalyzed hydroxylation of tyrosine is the rate-limiting step in melanogenesis, and most often there is an excellent positive correlation between changes in the rate of this reaction measured in situ and variations in the melanin content in melanocyte cultures of different racial origin or phenotype. Tyrosinase activity can be modulated through changes in the intracellular concentration of the enzyme or by modification of the specific activity of pre-existing enzyme molecules. Both types of regulation are operative in mammalian melanocytes. 4.4.2.1 Regulation of Tyrosinase Levels Intracellular protein levels reflect the balance between the rates of their synthesis and their degradation. As described above, the biosynthesis of tyrosinase and other melanosomal proteins is stimulated by the melanocortin hormones. On the other hand, the rate of tyrosinase degradation is regulated by both intracellular factors and external signaling molecules. Among the best-characterized intracellular factors contributing to the control of tyrosinase degradation, we will briefly discuss mutations in the tyrosinase gene affecting the structure of the protein [60], the intracellular composition of fatty acids [107–109], and interactions with other melanosomal proteins [58, 59]. Concerning external signaling molecules, the tyrosinase-destabilizing effects of cytokines normally present in the skin have been reported [110, 111].

4.4 Regulation of the Melanogenic Pathway

Intracellular traffic of melanosome-resident proteins and other proteins of the biosynthetic-secretory pathway is regulated by stringent quality control mechanisms, whereby misfolded or incorrectly assembled proteins are retained in the ER. Misfolded proteins accumulating in the ER are frequently retrotranslocated to the cytosol and degraded by proteasomes [112]. Proteasomal degradation following retrotranslocation involves ubiquitylation, a post-translational modification consisting of the covalent attachment of the 76-amino-acid ubiquitin polypeptide to the ε-amino group of a Lys residue in a protein substrate. A number of diseasecausing mutations in the tyrosinase gene found in OCA1 patients result in extensive intracellular retention in the ER and subsequent proteasomal degradation of tyrosinase [60]. This process involves aberrant N-glycosylation of the enzyme causing abnormal interaction with ER-resident chaperones [47, 113–115]. Aberrant processing with ubiquitylation and degradation of tyrosinase is also causally related to the depigmented phenotype of human melanomas [51] and also results in lack of pigmentation in normal melanocytes [56], thus showing that increased tyrosinase degradation can have profound effects on visible pigmentation. Recent findings suggest that the ubiquitin–proteasome system (UPS) plays a much more complex and sophisticated role in tyrosinase regulation than merely preventing the accumulation of misfolded and enzymatically inactive protein molecules in carriers of OCA1 mutations or in amelanotic melanomas (reviewed in [116]). Indeed, proteasomal degradation of wild-type tyrosinase appears also possible and susceptible of regulation. Fatty acids have been shown to modulate the stability of tyrosinase by modifying the activity of the UPS in opposing directions, depending on their structure. Thus, saturated palmitic acid stabilizes tyrosinase, whereas the monounsaturated and longer linoleic acid increases tyrosinase ubiquitylation and accelerates its degradation [108]. Accordingly, linoleic acid has been shown to exert a pharmacologic hypopigmenting effect [117]. More recently, it has been reported that the rate of tyrosinase ubiquitylation and hence proteasomal degradation of the enzyme is controlled at least partially by the activity of p38 mitogen-activated protein kinase (MAPK). p38 MAPK is activated by a variety of internal or environmental stresses including UV radiation and might in fact play a role in tyrosinase transcriptional activation during the tanning response [118]. Intriguingly, inhibition of p38 MAPK expression decreased tyrosinase ubiquitylation and consequently stimulated melanogenesis, suggesting that p38 might in fact behave as a negative regulator of tyrosinase intracellular stability [119]. p38 MAPK is activated by α-MSH in mouse melanocytes [120] and in human melanoma cells (our unpublished results), and is also activated following exposure to UV radiation [118]. This suggests that regulated tyrosinase ubiquitylation and proteasomal degradation can provide a mechanism to fine-tune the stimulatory effect of melanogenic stimuli such as the melanocortin peptides or UV radiation, whereby an excessive melanogenic response would be prevented via accelerated degradation of tyrosinase. It has been reported that tyrosinase quality control and degradation can also occur in a post-Golgi compartment probably related with the endosomal–lysosomal system, following complete maturation and independently of the UPS machinery

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[121]. This nonproteasomal degradative pathway could be induced by active-site directed inhibitors of tyrosinase such as phenylthiourea and might involve a specific Cys protease. The relative contributions of the proteasomal and nonproteasomal pathways to the degradation of normal and mutant tyrosinase variants remain to be determined. Using purified preparations of tyrosinase and Tyrp1 we showed that both proteins interact strongly and specifically in detergent solution to form heterodimeric complexes [59]. The functional consequences of this specific interaction have been analyzed. Hearing et al. first demonstrated that tyrosinase is less stable in mutant Tyrp1 mouse melanocytes than in cells expressing wild-type Tyrp1 and that forced expression of wild-type Tyrp1 in mutant melanocytes partly rescued the normal phenotype by increasing tyrosinase stability [58]. Direct interaction of tyrosinase and Tyrp1 in vivo, with significant stabilization of the former, was further confirmed by chemical cross-linking experiments [122]. The strong effect of Tyrp1 on tyrosinase maturation and stability might provide a basis for the albino phenotype of OCA3 patients, whereby a mutation in Tyrp1 might impair the maturation and stability of tyrosinase thus decreasing tyrosinase activity [60]. In summary, tyrosinase degradation appears to follow at least two different pathways. On the one hand, a UPS-dependent pathway might be particularly relevant for mutant or otherwise misfolded tyrosinase molecules. On the other hand, a post-Golgi proteasome-independent route most likely related with endosomal– lysosomal compartments and involving a specific Cys protease might act on fully glycosylated mature forms of the protein, and hence might be particularly relevant for the turnover of normal and correctly processed enzyme. The proteasomedependent pathway appears susceptible of regulation by internal factors (fatty acids) as well as by external stimuli (α-MSH, UV radiation) acting at least partially through p38 MAPK. Regulation of the UPS might contribute to the fine-tuning of pigmentation by preventing excessive melanin synthesis in cells following high intensity melanogenic stimuli. Moreover, the skin whitening effects of unsaturated fatty acids in vivo provide a proof of principle showing that pharmacological modulation of tyrosinase stability can be used as a strategy to treat pigmentary disorders. 4.4.2.2 Control of Tyrosinase-Specific Activity Several possible post-transcriptional mechanisms of regulation of tyrosinase specific activity have been reported, including activation by protein kinase C-dependent phosphorylation of residues located in the cytosolic tail of the protein [123], and changes in the melanosomal pH. Modulation of the melanosomal pH appears a particularly important mechanism, as it is likely one of the factors underlying racial differences in skin pigmentation and may also contribute to tyrosinase activation following stimulation of melanocytes with α-MSH. Melanocytes of black and Caucasian skin show very similar levels of tyrosinase mRNA and protein abundance, but “in situ” tyrosinase activity measured in intact cells is much higher in melanocytes from black donors [124]. Thus, the good correlation between melanin content and tyrosinase activity in melanocytes of

4.5 Conclusions and Perspectives

different racial origin is not accounted for by parallel differences in enzyme abundance, indicative of a major role for post-transcriptional regulatory mechanisms. This important difference in tyrosinase specific activity in melanosomes of different racial origin appears related with the pH of the organelle. Melanosomes are organelles of the endosomal–lysosomal lineage and as such they are expected to be acidic. This has been confirmed by estimations of the melanosomal pH in mouse melanocytes and melanoma cells [125, 126], as well as in normal human melanocyte cultures derived from Caucasian donors [127]. Interestingly, the melanosomes of melanocytes from black donors are more neutral than those derived from Caucasian skin. Moreover, treatment of melanocytes with lysosomotropic agents that increase the melanosomal pH such as ammonium chloride or the ionophores nigericin and monensin strongly stimulates tyrosinase activity in melanocytes from Caucasian donors, but has no effect in black melanocytes [127]. Similar increases in tyrosinase activity were obtained with selective inhibitors of vacuolar proton pump V-ATPase [128]. The optimal pH of mammalian tyrosinase is near neutral and tyrosinase-specific activity decreases dramatically at acidic pH [129, 130]. Accordingly, a model for racial pigmentation based on differences in melanosomal pH has been proposed, whereby the acidic environment in Caucasian melanosomes would keep tyrosinase largely inactive whereas the more neutral pH in black melanosomes would allow for full tyrosinase activity [127]. However, the interpretation of the activatory effect of agents expected to increase the melanosomal pH is complicated by the observation that tyrosinase processing and transport through the ER to the Golgi, and then to melanosomes via the endosomal sorting system, is enhanced in the presence of protonophore or proton pump inhibitors that increase the pH of intracellular organelles [131]. It has been shown that cAMP increases the pH of melanosomes and regulates the expression of several vacuolar ATPases and ion transporters [132]. Therefore, changes in the pH of melanosomes may partially underlie the melanogenic response to the melanocortin peptides. Accordingly, the melanosomal pH would be controlled by genetic determinants responsible for racial skin pigmentation traits, as well as by external signaling molecules such as the melanocortins. In turn, this modulation of the melanosomal pH would determine changes in tyrosinase specific activity thus contributing to the regulation of the rate of melanin biosynthesis.

4.5 Conclusions and Perspectives

The chemical analysis of natural melanin pigments and the kinetic and molecular characterization of new melanogenic enzymes have led to a profound modification of the melanogenic pathway as originally delineated by Raper and Mason. Melanins were originally considered relatively simple polymers containing essentially one type of decarboxylated DHI-derived monomeric units synthesized from

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l-tyrosine by the action of a single enzyme. Now, eumelanins are seen as complex and heterogeneous structures with both decarboxylated and carboxylated (DHICAderived) units formed by the concerted action of three enzymatic proteins, tyrosinase and the closely related Tyrp1 and Dct/Tyrp2. The relative proportions of both types of monomeric units determine the exact physicochemical properties of the pigment and may be highly variable depending on factors such as the activity of the melanogenic enzymes, the availability of substrates, and chemical properties of the melanosomal milieu like its metal ion content and its pH. Accordingly, a wide and quasicontinuous spectrum of physicochemical properties is expected for the natural pigments – a view much more attractive and consistent with our daily perceptions. However, whereas the biochemistry of eumelanogenesis has been basically worked out, current knowledge of the pheomelanogenic pathway remains incomplete. The participation of low-molecular-weight thiol compounds and the major role for 5-cysteynyldopa and alanyl-hydroxy-benzothiazine monomers has been established, but the structure of other biosynthetic intermediates is still unsolved. The involvement of enzymatic activities different from tyrosinase or nonenzymatic proteins such as membrane transporters also remains an open question. Therefore, it can be anticipated that the chemistry of pheomelanogenesis and the biochemical factors governing the type of pigment formed will be major areas of fruitful research in the near future. Moreover, recent evidence suggests that Dct/Tryp2 and maybe also Tyrp1 play intriguing and important roles in the regulation of cell proliferation and survival under certain stress conditions, in addition to their enzymatic activity in the melanogenic pathway. New and exciting findings concerning these nonmelanogenic roles of the tyrosinase family proteins can also be anticipated. The study of the regulation of mammalian melanogenesis has revealed a complex and sophisticated network of interconnected and cross-talking signals, including: (i) intramelanocytic factors such as fatty acids or the pH of intracellular organelles, (ii) paracrine effectors released by cells in the epidermal unit, (iii) systemic endocrine signals, and (iv) environmental influences such as UV radiation. All these factors trigger transcriptional and post-transcriptional regulatory mechanisms whose molecular details and relative importance in different physio(patho)logical situations will be worked out. This increased knowledge of the regulation of normal and aberrant melanogenesis will no doubt lead to better therapeutic approaches to the treatment of pigmentary disorders. Several of the signals that regulate melanin biosynthesis, such as UV radiation or the melanocortin hormones, act coordinately on various aspects of the cellular physiology, including DNA metabolism and cell cycle control, organelle biogenesis, subcellular trafficking, and the rate of a highly specialized metabolic pathway. Moreover, this simultaneous and integrated control of different cellular functions within melanocytes leads to a clear, visible and easily quantifiable phenotypic output. Accordingly, the biosynthesis of melanins provides a unique model for the holistic analysis of the regulation of metabolic processes.

References

Acknowledgments

Work in the authors’ laboratory is supported by grants SAF2009-10942 from the Comisión Interministerial de Ciencia y Tecnología (CICYT), Spain, FEDER funds (European Community), and 464/2008 from the CARM, Plan de Ciencia y Tecnología 2007/10. We thank Celia Jiménez-Cervantes for continuous help, support, and collaboration. We apologize to the many colleagues whose relevant work has not been explicitly cited due to space limitations.

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Valencia, J.C., Costin, G.E., Watabe, H., Yasumoto, K., Niki, Y., Kondoh, H., Ichihashi, M., and Hearing, V.J. (2006) Intracellular composition of fatty acid affects the processing and function of tyrosinase through the ubiquitin– proteasome pathway. Biochem. J., 394, 43–50. Martinez-Esparza, M., JimenezCervantes, C., Beermann, F., Aparicio, P., Lozano, J.A., and Garcia-Borron, J.C. (1997) Transforming growth factor-beta 1 inhibits basal melanogenesis in B16/ F10 mouse melanoma cells by increasing the rate of degradation of tyrosinase and tyrosinase-related protein-1. J. Biol. Chem., 272, 3967–3972. Martinez-Esparza, M., JimenezCervantes, C., Solano, F., Lozano, J.A., and Garcia-Borron, J.C. (1998) Mechanisms of melanogenesis inhibition by tumor necrosis factoralpha in B16/F10 mouse melanoma cells. Eur. J. Biochem., 255, 139–146. Helenius, A., Marquardt, T., and Braakman, I. (1992) The endoplasmic reticulum as a protein-folding compartment. Trends Cell. Biol., 2, 227–231. Petrescu, S.M., Petrescu, A.J., Titu, H.N., Dwek, R.A., and Platt, F.M. (1997) Inhibition of N-glycan processing in B16 melanoma cells results in inactivation of tyrosinase but does not prevent its transport to the melanosome. J. Biol. Chem., 272, 15796–15803. Petrescu, S.M., Branza-Nichita, N., Negroiu, G., Petrescu, A.J., and Dwek, R.A. (2000) Tyrosinase and glycoprotein folding: roles of chaperones that recognize glycans. Biochemistry, 39, 5229–5237. Xu, Y., Bartido, S., Setaluri, V., Qin, J., Yang, G., and Houghton, A.N. (2001) Diverse roles of conserved asparaginelinked glycan sites on tyrosinase family glycoproteins. Exp. Cell. Res., 267, 115–125. Ando, H., Ichihashi, M., and Hearing, V.J. (2009) Role of the ubiquitin proteasome system in regulating skin

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pigmentation. Int. J. Mol. Sci., 10, 4428–4434. Shigeta, Y., Imanaka, H., Ando, H., Ryu, A., Oku, N., Baba, N., and Makino, T. (2004) Skin whitening effect of linoleic acid is enhanced by liposomal formulations. Biol. Pharm. Bull., 27, 591–594. Galibert, M.D., Carreira, S., and Goding, C.R. (2001) The Usf-1 transcription factor is a novel target for the stressresponsive p38 kinase and mediates UV-induced tyrosinase expression. EMBO J., 20, 5022–5031. Bellei, B., Maresca, V., Flori, E., Pitisci, A., Larue, L., and Picardo, M. (2010) p38 regulates pigmentation via proteasomal degradation of tyrosinase. J. Biol. Chem., 285, 7288–7299. Smalley, K. and Eisen, T. (2000) The involvement of p38 mitogen-activated protein kinase in the alpha-melanocyte stimulating hormone (alpha-MSH)induced melanogenic and antiproliferative effects in B16 murine melanoma cells. FEBS Lett., 476, 198–202. Hall, A.M. and Orlow, S.J. (2005) Degradation of tyrosinase induced by phenylthiourea occurs following Golgi maturation. Pigment Cell Res., 18, 122–129. Kobayashi, T. and Hearing, V.J. (2007) Direct interaction of tyrosinase with Tyrp1 to form heterodimeric complexes in vivo. J. Cell. Sci., 120, 4261–4268. Park, H.Y., Perez, J.M., Laursen, R., Hara, M., and Gilchrest, B.A. (1999) Protein kinase C-beta activates tyrosinase by phosphorylating serine residues in its cytoplasmic domain. J. Biol. Chem., 274, 16470–16478. Iozumi, K., Hoganson, G.E., Pennella, R., Everett, M.A., and Fuller, B.B. (1993) Role of tyrosinase as the determinant of pigmentation in cultured human melanocytes. J. Invest. Dermatol., 100, 806–811. Bhatnagar, V., Anjaiah, S., Puri, N., Darshanam, B.N., and Ramaiah, A. (1993) pH of melanosomes of B 16 murine melanoma is acidic: its physiological importance in the regulation of melanin biosynthesis. Arch. Biochem. Biophys., 307, 183–192.

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130 Martinez, J.H., Solano, F., Garcia-

M.H. (2000) Aberrant pH of melanosomes in pink-eyed dilution (p) mutant melanocytes. J. Invest. Dermatol., 115, 607–613. 127 Fuller, B.B., Spaulding, D.T., and Smith, D.R. (2001) Regulation of the catalytic activity of preexisting tyrosinase in black and Caucasian human melanocyte cell cultures. Exp. Cell Res., 262, 197–208. 128 Ancans, J., Tobin, D.J., Hoogduijn, M.J., Smit, N.P., Wakamatsu, K., and Thody, A.J. (2001) Melanosomal pH controls rate of melanogenesis, eumelanin/ phaeomelanin ratio and melanosome maturation in melanocytes and melanoma cells. Exp. Cell. Res., 268, 26–35. 129 Hearing, V.J. and Ekel, T.M. (1976) Mammalian tyrosinase. A comparison of tyrosine hydroxylation and melanin formation. Biochem. J., 157, 549–557.

Borron, J.C., Iborra, J.L., and Lozano, J.A. (1985) The involvement of histidine at the active site of Harding-Passey mouse melanoma tyrosinase. Biochem. Int., 11, 729–738. 131 Watabe, H., Valencia, J.C., Yasumoto, K., Kushimoto, T., Ando, H., Muller, J., Vieira, W.D., Mizoguchi, M., Appella, E., and Hearing, V.J. (2004) Regulation of tyrosinase processing and trafficking by organellar pH and by proteasome activity. J. Biol. Chem., 279, 7971–7981. 132 Cheli, Y., Luciani, F., Khaled, M., Beuret, L., Bille, K., Gounon, P., Ortonne, J.P., Bertolotto, C., and Ballotti, R. (2009) αMSH and cyclic AMP elevating agents control melanosome pH through a protein kinase A-independent mechanism. J. Biol. Chem., 284, 18699–18706.

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5 Inhibitors and Enhancers of Melanogenesis Alain Taïeb, Muriel Cario-André, Stefania Briganti, and Mauro Picardo

5.1 Introduction

In humans, normal levels of pigmentation differ among races, and reflect quantitative and qualitative differences in amount, size, and type of melanins synthesized and stored by melanosomes, which are specific melanin-containing lysosome-like intracellular organelles located inside melanocytes [1–3]. Each epidermal melanocyte is surrounded by approximately 36 keratinocytes, forming the so-called epidermal melanin unit, which plays a key role in the distribution of melanin pigments, but which is also under strong dermal influences [4, 5]. Phenotypic differences in constitutive pigmentation are not related to melanocyte numbers, which are almost the same in all skin types [6], but are determined by several other factors, including the level of melanogenic activity [7], the ratio of black/brown eumelanin to red/yellow pheomelanin [8, 9], melanosome transport, and distribution into neighboring keratinocytes [10] (see also Chapter 10). Thus, the melanin content and distribution inside the epidermal layers can be considered a sign of the healthiness of the skin. However, despite its photoprotective function in human skin, abnormal increased production and accumulation of melanin characterizes a large number of skin diseases, including acquired hyperpigmentation, such as melasma, postinflammatory melanoderma, freckles, ephelides, senile lentigines, and so on [11, 12]. On the contrary, a reduction of skin pigmentation is associated with degenerative processes, such as depigmentation in aged skin, which is considered a marker of melanocyte senescence, or with pathological conditions, such as vitiligo. The impact of hyper/hypopigmentation disorders is considerable across most human cultures and higher in general in dark-skinned populations, even for hyperpigmentation. The resulting psychosocial and cosmetic problems, and large profitable market, have led to the more thorough investigation of melanogenic pathways and to the screening of the activity of a number of recognized and putative melanogenesis modulators.

Melanins and Melanosomes: Biosynthesis, Biogenesis, Physiological, and Pathological Functions, First Edition. Edited by Jan Borovanský and Patrick A. Riley. © 2011 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2011 by Wiley-VCH Verlag GmbH & Co. KGaA.

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5.1.1 Melanin Biochemistry 5.1.1.1 Melanin Biosynthesis Epidermal melanogenesis is a dynamic process, involving several regulating factors, which results in a peculiar composition of melanin pigments in accordance with skin type. Inside the melanosomes, the biosynthetic pathway for melanin formation is controlled by a combination of enzymatically catalyzed and chemical reactions. The first and rate-limiting step of melanogenesis is the oxidation of the amino acid l-tyrosine to dopaquinone catalyzed by tyrosinase or polyphenoloxidase (EC 1.14.18.1, o-diphenol : O2 oxidoreductase). The enzyme uses molecular oxygen to catalyze the hydroxylation of the monophenol l-tyrosine to the o-diphenol 3,4-dihydroxyphenylalanine (dopa) and the oxidation of dopa to the o-quinone dopaquinone, which subsequently serves as precursor of both eumelanin and pheomelanin (see Chapters 3, 4, 6) [13, 14]. Dopaquinone is spontaneously converted to the monomeric indolic precursors (5,6-dihydroxyindole (DHI) and DHI2-carboxylic acid (DHICA)) of the black/brown pigment eumelanin. Moreover some other enzymes, like the tyrosinase-related protein (TRP-1/Tyrp1) and dopachrome tautomerase (Dct; also known as tyrosinase-related protein 2 (TRP-2/ Tyrp2)) may also play an important role in melanogenesis in vivo. TRP-2 catalyzes the tautomerization of dopachrome to DHICA, which is later oxidized to DHICAmelanin subunits. TRP-1 has been reported to catalyze the oxidation of DHICA to eumelanin. Subunits of eumelanin are composed by DHICA in conjunction with DHI, which is generated by the spontaneously lost carboxylic acid moiety of dopachrome. Moreover, TRP-1 is important for the correct trafficking of tyrosinase to the melanosomes [15] and stabilization of its enzymatic activity [16, 17], and TRP-2 seems to be involved in the tyrosinase detoxification inside melanosomes [18]. In the presence of thiol compounds, such as cysteine of glutathione, dopaquinone forms 2- or 5-S-cysteinyldopa that generates the benzothiazine precursors of the yellow/red soluble melanins, known as pheomelanins [19, 20]. In general, a mixed type of pheo- and eumelanin polymer is produced and deposited onto the melanosomal matrix proteins. 5.1.1.2 Tyrosinase Maturation and Degradation Tyrosinase contains six N-linked glycosylation sites that are conserved in human and mouse tyrosinases, and mutations of these critical protein maturation sites reduce tyrosinase catalytic function [21]. The altered ability to glycosylate tyrosinase induces the inhibition of its folding and maturation in the endoplasmic reticulum (ER) and Golgi, resulting in hypopigmentation [22]. Tyrosinase is degraded endogenously, at least in part, via the ubiquitin–proteasome system (UPS) [23], which selectively degrades intracellular ubiquitylated proteins, such as proteins misfolded in the ER and short-lived proteins [24–26]. Proteasome proteolyses tyrosinase via ER-associated protein degradation, a mechanism for quality control that involves retention in the ER and retro-translocation into the cytosol of misfolded or unassembled secretory proteins [27].

5.1 Introduction

5.1.1.3 Catalytic Site Tyrosinase is widely distributed throughout the phylogenetic scale from bacteria to mammals, and despite the high heterogeneity concerning the length and overall identity of the published sequences, its catalytic site is highly conserved among different species and shows high homology with TRP-1 and TRP-2 [28]. In particular, tyrosinase shows an active site consisting of a hydrophobic pocket, with a central copper-binding domain, which contains two peptidic segments, composed of strictly conserved amino acid residues, including three histidines [29]. Moreover, human tyrosinase shows the N-terminal signal peptide, which plays a relevant role in trafficking and processing the enzyme, and the C-terminal hydrophobic transmembrane segment, which is rich in cysteine residues and is necessary for targeting tyrosinase to the melanosome [29–32]. The mechanism of catalysis of tyrosinase has been extensively investigated, due to its complexity and the peculiarity of the existence of two catalytic activities (tyrosinase hydroxylase and dopa oxidase) at the same active site [33, 34] (see Chapter 4). The link between copper ions and the three histidines is needed to effectively bind l-tyrosine to the active site and initiate melanin synthesis [35]. Thus, every alteration of peptide segments or copper ions can result in deregulation of tyrosinase activity. 5.1.2 Paracrine Signaling and Regulation of Epidermal Melanogenesis

Melanocytes work in close harmony with their neighboring cells in the epidermis. They are influenced by a variety of biological factors, including cytokines, growth factors, vitamins, and prostaglandins, which determine not only whether melanin is synthesized, but what type of melanin is produced. Presumably, these factors provide the complex signals that stimulate pigmentation after trauma, UV exposure, or other environmental stimuli that induce alterations in the levels of pigment production [36–38]. The expression of tyrosinase, related melanogenic enzymes, and melanosome structural proteins (MART-1 and PMEL17) may be modulated at the transcriptional level by the microphthalmia-associated transcription factor (MITF) [39, 40]. The binding of α-melanocyte-stimulating hormone (α-MSH) to the melanocortin 1 receptor (MC1R) activates adenyl cyclase and the subsequent release of cAMP, leading to activation of the protein kinase A (PKA) pathway. PKA induces the phosphorylation of cAMP-responsive element binding protein (CREB) transcription factors, which mediate MITF-M promoter activation to induce melanogenesis [41]. Interleukin-6 (IL-6) and the Wnt signaling pathway also regulate MITF [42–44]. Post-transcriptional MITF activation involves its transient phosphorylation via ribosomal S6 kinase (RSK), glycogen synthase kinase-3β (GSK3β ), p38 stress signaling, and mitogen-activated protein kinase (MAPK) pathways [45–47]. Moreover, paracrine linkages among keratinocytes, fibroblasts, and melanocytes within the skin play important roles in regulating epidermal melanization. In response to various stimuli, human keratinocytes secrete various cytokines that serve as mitogens or melanogens for human melanocytes, including endothelin (ET)-1, stem cell factor (SCF), basic fibroblast growth factor (bFGF), and α-MSH

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[48–51]. Overexpression of SCF and c-kit in the dermis has been proposed to play an important role in the mechanism of hyperpigmentation in melasma [52]. 5.1.3 Methods of Study

There has been great variation in results reported for different bioactive compounds targeted at regulating melanin production; in part, this variation derives from the diverse conditions of melanogenic assays employed, the different cell lines (including melanoma cells) used as targets, and the different endpoints used to establish efficacy. In an attempt to standardize the assessment of novel bioactive agents for therapy of pigmentary lesions, a method called STOPR (standardized testing of pigmentary regulators) has been developed [53]. This protocol involves initial screening of putative melanogenic compounds using purified tyrosinase, followed by evaluation of cell proliferation, total melanin accumulation, and melanogenic potential using cultured pigmented murine melanocytes. However, even if the results obtained with the STOPR method are reproducible and economic, and can provide important insights into the mechanism(s) of depigmenting agents, more details about the effects toward human pigmentation process are needed. Up to now, in fact, the majority of in vitro testing studies have been performed by using mushroom tyrosinase, whereas the use of human tyrosinase can afford results more comparable to those observable in human skin [54]. To evaluate the active role of keratinocytes in melanogenesis modulation mammalian skin or keratinocyte–melanocyte cocultures should be preferred as testing models rather than only pure melanocyte cultures. For instance, arbutin is a more potent inhibitor of melanin formation on melanocytes cocultured with keratinocytes than on pure melanocyte cultures [55]. Furthermore, to analyze the contribution of dermal cells to the pigmentation process skin reconstructs including fibroblasts should be used [5]. However, no experimental models are available for evaluating the effect of compounds able to induce hypopigmentation not only by inhibiting de novo melanin production, but also by increasing desquamation or bleaching pigment. Recently, new approaches have been proposed to discover new depigmenting agents. Chemical genetic screening performed in cultured Melan-A cells has been successfully employed to show depigmenting properties in vitro and shed new light on pathways influencing mammalian pigmentation [56, 57]. Potent pigmentation enhancers or inhibitors have been identified by screening tagged triazine-based combinatorial libraries in immortalized murine melanocytes, albino melanocytes, or zebrafish. The mechanism of action of the identified compounds were investigated in zebrafish and in skin equivalents, as useful tools to address the ability of these molecules to modulate pigmentation in a model more closely representative of human skin. In particular, zebrafish may represent a useful and cost-effective alternative animal model, and can afford a good method to identify and characterize mutations that alter pigmentation or influence the development or subsequent migration of pigmentation precursor cells in the neural crest [58, 59].

5.2 Depigmenting Agents

5.2 Depigmenting Agents

Table 5.1 summarizes the agents discussed in the text according to the main pharmacological target and Table 5.2 indicates the major clinical indications. 5.2.1 Agents Acting Prior to Melanin Synthesis 5.2.1.1 Transcriptional Inhibition of Melanogenic Enzymes Considering the relevant role played by MITF in regulating melanogenesis, modulation of signaling pathways involved in its activity may have potential therapeutic use. Substances able to inhibit MITF expression and activity, as well as the extracellular signal-regulated kinase (ERK) and serine/threonine kinase (Akt)/protein kinase B (PKB) pathways could represent depigmenting agents [82, 83]. Among these, all-trans-retinoic acid (ATRA) is able to interfere with melanocyte development and melanogenesis. ATRA has a lightening effect on hyperpigmented lesions of photodamaged human skin [74, 75], but its depigmenting mechanism is not well known. This compound exerts controversial effects on melanocytes: it induces the transcription of tyrosinase by protein kinase C (PKC) activation and MITF expression, leading to melanocyte differentiation, and it promotes apoptosis of differentiated melanocytes via the caspase-3 pathway and Bcl-2 downmodulation [84]. In B16 melanoma cells, ATRA caused growth arrest and differentiation, and increased the level of PKC, which activates tyrosinase via phosphorylation of serine residues of cytoplasmic domain, thus enhancing melanin production [85]. Several researchers reported that ATRA and other vitamin A derivatives are able to increase pigmentation both in vitro and in animal models [86, 87]. However, other data in the literature suggest that ATRA activate melanogenesis only in cells or subjects with low amounts of melanin, whereas it significantly improves the irregular hyperpigmentation associated with photoaging of inflammation [88]. Solar lentigo, a common component of photoaged skin, has been reported to represent an impaired homeostasis of the epidermal unit due to chronic sun exposure [89]. In the pathogenetic model suggested by the authors based on immunohistochemistry and electron microscopy, keratinocytes are more altered than melanocytes, suggesting that abnormal pigment retention in keratinocytes is the primary defect in solar lentigo. This evidence may partly explain the therapeutic effect of retinoids, which firstly targeted keratinocytes. Recently, ATRA and retinol have been found to inhibit cAMP-mediated melanin production in B16 melanoma cells and to downregulate tyrosinase, as well as TRP-1 expression prior to translation [90]. Although the role of PKC in the induction of melanogenesis remains controversial the existence of an interaction between the PKC pathway and the cAMPdependent PKA pathway has been suggested by the evidence that PKC inhibitors sphingosine and calphostine also inhibit the melanogenic response dependent on α-MSH [77]. The link between PKC- and cAMP-dependent pathways in regulating

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Transcription inhibition 4-tertiary butylphenol

hydroquinone

4-hydroxyanisole N-acetyl-4-S cystaminylphenol monobenzyl ether of hydroquinone 4-n-butylresorcinol α-arbutin

C2-ceramide

zeolite

diosgenin

L. apetalum extract

dihydrolipoic acid

4-tertiary butylcathecol

Interference with tyrosinase activity

lysophosphatidic acid

protein kinase C inhibitors

all-trans-retinoic acid

During melanin synthesis

Classification of depigmenting agents according to main pharmacological impact.

Before melanin synthesis

Table 5.1

reduction of melanosome transfer

inhibition of melanocytic dendrite formation

niacinamide

methylophiopogonanone B centaureidin

Inhibitors of melanosome transfer

After melanin synthesis

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Post-translational modification of melanogenic enzymes

kojic acid gentisic acid and methyl ester of gentisic acid azelaic acid aloesin ellagic acid resveratrol oxyresveratrol

N-butyldeoxynojircimicin

deoxymannojirimycin

d-pantetheine-S-sulfonate

resveratrol

quinolines

linoleic acid

phospholipase D2

TRP-2 (Dct) modulation

deoxyarbutin

glucosamine

pyrroloquinoline quinone

all-trans-retinoic acid

adapalene

β-carotene

tarazotene

(Continued)

all-trans-retinoic acid

retinoids

2,6-dimethoxy-N-(4methoxyphenyl) benzamide

lectins neoglycoproteins

RWJ-50353 soybean extracts (soybean trypsin inhibitor and Bowman– Birk protease inhibitor)

Acceleration of epidermal turnover

inhibition of membrane glycosylation

inhibition of protease-activated receptor 2

After melanin synthesis

oxyresveratrol derivative

gnetol

arbutin

During melanin synthesis

tunicamycin

Before melanin synthesis

5.2 Depigmenting Agents 123

(Continued)

Before melanin synthesis

Table 5.1

peroxidase inhibitors

interference with the melanogenic pathway

interference with byproduct production

methimazole

N,N′-dilinoleylcystamine

cystamine

thioctic acid

gallic acid

pycnogenol

piceatannol

phytoncide

licorice extracts

salicylic acid

α-tocopherol ferulate

linoleic acid

lactic acid

α-tocopherol

6-hydroxy-3,4dihydrocumarins

glycolic acid

disodium isostearyl 2-O-lascorbyl phosphate

trichloroacetic acid

magnesium l-ascorbate-3-phosphate

chemical peels

retinaldehyde

After melanin synthesis

ascorbic acid

During melanin synthesis

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5 Inhibitors and Enhancers of Melanogenesis

5.2 Depigmenting Agents Table 5.2

Clinical applications of depigmenting agents.

Melanogenic step

Treatment

Clinical indication

Main references

Interference with tyrosinase activity

hydroquinone

melasma

reviews: [60, 61] (Cochrane database)

postinflammatory hyperpigmentation solar lentigo 4-n-butylresorcinol

melasma

[62, 63]

solar lentigo deoxyarbutin

solar lentigo

[64, 65]

azelaic acid

melasma

reviews: [60, 61] (Cochrane database)

postinflammatory hyperpigmentation

Interference with byproducts production

ellagic acid + arbutin

melasma

aloesin

postinflammatory hyperpigmentation

aloesin + arbutin

postinflammatory hyperpigmentation

[67]

kojic acid

melasma

[68]

ascorbic acid

melasma

[69]

[66]

postinflammatory hyperpigmentation solar lentigo

Peroxidase inhibitors

α-tocopherol

melasma

thioctic acid

melasma

pycnogenol

melasma

methimazole

melasma

[70, 71]

postinflammatory hyperpigmentation Inhibitors of melanosome transfer

niacinamide

postinflammatory hyperpigmentation

[72, 73]

solar lentigo (Continued)

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5 Inhibitors and Enhancers of Melanogenesis Table 5.2

(Continued)

Melanogenic step

Treatment

Clinical indication

Main references

Acceleration of epidermal turnover

all-trans-retinoic acid

melasma

reviews: [60, 61, 74, 75] (Cochrane database)

solar lentigo tarazotene

postinflammatory hyperpigmentation

[52, 76]

solar lentigo adapalene

melasma

[52, 74, 77]

solar lentigo β-carotene

melasma

[78]

trichloroacetic acid

melasma

reviews: [60, 61] (Cochrane database)

solar lentigo glycolic acid

melasma solar lentigo

salicylic acid

melasma

reviews: [60, 61] (Cochrane database) [79]

postinflammatory hyperpigmentation

Combined therapy

linoleic acid

melasma

[80]

licorice extracts

melasma

[81]

hydroquinone + alltrans-retinoic acid + a steroid

melasma

reviews: [60, 61] (Cochrane database)

postinflammatory hyperpigmentation solar lentigo

hydroquinone + alltrans-retinoic acid

melasma

reviews: [60, 61] (Cochrane database)

melanogenesis has been confirmed by the evidence that MITF-M is a key factor for PKC-β [91]. Bisisolylmaleimide GF 109203X, a selective inhibitor of PKC, was found to reduce melanin production in human melanocytes, and to decrease both basal and UV-induced skin pigmentation in guinea, pigs, suggesting its possible application for the treatment of unwanted pigmentation [92]. Moreover, lysophosphatidic acid (LPA), an intercellular lipid mediator, which is known to have growth factor-like activity, contributes to reduced melanin synthesis via the downregulation of MITF [93]. Sphingomyelin is the most common sphingolipid in the skin

5.2 Depigmenting Agents

and ceramides are products of sphingomyelin hydrolysis by sphingomyelinases [94], suggesting that sphingolipid breakdown products play important roles in the regulation of epidermal proliferation, differentiation, and melanin synthesis. C2ceramide was found to inhibit cell proliferation and melanogenesis in human melanocytes, and in a mouse melanocyte cell line [83, 95, 96]. The suggested mechanism by which C2-ceramide decreases the pigmentation of melanocytes involves a sustained and long-lasting activation of ERK1/2 and Akt kinase, resulting in a reduction of MITF and inhibiting tyrosinase activity. Although rapid activation of ERK induces MITF phosphorylation and degradation, its delayed activation may lead to the inhibition of MITF expression Zeolites have crystalline open framework structures constructed from SiO4 and AlO4 tetrahedra linked through oxygen bridges. Each oxygen atom is shared by two silicon or aluminum atoms. Silicates and aluminosilicates possess biological activity and also are nontoxic agents. Zeolite inhibits MITF-mediated tyrosinase expression and melanin synthesis via increasing ERK activity in B16F10 melanoma cells [97]. Recently, heterocyclic pyrimidine compounds have been reported to protect epidermal cells from UV-B-induced damage and also to suppress melanogenesis via an MITF-mediated decrease of tyrosinase expression [98]. Compounds able to interfere with MITF post-transcriptional regulation can modulate the pigmentation process. Diosgenin, a steroidal saponin that is found in several plants, shows inhibitory effects on melanogenesis by activating phosphatidylinositol-3-kinase (PI3K) signaling. Akt and GSK3β are downstream molecules of PI3K-mediated signaling, and have been widely implicated in cell homeostasis by its ability to phosphorylate a broad range of substrates [99]. Diosgenin increases phosphorylated levels of both Akt and GSK3β, leading to a subsequent activation of MITF [80]. Cytokines reportedly regulate melanogenesis, and are major regulators of tyrosinase and related proteins. In Mel-Ab cells, transforming growth factor (TGF)-β1 inhibits pigment formation, and is able to interfere with tyrosine synthesis and possibly with the intracellular stability of the protein itself [100–102]. The proposed mechanism is a delayed ERK activation and subsequent reduction of MITF and tyrosinase production. In addition, IL-6 and tumor necrosis factor (TNF)-α are able to decrease pigmentation by acting on tyrosinase activity, although they exhibit an inhibitory effect in much higher concentrations [103, 104]. An extract of Lepidium apetalum (ELA) was reported to decrease UV-induced skin pigmentation in brown guinea pigs and melanogenesis of HM3KO human melanoma cells. The observed decrease of tyrosinase mRNA and protein expression as well as melanin content is proposed to be connected to ELA-mediated increase of IL-6 in keratinocytes, and the subsequent decreased expression of MITF [105]. The central role of MITF in melanogenesis is confirmed by the evidence that some antioxidants, such as dihydrolipoic acid, α-lipoic acid (LA), and resveratrol, acts as depigmenting agents by a mechanism that does not involve the antioxidant activity per se. These compounds affect MITF and tyrosinase promoter activities, and reduce both endogenous and induced MITF mRNA and protein levels. Moreover, these agents induce depigmentation in melanocyte cultures, by counteracting

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the forskolin- and UV-B-stimulated promoter activities of these genes, and significantly reduce tyrosinase activity [46]. Dihydrolipoic acid was found to depigment dark-skinned swine and to prevent UV-B-induced tanning in vivo. However, due to the fact that MITF is essential in regulating melanocyte differentiation, development, and survival [40, 106], to introduce compounds able to reduce MITF expression and activity as therapy for skin hyperpigmentation can lead to melanocyte death, as suggested by the decreased resistance to UV-induced apoptosis in melanocytes deficient in MITF expression [107]. 5.2.1.2 Post-Translational Modification of Melanogenic Enzymes The major post-translational modification of melanogenic enzymes is asparaginelinked glycosylation, a process critical for the proper maturation of enzymes and the release of their soluble form [21, 108]. Melanogenic proteins show different glycosylation patterns, probably due to the different three-dimensional structures of tyrosinase, TRP-1, and TRP-2 [109]. Agents able to inhibit N-glycosylation result in improper protein folding and reduction of melanosome maturation, inhibition of melanosomal enzyme activities, or suppression of melanogenesis [110, 111]. In murine as well as in human melanoma cells, treatment with specific inhibitors of lipid carrier-dependent glycosylation of protein, such as tunicamycin or glucosamine, is able to decrease pigmentation and alter melanosome structure and biochemical functions [112, 113]. N-Butyldeoxynojircimicin inactivates tyrosinase and dramatically decreases melanin content in B16 melanoma cells, by inhibiting α-glycosidase I, an early step of N-glycosylation [114]. The same compound induced only a slight decrease of dopa oxidase activity of TRP-1, suggesting that this enzyme is able to bypass the glycosidase blockade. Recently, it was found that the pH-sensitive liposomes increased the glycosylationinhibiting and, thus, pigment-lightening effects of N-glycosylation inhibitors in vitro [115]. Treatment of HM3KO melanoma cells with deoxymannojirimycin, an inhibitor of α-1,2-mannosidase, which is thought to be responsible for late glycan processing, showed inhibition of glycosylation, transportation of tyrosinase to the melanosome and melanin synthesis [116]. In B16 melanoma cells, BMY-28565, a tyrosinase inhibitor, inhibited melanogenesis by depressing tyrosinase activity with no impact on tyrosinase mRNA levels and it has been suggested that this compound inhibited tyrosinase by modifying the sugar moieties of the enzyme [117]. Several compounds, including calcium d-pantetheine-S-sulfonate [118], ferritin [119], and glutathione [120], have been found to act as depigmenting agents by interfering with melanogenic enzyme glycosylation. Regarding calcium d-pantetheine-S-sulfonate, the similarity with the chemical structure of coenzyme A, which is involved in intracellular transport of protein, can play a relevant role in its mechanism of action [121]. Ferritin downmodulation and the subsequent intracellular stress conditions have been reported to substantially influence proper maturation of tyrosinase [119]. Resveratrol treatment of melanocytes determined depigmentation by increasing the retention inside of ER of immature tyrosinase and disrupting the trafficking of the enzyme from the ER to the Golgi, leading to a dramatic decrease of functional tyrosinase [122].

5.2 Depigmenting Agents

Apart from the inhibition of tyrosinase glycosylation and the blockade of maturation and transfer processes of the enzyme, the bleaching effect of glutathione involves other mechanisms, such as chelation of copper ions at the active site, switch from eumelanogenesis to pheomelanogenesis, quenching of free radicals produced during melanogenesis, and modulation of depigmentation induced by melanocyte toxic compounds [54, 120]. Furthermore, intracellular pH is an important factor in the regulation of tyrosinase function, dramatically affecting tyrosinase enzymatic activity, and it is critical to the sorting of proteins to melanosomes [123, 124]. In Melan-A cells, quinoline analogs seem to disrupt the intracellular trafficking of tyrosinase gene family proteins, probably with a mechanism involving their ability to modify pH of lysosomal compartment, leading to an alteration of subcellular localization of lysosome-associated membrane protein-1 and the subsequent reduction of pigmentation [125]. 5.2.1.3 Increased Tyrosinase Ubiquination Activation in vitro of tyrosinase degradation has been reported after treatment in vitro with different factors regulating the UPS, such as basic polypeptides, sodium dodecyl sulfate, guanidine-HCl, amino acids, or fatty acids [27, 126, 127]. Fatty acids, which are major components of cell membranes and are highly involved in intracellular signaling and in the binding of nuclear receptors [128], have been identified as intrinsic regulators of the UPS. There are several reports on the ability of fatty acid to induce hypopigmentation, by interfering at different steps of melanogenic pathway [129, 130]. Linoleic acid determined a reduction of melanin content without relevant changes of melanosome numbers or tyrosinase mRNA levels, suggesting a mechanism of action involving maturation and/or degradation of tyrosinase [131]. Fatty acids have been shown to have contrasting effects on the UPS, as unsaturated fatty acids, such as linoleic acid, increases the ubiquitination of tyrosinase, while palmitic acid decreases this process, leading to accelerated or decelerated degradation of tyrosinase by proteasomes, respectively [128, 129]. Topical application of linolenic, linoleic, and oleic acids (efficiency in decreasing order) has been shown to decrease UV-induced hyperpigmentation, suggesting that specific regulators of the UPS could be useful for the prevention and/or treatment of hyperpigmentary disorders. As with unsaturated fatty acids, phospholipase D2 also decreases melanogenesis through the same ubiquitin-mediated degradation of tyrosinase [132]. However, it has been also described that phospholipases and arachidonic acid produced a stimulation of melanogenesis [133], and it is possible that hyperpigmentation associated with the UV light response are mediated by those agents. If so, phospholipases and unsaturated fatty acids do not always exhibit hypopigmenting effects. 5.2.2 Agents Acting During Melanin Synthesis 5.2.2.1 Interference with Tyrosinase Due to its central role in regulating pigmentation, one of the most obvious cellular targets for depigmenting agents is the enzyme tyrosinase. Considering that the

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tyrosinase active site is characterized by a hydrophobic pocket into which the phenyl ring of the substrate is inserted, high stereo-specificity for l-isomers, and spatial restriction imposed by the distance between the phenyl ring and the groups on the side-chain, this suggests that a putative inhibitor should have a correspondent structure. Inhibition of tyrosinase activity can be obtained in different ways, including: (i) competitive inhibition, (ii) noncompetitive inhibition, and (iii) chelation of the copper atoms at the active site. Inhibitor strength is usually expressed as the inhibitory IC50 value, which is the concentration of an inhibitor needed to inhibit half of the enzyme activity in the tested condition. However, the classification of tyrosinase inhibitors based on the structure and mechanism of action is not easy due to the large amounts of products reported and the controversial results of the kinetics performed employing different testing systems from mushroom tyrosinase, mammalian tyrosinase, melanocytic cultures, cocultures of keratinocytes and melanocytes, and finally in vivo application to animal skin. Furthermore, several compounds are able to inhibit tyrosinase activity with multiple mechanisms, such as interfering with residues of both catalytic and regulatory sites or being metabolized to a product able to act as a competitive as well as a noncompetitive inhibitor. A competitive inhibitor might be a copper chelator, nonmetabolizable analog, or derivative of the true substrate and is characterized by the capacity to bind the active site of the enzyme in a reversible manner, avoiding the interaction with the natural substrate and decreasing the enzyme–substrate affinity, as demonstrated by the increase of Michaelis–Menten constant (Km). In contrast, an uncompetitive inhibitor can bind only to the enzyme–substrate complex. A mixed (competitive and uncompetitive mixed) type inhibitor can bind not only with the free enzyme, but also with the enzyme–substrate complex. For most mixed-type inhibitors, their equilibrium binding constants for the free enzyme and the enzyme–substrate complex, respectively, are different. However, a special case among the mixed inhibitors is the noncompetitive inhibitors, which bind to the free enzyme and an enzyme–substrate complex with the same equilibrium constant, inducing a decrease of enzymatic activity maximum velocity (Vm) and not modifying Km. As final result both classes of inhibitors determined a reduction of tyrosinase kinetics and melanin synthesis. Compounds with electron donor groups may act as substrates and those with powerful electron acceptor groups may induce a competitive inhibition of tyrosinase. Inhibitors of tyrosinase may be grouped into two general categories. The first includes compounds that possess an aromatic ring with functional groups, which form hydrogen bonds. The second category includes anions and molecules that form complexes with copper. In the first category are included compounds containing either a 4-substituted phenol moiety or catechols, structurally similar to tyrosine or dopa, which, as alternative substrates of tyrosinase, are oxidized by the enzyme without producing melanin pigment [134, 135]. The 4-substituted phenol group has the ability to bind to the binuclear active site of the enzyme and inhibit its catalytic activity. The catechol structure, with two hydroxyl (OH) groups at the ortho positions, may behave as a chelator to the copper ions of tyrosinase. However, the

5.2 Depigmenting Agents

quinones generated can react with sulfydryl group of melanosomal proteins and/ or essential enzymes, interfering with cell growth and proliferation, and irritating the skin [134]. The first compound introduced as a skin-lightening agent was hydroquinone (HQ), a phenolic compound chemically known as 1,4-dihydroxybenzene, which is considered one of the most effective inhibitor of melanogenesis in vitro and in vivo, and it has been defined as the gold standard to compare the effectiveness of new depigmenting molecules. This compound has been widely used for the treatment of skin pigmentation disorders, such as melasma, or postinflammatory hyperpigmentation [61, 136–138]. Due to its structural homology with melanin precursors, HQ covalently binds to histidine or interacts with copper at the active site of tyrosinase, leading to the inhibition of the enzymatic oxidation of tyrosine and phenol oxidases. It also causes inhibition of RNA and DNA synthesis, and may alter melanosome formation and selectively damage melanocytes [139]. The decrease of melanin production is a direct consequence of HQ-induced suppression of melanocyte metabolic processes. Moreover, the ability of HQ to produce reactive oxygen species (ROS), inducing oxidative damage of membrane lipids and proteins, such as tyrosinase, and to deplete glutathione has been suggested to contribute in inhibiting pigmentation [54, 140–142]. Due to its effectiveness, the use of this molecule for whitening skin treatments was rapidly extended. The efficacy and adverse effects of 4% HQ were evaluated in a double-blind placebocontrolled trial [143] and it has been reported that HQ is generally considered very safe [144]. However, due to its behavior as strong oxidant and its rapid transformation to quinones, which are melanocytotoxic compounds, HQ has been suggested to be toxic when applied on the skin in high concentration [145]. Commonly reported side-effects of HQ are skin irritation or contact dermatitis, which can be easily treated with topical steroids, but in a few patients, after a prolonged topical application of HQ in high concentration, permanent hypomelanosis or exogenous ochronosis, a quite irreversible hyperpigmentation in the treatment area, have been reported. To limit its cytotoxicity and side-effects the HQ dosage was reduced to 2%, a concentration that has been reported to improve hypermelanosis in 14– 70% of patients [146]. Moreover, it has been frequently used for many years, mixed with other compounds to increase its efficiency [61, 147, 148] in a number of melasma treatments. However, due to the hazard of long-term treatments, predisposing even to carcinogenicity [149], today the human use of HQ has important legal restrictions, and the addition of this compound in cosmetics has been banned by the European Commission (24 th Directive 2000/6/EC) and formulations containing HQ are available only by prescription of physicians or dermatologists. Several other phenolic compounds have been screened or used, and studies on the relationship between chemical structure and tyrosinase inhibitory activity have been performed. Methylation of one hydroxyl of para-hydroxylated monophenols favored the nucleophilic attack at the active site, enhancing the catalytic efficiency [150]. 4-Tertiary butylphenol has been reported to induce in human melanocytes a competitive inhibition of both tyrosine hydroxylase and dopa oxidase activities of tyrosinase without a cytotoxic response [138]. However, exposure to 4-tertiary

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butylphenol and 4-tertiary butylcathecol, which are used in the polymer industry, may cause leucoderma [151]. These substances generate reactive quinones and activate apoptotic process, leading to a loss of functional melanocytes [152]. Melanocytes from subjects with different phototypes demonstrated comparable sensitivity to 4-tertiary butylphenol, suggesting that the cytotoxic effect is completely independent from the tyrosinase activity and melanin content. Due to the presence of lypophilic groups, 4-hydroxyanisole (p-hydroxy-methoxybenzene) and N-acetyl-4-S-cystaminylphenol (4-SCAP) penetrate within the epidermis and target pigmented cells, showing depigmenting properties [142, 153–155]. 4-Hydroxyanisole is an alternative substrate for tyrosinase, being oxidized by the enzyme and in melanoma cell lines exhibits melanocytotoxic effects stronger than those exerted by HQ [156, 157]. Clinical effectiveness of 4-hydroxyanisole in skin hyperpigmentation has been demonstrated in combination with treinoin in solar lentiges and related hyperpigmented lesions [158]. Topical treatment with 4-SCAP of Yucatan pigs induces a stable but reversible depigmentation with less irritant effects as compared to HQ [159]. The activity seems to be related to: (i) a marked decrease in the number of functioning melanocytes and melanized melanosomes, (ii) a decreased number of melanosomes transferred to keratinocytes, and (iii) a selective degeneration of melanocytes and deposition of melanin-like material in the Golgi apparatus, where tyrosinase is located [160]. Like HQ, the monobenzyl ether of HQ (MBEH) belongs to the phenol/catechol class of chemical agents. However, this molecule causes a permanent depigmentation of the skin by its ability to be metabolized inside cells to a reactive quinone, which is highly cytotoxic [140]. This property makes MBEH suitable for eliminating residual areas of normally pigmented skin in patients with diffuse vitiligo [161]. In general, all diphenols leading to a reactive quinone are putative cytotoxic agents. This type of hypopigmenting agent does so by its cytotoxic action on melanocytes rather than by tyrosinase inhibition. This is the case of 4-(phydroxyphenyl)-2-butanone [162] and 4-n-butylresorcinol [163, 164]. Moreover, 4-nbutylresorcinol has been shown to inhibit the activity of both tyrosinase and TRP-1, and to be a molecule well tolerated and significantly effective in the treatment of melasma [62, 63, 165]. 4-Hydroxyphenyl α-d-glucopyranoside (α-arbutin) is a naturally occurring HQ β-d-gluconopyranoside, which is enzymatically synthesized from HQ and saccharides [166, 167]. This compound shares with HQ the ability of acting as an alternative substrate of mammalian tyrosinase at noncytotoxic concentrations [168] and its inhibitory activity has been tested against tyrosinase from different sources, such as mushroom, B16 mouse melanoma, and human melanoma cells [169]. The treatment with α-arbutin determined a reduction of melanin synthesis also in human epidermal equivalents containing melanocytes [170]. Interestingly, in the medium of this experimental model about 70% of the applied α-arbutin was recovered, but HQ was not detected, suggesting that the activity of α-arbutin is not due to HQ release. In addition, arbutin, a natural β-glycoside optical isomer of α-arbutin, usually found in cranberries, blueberries, wheat, and pears [171], is a good inhibitor of tyrosinase, without affecting expression and synthesis of the

5.2 Depigmenting Agents

enzyme in human melanocyte cultures [54, 172]. Moreover, this molecule influences DHICA polymerase activity and PMEL17/silver protein, and exerts an inhibitory effect on the maturation of melanosomes [172]. However, the depigmenting effect of arbutin has not been confirmed in clinical trials [173] and its mild effectiveness has been attributed to a controlled release of HQ as a result of in vivo cleavage of the glycosidic bond. Higher concentrations of arbutin are more efficacious than lower concentrations, but may cause paradoxical hyperpigmentation [141]. To increase its efficiency, α-glucosides of arbutin have been chemically synthesized [174], possibly since they are easier hydrolyzed to release HQ due the higher availability of α-glycosidases. Recently, deoxyarbutin, synthesized by removing every hydroxyl group of arbutin, has been identified as an excellent tyrosinase inhibitor in the screening of a number of candidate compounds [64]. In a hairless, pigmented guinea pig model and in human skin deoxyarbutin induced reversible inhibition of in situ tyrosinase activity, and rapid and sustained skin-lightening. This compound, as well as its derivatives, has been found to act as an effective competitive inhibitor of tyrosinase, leading to decreased melanin content in melanocytes [175]. This depigmenting effect is completely reversible, indicating no permanent suppression of tyrosinase activity and melanin synthesis. The presence of hydrophobic and less bulky groups at the para position of phenol increases binding to tyrosinase and decreases the potential for oxidation, leading to effective inhibition and reducing cytotoxicity [65, 176]. Deoxyarbutin and its derivatives can enter viable melanocytes at nontoxic concentrations, blocking enzyme activity of pre-existing tyrosinase, more than 90% of which is bound to the melanosome membrane, determining a post-translational regulation that can be reverted after the compounds are removed [54, 175]. Due to the presence of two copper ions at the active site of the enzyme, chelating compounds can be considered specific for tyrosinase. Kojic acid (5-hydroxy-2hydroxymethyl-4H-pyran-4-one) is an antibiotic produced by many species of Aspergillus and Penicillium with a high capacity for chelating transition metal ions such as Fe3+ and Cu2+. Kojic acid is a good tyrosinase inhibitor, but it has some undesirable side-effects. It may cause allergy [177] and it also has been related so some hepatic tumors in heterozygous mice deficient in p53 [178]. The depigmenting action of kojic acid is attributed to its chelator ability [68], even if it could also interfere at different steps of melanin synthesis [179] and inhibit NF-κB activation in keratinocytes, contrasting the hyperpigmentation associated with inflammatory response [180]. Although in monotherapy kojic acid showed only a modest effectiveness, clinical trials have reported a skin-lightening effect of this compound in combination with other agents [60, 181]. Recently, some stable derivatives have been synthesized having more efficiency because their penetration through the skin is increased [182–184]. In this group, the most important ones are a derivative synthesized by joining two pyrone rings through and ethylene linkage [185] and kojyl-APPA (5-((3- aminopropyl)-phosphino-oxy)-2-(hydroxymethyl)-4H-1-pyran-4one), tested in melanoma cells and normal human melanocytes [186]. Gentisic acid (2,5-dihydrobenzoic acid; GA), a component of gentiana roots, showed good melanogenesis inhibition properties [187] and it has been explored

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for use in topical cutaneous applications. The methyl ester of GA (MG) has been found to inhibit mammalian tyrosinase in pure form, in crude cell-free extract, in addition to inhibition of melanin formation in cultured melanocytes. MG and GA are structurally similar to HQ, and it has been postulated that MG acts as a prodrug and inhibits tyrosinase activity following the liberation of HQ. Kinetics studies, however, have found a direct effect of MG on enzyme function, suggesting a binding of the copper ions in the active site, possibly in the presence of oxygen. MG can enter viable melanocytes at sufficient concentrations to block tyrosinase activity, and thus de novo melanin biosynthesis, and it does not appear to inhibit TRP-2 or TRP-1 at concentrations higher than needed to inhibit tyrosinase [173]. MG was also less cytotoxic and mutagenic than HQ, although some low adverse side-effects were reported. Thus, this compound is proposed as a good candidate for skin-lightening, even if a test in skin models is needed to postulate its effectiveness in vivo. Compounds able to bind either amino or carbonyl groups may block the access of tyrosine to the active site, behaving as competitive inhibitors. Among these, azelaic acid (AZA), a naturally occurring 9-carbon dicarboxylic acid, derived from cultures of Pityrosporum ovale, is included [188, 189]. The depigmenting activity of AZA is mediated by inhibition of mitochondrial oxidoreductase activity and interference with DNA synthesis as well as competitive and reversible inhibition of tyrosinase activity in vitro (Ki = 2.73 × 10−3 M) [190–192]. Its lightening effect appears to be selective and most apparent in highly active melanocytes, with minimal effects in normally pigmented skin [193]. AZA has been used to treat melasma and postinflammatory hyperpigmentation [72, 194], and to arrest the progression of lentigo maligna to melanoma [195]. Topical AZA 15–20% has been reported to be as efficacious as HQ improving melasma skin [196], even if not all authors agree with these therapeutic effects. Moreover, this compound has been reported to produce clinical and histological resolution in facial lentigo maligna, and to induce good results in the treatment of rosacea, solar keratosis, and hyperpigmentation associated with burns and herpes labialis. Furthermore, due to its mild and transient side-effects (i.e., erythema and cutaneous irritation) AZA is generally well-tolerated and can be used for extended periods [197]. Flavonoids are plant polyphenols classified as benzo-γ-pyrane derivatives, due to the presence of phenolic and pyrane rings in their chemical structures. These compounds include more than 4000 members identified in leaves, bark, and flowers. Several natural medical treatments contain these polyphenols, due to their beneficial effects, including anticancer, antiviral, and anti-inflammatory protection against UV damage [188]. Some of the therapeutic effects are related to the antioxidant and ROS scavenger properties of flavonoids [198]. Moreover, their ability to chelate metals at the active site of metalloenzymes can explain melanin reduction induced by these compounds, even if their action at the distal part of the melanogenesis oxidative pathway can contribute to the hypopigmenting effect. The chemical structure of flavonoid groups can influence their mechanism of action: flavonoids with a α-keto group are similar to dopa and thus they show potent tyrosinase inhibition [188]; the presence of a free 3-hydroxy group improves

5.2 Depigmenting Agents

the flavonoid’s capability to chelate copper in tyrosinase’s active site [199]. Aloesin, a natural hydroxymethyl chromone compound isolated from aloe vera, was demonstrated to be a competitive inhibitor of tyrosinase [200] and showed a dosedependent depigmenting effect in melanocytes alone in vitro. Due to the hydrophilic nature of aloesin and low penetration rate across skin layers, combined treatment with this compound and arbutin has been evaluated to assess the synergistic effects on tyrosinase activity. The two compounds act in a synergistic manner and interfere with tyrosinase function by different mechanisms: aloesin exhibits noncompetitive inhibition, while arbutin inhibits competitively [67, 201]. In pigmented human skin equivalents, aloesin showed dose-dependent reductions in tyrosinase activity and melanin content, and demonstrated a good safety profile [202]. Considering that aloesin revealed no cytotoxicity, it has been suggested as a good alternative to HQ [142]. Ellagic acid is a naturally occurring polyphenol widely distributed in plants, and it has been reported to have anticarcinogenic, antifibrosis, and antioxidative properties. This compound shows high affinity to copper at the active site of tyrosinase and inhibits its activity by binding to the copper [203]. Furthermore, in brownish guinea pigs, as well as in human skin, topically applied ellagic acid induced a reversible inhibition of melanin synthesis only in UV-activated melanocytes [204]. Moreover, oral administration to humans of ellagic acid-rich pomegranate extract has a protective effect on slight sunburn caused by UV irradiation [205]. A recent study compared the effectiveness of gel formulations containing arbutin, synthetic ellagic acid, and plant extracts that contain ellagic acid on patients with melasma. The formulation prepared with plant extracts was suggested as an alternative to mono- or combined treatment of melasma, because it is nontoxic and has an effect of tyrosinase enzyme inhibition as demonstrated experimentally, as well as having antioxidant and UV-preventive effects [66]. Most of the hydroxystilbene compounds, like resveratrol (3,4,5-trihydroxystilbene), are derived from natural products found in herbal medicines and showed efficient skin-lightening properties, due to their high affinity for tyrosinase [206]. Apart from resveratrol and its derivatives, such as oxyresveratrol or gnetol, this group includes methoxylated or glucosylated derivatives (piceid-glucoside, rhapontigenin, and rhaponticin) with a structural skeleton comprised of two aromatic rings linked by an ethylene bridge [207, 208]. The number and position of hydroxyl substituents of hydroxystilbene compounds seem to play an important role on the inhibition of tyrosinase activity [206]. Kinetics studies revealed that oxyresveratrol and gnetol are more efficient tyrosinase inhibitors than resveratrol [207]. Also, the trans-olefin structure of the stilbene skeleton plays a relevant role in inhibiting tyrosinase, as suggested by the fact that trans-resveratrol is much more potent than the cis isomer [208]. In all cases, the inhibition of the enzyme is reversible and in vivo treatments should maintain high intracellular levels of the hydroxylated stilbene inside melanocytes. Some data indicated the ability of resveratrol to reduce MITF and tyrosinase promoter activation in B16 mouse melanoma can contribute to its effect on pigmentation [46, 54]. However, such evidence is controversial due to other data suggesting that resveratrol-treated human melanocytes displayed a

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steady-state tyrosinase RNA and consequently regulation of enzyme transcription did not induce depigmentation [122]. Additionally, further analysis of the resveratrol-treated normal human melanocytes showed ER-retained immature tyrosinase, suggesting disrupted trafficking of tyrosinase in the Golgi–ER– lysosome and elevated proteolytic degradation [122]. In a study with 285 different plant extracts from oriental herbs, the best one seemed to be the extract from Morus alba L., which contains 2-oxyresveratrol. The extract inhibited tyrosinase activity remarkably, and showed no toxicity and no skin irritation effect [209]. 5.2.2.2 TRP-2 Modulation TRP-2 (Dct) is another major enzyme correlating with the melanogenesis pathway, even if it role in controlling melanin biosynthesis is not as crucial as the function of tyrosinase. This enzyme catalyzes the transformation of dopachrome into DHICA, acting as a “dopachrome conversion” factor [210]. In the melanization pathway, the conversion of dopachrome is a turning point that determines if DHIeumelanin or DHICA-eumelanin will be produced. Spontaneous conversion of dopachrome to DHI is faster than TRP-2-induced generation of DHICA, thus the main pathway is generally the production of DHI-eumelanin [211]. However, when generated, DHICA-eumelanin is able to inhibit the production of DHI-eumelanin. Due to the fact that DHICA-eumelanin pigment has a yellow/light brown color, which is milder than the light brown/black of DHI-eumelanin, the stimulation of TRP-2 is consequently described as having lightening effects. An oxyresveratrol derivative showed depigmenting properties by activating TRP-2 [212]. 2,6-DimethoxyN-(4-methoxyphenyl)benzamide, which was synthesized using a combination of benzoic acid and aniline, has been reported to accelerate dopachrome transformation into DHICA in the presence of TRP-2 and to exhibit significant depigmentation ability on the UV-B-induced hyperpigmentation of brown guinea pig skin [213]. Compounds able to inhibit tyrosinase activity without interfering with TRP-2 functionality can also result in DHICA-eumelanin accumulation, leading to an improvement of skin hyperpigmentation. ATRA and pyrroloquinoline quinone, which functions as a ROS scavenger and protects cells against injury and DNA fragmentation, increased the generation of DHICA-eumelanin, even in the absence of a direct activation of TRP-2 activity, resulting in a skin-lightening effect [90, 214]. 5.2.2.3 Interference with Byproduct Production (Antioxidant and Reducing Agents) Skin exposure to sunlight generates ROS, which are able to stimulate epidermal melanogenesis either directly, acting as second messengers, or indirectly, through the induction of the production of cytokines by epidermal and dermal cells [215]. Compounds with redox properties can interfere with pigmentation process by neutralizing highly reactive o-quinone intermediates, and thus avoiding the oxidative polymerization during melanin synthesis, or by scavenging ROS. Ascorbic acid (AsA) interferes with the different steps of melanization, by reducing dopachinone and by blocking DHICA oxidation [140]. Moreover, AsA is highly unstable,

5.2 Depigmenting Agents

being quickly oxidized and decomposed in aqueous solution, and the degree of penetration into the skin is low, due to its prevalent hydrophilic nature. Different esters of AsA, such as magnesium l-ascorbate-3-phosphate (MAP), which is stable in water and in alkaline medium, have been used. MAP is absorbed percutaneously, reaches the epidermal layer, where can be hydrolyzed in AsA, and induces a lightening effect in both normal and hyperactive melanocytes [216]. Application of 10% MAP cream was shown to suppress melanin formation. A significant lightening effect was seen clinically in 19 of 34 patients with melasma and solar lentigo. Furthermore, MAP has been shown to have a protective effect against skin damage induced by UV-B irradiation, by reducing, regardless of the drug administration route, lipid peroxidation and inflammation reaction in hairless mice [216, 217]. In vitro studies showed that MAP was converted to AsA as it crossed the epidermis, but that sodium ascorbate (AS-Na) did not pass through the epidermis. Furthermore, MAP was also converted to AsA in serum. These results suggest that the protective effect of MAP on UV-B-induced cutaneous damage is due to the conversion of MAP to AsA [218]. A novel amphiphilic ascorbic derivative, disodium isostearyl 2-O-lascorbyl phosphate (VCP-IS-2Na), significantly suppressed the cellular tyrosinase activity of melanoma cells and melanocytes [219]. Treatment of human skin models with 1.0% VCP-IS-2Na decreased melanin synthesis, without inhibiting cell viability. A recent study compared both in vitro and in vivo the depigmenting effect of AsA or multivitamin [69]. In the melanoma cells stimulated by α-MSH multivitamin, which is composed by various vitamins, decreased melanin content more than AsA did. Multivitamin also revealed skin-lightening action as effective as AsA, clinically. The combination of hypopigmenting vitamins has an additive antimelanogenic effect with low cytotoxicity and downregulation of MITF, which gives enhanced efficacy. In vitro, vitamin E (α-tocopherol) derivatives induce the inhibition of melanogenesis in epidermal melanocytes and show a potent inhibitory effect on tyrosinase [54]. The ability of α-tocopherol to act as a scavenger of ROS and chain-breaking antioxidant, interfering with lipid peroxidation of the melanocyte membrane, and to increase the intracellular glutathione content could explain its depigmenting effect [140]. However, during the antioxidant reaction α-tocopherol undergoes auto-oxidative processes and produces quinone radicals, leading to a loss of its antioxidative effect. The activation of the antioxidant recycling mechanism in which AsA, through the transfer of one electron, is able to regenerate α-tocopherol from its radicalic form, results in the more effective and long-lasting antioxidant response observed for coadministration of these compounds. In vivo, topical application of α-tocopherol and AsA reduced the tanning response, through an inhibition of UV-induced melanogenesis and proliferation of melanocytes. α-Tocopherol ferulate is a related compound in which α-tocopherol is linked by an ester bond to ferulic acid, an antioxidant that stabilizes α-tocopherol [220]. This molecule has been reported to inhibit melanin synthesis in normal human melanocytes, without interfering with the tyrosinase pathway. Moreover, in an attempt to provide an efficient penetration into the skin, a formulation in which α-tocopherol ferulate is incorporated in liposomes has been synthesized [221]. The association between

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the protective effect of ferulic acid against α-tocopherol oxidation and the good percutaneous penetration of the liposomal preparation leads to the hypothesis that α-tocopherol ferulate added to liposomes may exert its lightening effects in active melanocytes providing an efficient intracellular distribution of α-tocopherol. A study investigating the biochemical effect of α-tocopherol ferulate in human melanoma cells suggests the whitening effect is due to tyrosinase inhibition at the post-transcriptional level, possibly by an unidentified secondary molecule [142]. Tocopherols conjugated with phenols at position 4, such as resorcinol, seem to have a strong effect [222]. These compounds could exert a double action – the inhibition of tyrosinase by the phenol moiety and the lipophilicity and ROS scavenger activity of the α-tocopherol moiety. 6-Hydroxy-3,4-dihydrocumarins, which are antioxidants with α-tocopherol-like chemical structures [223], have been reported to have antimelanogenic activity in cultured normal human melanocytes at noncytotoxic concentrations and without interfering with tyrosinase activity. A possible mechanism is the acceleration of the biosynthesis of glutathione (GSH) within melanocytes, leading to the inhibition of tyrosine transfer to premelanosomes [224]. In an effort to find new skin-lightening agents, natural plant extracts that contain compounds that have antioxidant and free-radical scavenger properties have been tested. Several studies identified substances, such as phytoncide [225], piceatannol [226], pycnogenol [227], and gallic acid [228], that exert antimelanogenic activity via their antioxidative property and/or their ability to suppress ROS while increasing the ratio between glutathione and oxidized glutathione (GSSG). LA (also known as thioctic acid), a disulfide derivative of octanoic acid, exhibits several biological effects, which include the quenching of ROS, metal chelation, interaction and regeneration of other antioxidants, redox regulation of protein thiol groups, and effects on gene expression and apoptosis [229, 230]. Lipoamide dehydrogenase, located exclusively in mitochondria, reduces free LA in dihydrolipoic acid, which is a potent antioxidant, being able to reduce ubiquinone and oxidized glutathione. LA has been reported to prevent UV-induced photo-oxidative damage, mainly through the downmodulation of NF-κB activation, and to inhibit tyrosinase activity, probably by chelating the copper ions at the active site [46, 231]. Recently, LA has been conjugated to poly(ethylene glycol) (PEG) of molecular weight 2000 (LA-PEG 2000) and its effects on melanogenesis has been evaluated [232] LA-PEG 2000 is a highly water-soluble molecule, which has lower cell cytotoxicity than LA. This compound significantly suppressed the biosynthesis of melanin, and reduced both the activity and expression of tyrosinase in B16F10 melanoma cells. 5.2.2.4 Interference with the Melanogenic Pathway Reaction of thiol compounds with quinone intermediates of melanin can switch pigment synthesis from eumelanin to pheomelanin [233]. Elevated amounts of thiols, including glutathione, have been associated, in fact, with a prevalence of pheomelanin, leading to a lack of pigmentation observable both in humans and in mice [234]. Cystamine has been reported to be effective in reducing epidermal pigmentation in combination with HQ [147]. Cystamine as well as cysteamine, its

5.2 Depigmenting Agents

reduced form, can shift the melanin biosynthetic pathway in favor of pheomelanin by nucleophilic addition to dopaquinone, which is much faster than conversion of dopaquinone to dopachrome [235]. N,N′-dilinoleylcystamine, a compound of cystamine and linoleic acid connected by an ester bond, has a pigment-lightening effect on HM3KO melanoma cells by reducing the level of eumelanin and increasing the level of phaeomelanin [236]. 5.2.2.5 Peroxidase Inhibitors In the 1970s, Okun, Edelstein, Or, Hamada, and Donnellan suggested that peroxidase, an iron-containing enzyme, alone or in conjunction with tyrosinase catalyzes the oxidation of l-tyrosine to melanins [237, 238]. Hydrogen peroxide (H2O2) is produced as by product of DHI and DHICA oxidation, and the involvement of peroxidase–H2O2 systems in later stages of melanogenesis, during the oxidation of indolequinone precursors of eumelanin and benzothiazinylalanine precursors of pheomelanin, has been suggested [239, 240]. Recent studies have identified new physiological functions of tyrosinase, namely catalase (conversion of H2O2 to ½O2 and H2O) and peroxygenase (H2O2-dependent oxygenation of substrates), leading to scavenging of tyrosyl radicals, which are toxic oxidants of melanocytes. The role of H2O2 in melanogenesis has not been clearly defined, with some reports indicating it functions to enhance pigment formation by regulating levels of tyrosinase [241], others suggesting the molecule serves as a potent inhibitor of tyrosinase [242]. Moreover, H2O2 is released by cytokines, such as TNF-α or TGF-β, which induce depigmentation [243–245], and it has been reported to produce a transient reduction of tyrosinase and other melanogenic proteins, through the downregulation of the MITF transcription factor [246]. The implication of H2O2 in the first steps of melanogenesis has been supported by a study showing that H2O2 was generated during the oxidation of dopa to dopaquinone and that tyrosinasemediated diphenolase activity was enhanced by the endogenously generated H2O2 [247]. These data afford a possible explanation of depigmentation and reduction of the polymerization rate of eumelanin induced by inhibition of peroxidase [248]. Methimazole, an antithyroid agent belonging to the thionamide group, exerts inhibitory action towards both mushroom tyrosinase and peroxidase, and in brown guinea pigs induces mild-to-moderate inhibition of melanization, with morphologic changes of melanocytes [249]. This molecule showed whitening action also in humans and it has been suggested as a safe skin-depigmenting compound for topical treatment of hyperpigmentary disorders [70, 71]. 5.2.3 Agents Acting After Melanin Synthesis 5.2.3.1 Inhibitors of Melanosome Transfer A new approach to achieve depigmentation is to study the possible application as depigmenting agents of molecules able to interfere with melanosome maturation and transfer. Reduction of skin pigmentation can be achieved, in fact, also by decreasing melanosomal transfer from melanocytes to surrounding keratinocytes

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and subsequent melanosome processing inside recipient cells [250]. Several compounds have been found to inhibit melanosome transfer by interfering with different steps of this process. 5.2.3.1.1 Inhibition of Melanocytic Dendrite Formation To effectively transfer melanosomes, melanocytes should have a good dendrite generation and extension towards surrounding keratinocytes. The reorganization of the melanocyte cytoskeletal elements is needed for the extension of melanocyte dendrites [251]. Agents able to activate Rho, a small GTPase, which is involved in cell morphology and dendrite formation, can interfere with melanosome transfer. In melanocyte and keratinocyte cocultures, methylophiopogonanone B (5,7-dihydroxy-6,8-dimethyl-3-(4-methoxybenzyl)chroman-4-one) and centaureidin (5,7,3′-trihydroxy-3,6,4′-trimethoxyflavone), a flavonoid glucoside, have been reported to activate Rho, inducing cytoskeleton disorganization, and to reduce melanosome transfer, by determining a reversible dendrite retraction [251–253]. These compounds did not influence melanin synthesis or the expression of melanogenic enzymes and can be considered as promising skin-lightening compounds, even if more studies are needed to verify their applicability in vivo. 5.2.3.1.2 Reduction of Melanosome Transfer Niacinamide, a biologically active form of niacin, is a precursor to the cofactors nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate, which participate in numerous enzymatic reactions [73, 201]. This compound has been proposed to have several actions in the skin, including anti-inflammation, prevention of photo-immunosuppression, and increased intercellular lipid synthesis [254]. Topical niacinamide is described to improve the appearance of photoaged facial skin (including texture, hyperpigmentation, redness, fine lines, and wrinkles) [255–257]. Moreover, this molecule was found to inhibit melanosome transfer to keratinocytes both in vitro and in humans. However, the associated mechanism of action remains to be elucidated [73]. 5.2.3.1.3 Inhibition of PAR-2 protease-activated receptor 2 (PAR-2) is G-protein-coupled receptor and is activated by serine proteases, including trypsin or mast cell tryptase [258, 259]. Due to its expression in keratinocytes, but not in melanocytes, PAR-2 is involved in the modulation of skin pigmentation through the interaction between melanocytes and keratinocytes [260–262]. The inhibition of serine protease results in an impaired activation of PAR-2 on the keratinocyte leading to the accumulation of melanosomes within the melanocyte, and blocking the melanosomal transfer and melanin dispersion inside keratinocytes, subsequently inducing skin-lightening [263–265]. In epidermal reconstructs, RWJ-50353, a serine protease inhibitor, has been reported to induce accumulation of melanosomes in melanocytes and a concomitant negative feedback mechanism that slows pigment production [259].

5.2 Depigmenting Agents

Moreover, Yucatan swine skin treated with RWJ-50353 for an 8-week period demonstrated a dose-dependent, reversible skin-lightening effect [259]. Soybean extracts have been explored as depigmenting agents. Kunitz-type trypsin inhibitor (soybean trypsin inhibitor (STI)) and the Bowman–Birk protease inhibitor (BBI), two protein proteinase inhibitors isolated from soybean seeds, have been reported to reduce pigmentation in dark-skinned Yucatan swine, probably by inhibiting PAR-2 activation, leading to a reduction in keratinocyte phagocytosis and melanin distribution [265]. Other components of soybean can also play a role in the mechanism of action of STI and BBI: free fatty acids and their acyl-coenzyme A esters can inhibit trypsin and participate in PAR-2 inhibition; isoflavones may reduce dopa oxidase activity of tyrosinase; and phospholipids can assist the epidermal delivery of STI and BBI. 5.2.3.1.4 Inhibition of Membrane Glycosylation To transfer melanosomes, recognition between melanocytes and keratinocytes is needed. Due to their influence in intracellular trafficking, endocytosis, and cell– cell recognition, lectins and neoglycoproteins have been investigated as compounds involved in this phenomenon. In an in vitro model, the role of glycosylated residues on melanocyte and keratinocyte membranes as part of receptor-mediated endocytosis facilitating melanosome transfer has been identified [250]. In the same paper, the authors suggest that lectins and neoglycoproteins play an inhibitory role in this process. In keratinocyte–melanocyte cocultures lectins were found to induce a reversible inhibition of melanosome transfer and this effect was enhanced by the presence of niacinamide [254]. 5.2.3.2 Acceleration of Epidermal Turnover Skin-lightening can also result from an increased rate of epidermal layer renewal and removal of pigmented upper layer keratinocytes. Thus, compounds able to activate skin desquamation can effectively act as depigmenting agents [140, 266]. 5.2.3.2.1 Retinoids Due to their capacity of inducing dispersion of keratinocyte pigment granules, interference with pigment transfer, acceleration of epidermal turnover, reduced cohesiveness of corneocytes, and desquamation [54, 267, 268], retinoids are considered hypopigmenting compounds. ATRA shows a depigmenting effect by inhibiting tyrosinase and TRP-1 transcription [90], and it has been reported to improve melasma, with some side-effects, such as erythema, peeling at the site of application, and postinflammatory pigmentation [54, 269–271]. This molecule is also used in combination in topical creams, such as a widely prescribed formulation proposed by Kligman and Willis containing 5% HQ, 0.1% tretinoin, and 0.1% dexamethasone [266]. However, ATRA requires long-term treatment (24 weeks) to achieve good clinical results; thus, other retinoids have been explored and the results of their application in pigmentary disorders have been reviewed [75]. Tarazotene, an

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acetylenic topical retinoid, was found to be effective towards irregular hyperpigmentation associated with photoaging [76], whereas isotretinoin did not show significant positive results in melasma patients [272]. Topical application of β-carotene in nanothalospheres, which are special vectors able to deliver intact β-carotene into the intracellular space of melanocytes, induced good skin-lightening action with minimal side-effects [78]. Adapalene is a naphthoic acid derivative with potent retinoid activity; it controls cell proliferation and differentiation, and has significant anti-inflammatory action. In a randomized clinical trial, the efficacy of adapalene 0.1% gel was found to be comparable to that of tretinoin 0.05% cream in the treatment of melasma and solar lentigines, but patients using adapalene showed fewer side-effects and greater acceptability [273]. The treatment of black guinea pig skin with the combination of retinoic acid with the monobenzylether of HQ produced a complete degree of depigmentation in the majority of treated sites and reduced the average number of melanocytes [274]. A combination of the retinoid retinaldehyde 0.1% and glycolic acid 6.4% showed a higher skin-depigmenting potential than retinoic acid 0.05% in the tail skin of C57BL/6 mice [275]. 5.2.3.2.2 Chemical Peels To treat pigmentary lesions such as solar lentigines, melasma, and postinflammatory hyperpigmentation, superficial and medium-depth peels with trichloroacetic acid (TCA), α-hydroxy acids (AHAs), such as lactic acid, glycolic acid, and salicylic acid, and tretinoin may be used mainly in fair-skinned individuals even if some clinical trials have proved their efficacy also in dark-skinned patients [276]. According to the concentrations used, AHAs can induce different effects: promotion of exfoliation by decreasing corneocyte cohesion and stimulating basal layer renewal, at low concentrations; promotion of epidermolysis and melanin dispersion in the basal layer, at high concentrations. Furthermore, AHAs showed a direct inhibition of tyrosinase, without influencing mRNA or protein expression [266]. A clinical trial on TCA peels reported 40% complete regression and 50% partial regression of lentigines [277]. Lactic acid supplemented with AsA determined a general skin-whitening effect in subjects with medium to dark skin [278]. Salicylic acid is effective in inducing skin desquamation in dark-skinned individuals [79], possibly acting also by a noncompetitive inhibition of tyrosinase [279]. Owing to the low risk of inflammatory hyperpigmentation, glycolic acid peels can be used safely in dark-skinned individuals. A 1% tretinoin peel has been suggested as a well-tolerated and effective chemical peel for melasma as well as 70% glycolic acid [280]. According to Kligman’s opinion, however, 1% tretinoin could act as a modulator of gene expression, leading to acceleration of epidermal cell turnover, rather than as a peeling agent [281]. 5.2.3.2.3 Linoleic Acid Apart from its effect on tyrosinase degradation, linoleic acid also influences skin pigmentation by stimulating epidermal turnover and increased desquamation of melanin pigment from the epidermis. Skin-lightening capabilities of unsaturated fatty acids, linoleic acid or α-linoleic acid, have been evaluated on UV-induced

5.3 Enhancers of Melanogenesis

hyperpigmentation of brown guinea pig skin [131]. Inhibition of melanogenesis in vivo has been proposed to be related to the peroxidation of the unsaturated bonds of these molecules and to the increased epidermal turnover. 5.2.3.2.4 Licorice Extracts The compounds extracted from the roots of licorice (Glycyrrhiza glabra L.) are widely used in traditional Chinese medicine for the treatment of a number of diseases [54, 171, 282]. Some of these molecules show depigmenting properties. Among hydrophilic extracts of licorice, liquiritin, a glucoside-derived flavonoid, was found to be effective for prolonged treatments of epidermic melasma [81]. The proposed mechanism of action is an acceleration of epidermis turnover and melanin dispersion induced by flavone rings contained in liquiritin and its homolog isoliquiritin. Glabridin is the main component of the hydrophobic fraction of licorice, and it has been reported to inhibit UV-B-induced pigmentation and tyrosinase activity [283], although no evaluation of its efficacy as a depigmenting agent has been carried out. Moreover, glabridin is able to reduce superoxide production and cyclooxygenase activity, suggesting that this compound can counteract skin inflammation as well as melanin biosynthesis induced by UV exposure. Licorice extracts interfere with different steps of the pigmentation process, by inhibiting tyrosinase activity in a dose-dependent manner and by removing epidermal melanin, and they have been tested for treating melasma patients with good results and very mild irritation [142].

5.3 Enhancers of Melanogenesis

Constitutive pigmentation is the result of genetic individual background that determines the phenotype of the different skin types (see Chapter 11). The facultative pigmentation is a mechanism that has evolved to protect human skin from high levels of terrestrial UV rays. Increased cutaneous eumelanin production results in skin pigmentation, providing a partial barrier to the penetration of UV and visible light. In particular, it forms a protective “nuclear cap” of melanin over the nuclei of basal keratinocytes. Eumelanin also scavenges UV-induced ROS that can damage DNA, protein, and lipids. Thus, when the melanin shield is compromised individuals are exposed to increased skin aging and cancer risk. While the photoprotective benefit of tanning is quite modest, there may be noncarcinogenic pharmacological opportunities to stimulate eumelanogenesis, avoiding UV-induced genotoxic damage. To define the optimal approach for enhancing eumelanin production the signaling pathways and intrinsic/extrinsic factors that influence melanocyte proliferation or metabolism should be taken in account. After UV exposure melanin synthesis is activated via the increase of MITF, which is in turn regulated by the transcription factor SOX-9. Several signaling molecules, such as bFGF, hepatocyte growth factor (HGF), SCF, ET-1, ACTH, and α-MSH, bind their respective receptors present on melanocytes, stimulating pigment

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production. Other pathways involving p53, peroxisome proliferator-activated receptor (PPAR) or ERK are also involved in the melanogenic response (see Chapter 11). 5.3.1 Activation Through Receptor Mechanisms 5.3.1.1 Melanotropic Peptides MC1R is one of several proteins involved in the normal biosynthesis of melanin. α-MSH binds MC1R to stimulate both eumelanogenesis, by upregulating tyrosinase activity, and melanocyte proliferation, through activation of adenylate cyclase. Although α-MSH stimulates natural skin protection, the process requires harmful UV radiation and it is highly susceptible to proteolytic degradation. The development of a synthetic analog was therefore suggested as a suitable strategy to obtain a tanning agent, which stimulated the protective effect of eumelanogenesis without requiring UV and with a long duration of action. [Nle4-d-Phe7]-α-MSH, also called NDP-MSH or Melanotan, is one of several potent analogs of α-MSH, which has been developed by replacing the methionine at position 4 with norleucine, and the phenylalanine is racemized to the d-isomer at position 7. NDP-MSH exhibits a 10- to 100-fold increased activity respect to the endogenous α-MSH molecule, increases melanogenesis and tyrosinase activity in human melanocytes, and more specifically induces significant increases in the eumelanin content of melanocytes while having a lesser and more varied effect on the levels of pheomelanin. Subcutaneous injection of NDP-MSH increases skin pigmentation and activates eumelanin expression, without any significant change in pheomelanin expression, reproducing, thus, the results of in vitro studies. In humans, increased skin pigmentation occurs despite minimization of sun exposure and concomitant use of sunscreens, although it was more evident in sun-exposed areas, than in covered skin sites [284]. However this study has been performed only in subjects with skin types III or IV, avoiding the opportunity to reach any conclusion about the potential increase of eumelanin in individuals with skin types I or II, which are characterized by high levels of pheomelanin and high susceptibility to UV-induced harmful effects. In a double-blind randomized placebo-controlled study involving 65 subjects with low or high minimal erythemal dose (MED) daily subcutaneous injection of NDP-MSH for 10 days in three monthly cycles preferentially increased cutaneous melanin density in subjects with the lowest baseline skin melanin levels, and also resulted in a 50% decrease in epidermal sunburn cells and a 59% reduction in thymine dimers formation in the low MED group [285]. Considering that in humans, as in other animals, MC1R is a key point in the regulation of pigmentation phenotype and that variations of gene sequence of this protein are associated with a poor tanning response, to investigate whether the presence of alleles with diminished function would affect the binding affinity and efficacy of NDP-MSH represents an important topic. Earlier studies investigating key receptor residues involved in α-MSH–MC1R binding found that while the mutation of

5.3 Enhancers of Melanogenesis

conserved amino acids reduced the binding affinity of α-MSH, that of NDP-MSH remained unchanged or only slightly reduced. More recently it was found that both homozygous or heterozygous cultures for several alleles, including the diminished function variants Arg142His, Arg151His, and Leu93Arg, demonstrated a significantly reduced response to normal α-MSH and superpotent NDP-MSH compared to wild-type receptors. Fitzgerald et al. [286] investigated for the first time the effect of NDP-MSH in human skin with a variant MC1R genotype. Their data demonstrated that NDP-MSH causes the greatest increase in skin melanin density in MC1R variant carriers (i.e., alleles associated with fair skin and red hair), suggesting that individuals with a probable increased risk of skin cancer can benefit from a drug suspected to only work in wild-type MC1R individuals. Other studies have confirmed that NDP-MSH treatment induces skin pigmentation both in the presence and absence of UV radiation, although UV-B radiation or sunlight appear to have a synergistic effect on the tanning response [287]. 5.3.1.2 Cytokines and Growth Factors Many studies have demonstrated that melanocyte growth and survival is regulated by keratinocyte-derived factors, such as bFGF, SCF, and ETs. In particular, SCF activates its transmembrane tyrosine kinase receptor, c-kit, promoting dimerization, autophosphorylation, and transphosphorylation of several substrates at specific tyrosine residues. After associating with the adapter protein, Grb2, an activated c-kit induces tyrosinase phosphorylation of phospholipase C-γ, which mediates the production of diacylglycerol and activates the PKC pathway. Both the SCF/c-kit and the ET-1/type B ET receptor (ETBR) pathways are crucial not only during neural crest formation and early melanocyte development, but also for melanocyte function postnatally [36]. Intradermal injection of SCF or c-kit neutralizing antibody into human xenografts was shown to regulate the number of Ki67-postive melanocytes. The synergistic effect of SCF/c-kit and ET-1/ETBR pathways in enhancing the level of DNA synthesis and activating the MAPK pathway was previously evaluated using an in vitro system, in which the combination of these two cytokines was added to human melanocyte cultures. Downregulation of SCF in vitiliginous keratinocytes was reported to be responsible for keratinocyte and melanocyte apoptosis. The treatment with either SCF or ET-1 has been shown to influence cell adhesion and melanocyte cell migration in culture, implying the plausible role of these two cytokines in regulating melanocyte function in vitiligo [36]. In addition, it has been postulated that these cytokines may influence the recruitment of melanocytes out of pigmented hair follicles in vitiligo lesions to induce repigmentation. The effect of both SCF and ET-1 in living intact human skin has been evaluated in a xenograft model using fresh Caucasian cadaveric skin on mice. Intradermal injections of the combination of these two paracrine melanogenic cytokines was shown to induce proliferation, melanogenesis, and dendritogenesis of melanocytes, suggesting that SCF administered locally in combination with ET-1 may have a potential therapeutic effect for the treatment of melanocytopenic disorders [288]. Geniposide, which is an

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herbal extract used in traditional Chinese medicine for the treatment of generalized vitiligo, was found to increase pigmentation via SCF/c-kit in normal human melanocytes [289]. Due to the fact that in vitiligo the presence of cytotoxic T cells targeting melanocyte antigens and an imbalance of the cytokine network were described, therapeutic approaches able to counteract immune response mediators have been investigated. In recent years, vitamin D analogs, particularly calcipotriol and tacalcitol, have been used as topical therapeutic agents in vitiligo. Vitamin D ligands are designed to target the local immune response in vitiligo, acting on specific T cell activation, mainly by inhibiting the transition of T cells from early to late G1 phase and by inhibiting the expression of several proinflammatory cytokines genes, such as those encoding TNF-α and interferon-γ. Vitamin D3 compounds are known to influence melanocyte maturation and differentiation, and also to upregulate melanogenesis through pathways activated by specific ligand receptors, such as ET receptor and c-kit [290]. Bone morphogenetic proteins (BMPs) are secreted signaling molecules that belong to the TGF-β superfamily. Apart from their role as stimulators of bone formation, BMPs affect other tissues, inducing various effects like proliferation that oppose differentiation and apoptosis. To date, more than 20 different BMP proteins have been identified, all sharing structural homology and interaction with specific BMP receptors. BMP effects depend on many factors including tissue concentration, BMP type, presence of antagonists, embryonic stage of the target tissue, and the type of receptors expressed by the target cells. BMP-2 has been reported to stimulate melanogenesis, without having a direct effect upon melanocyte differentiation, as demonstrated by the BMP-2- induced increase of tyrosinase mRNA, without affecting MITF, TRP-1, or TRP-2 mRNAs, indicating that BMP-2 signaling selectively targets the transcription of the tyrosinase gene [291]. Noggin is a secreted BMP antagonist that competes for binding to BMP receptors. Mice overexpressing noggin in the hair follicle epithelium have been reported to display a darker coat color than wild-type mice, demonstrating that BMP signaling influence murine melanogenesis in vivo [292]. Yaar et al. reported that normal neonatal human melanocytes and keratinocytes express the BMP receptors and produce BMP-4, suggesting that BMP antagonists can enhance skin pigmentation [293]. The extract of the human placenta, named “melagenin,” has been reported to stimulate in vitro the proliferation of melanocytes a nd the synthesis of melanin. A pilot trial evaluated the effectiveness of the topical application of the melagenin in pediatric vitiligo patients. Sixty-two out of the 366 treated subjects, ranging from 4 to 15 years old and followed up for 1 year, were characterized by depigmentation involving more than 70% of the body. The authors reported that the treatment with melagenin proved to be effective in 83% of vitiligo patients [294]. Currently, the placenta extract is used in Cuba, even if the protocol lacks external validation. The possible mechanism of action of the extract has been reported to be associated with the presence of melanocyte growth factors in placenta, including ET.

5.3 Enhancers of Melanogenesis

5.3.2 Non-Receptor-Mediated Activation 5.3.2.1 Forskolin and cAMP The skin-tanning effectiveness of α-MSH analogs is affected by the technical difficulties of administering peptides to humans, as well as by the requirement of a functional MC1R to activate eumelanogenesis. In direct analogy to humans, MC1R mutations in mice lead to yellow coat color and inability to tan. The major intracellular messenger used by MC1R to induce pigmentation is cAMP. Forskolin directly activates adenylate cyclase thereby increasing cAMP levels and stimulates MITF activity, reproducing the intracellular signaling pathway activated by MC1R. Due to its ability to bypass MC1R, forskolin is hypothesized to enhance pigmentation regardless of MC1R genotype. In mice expressing nonfuctional MC1R daily topical application of forskolin has been reported to stimulate the tanning response, affording a pigmentation able to counteract UV-induced damage and carcinogenesis [295]. Forskolin was also found to increase the removal of cyclobutane pyrimidine dimers and 6,4-photoproducts in human keratinocytes treated with UV-B [296]. These data confirmed the ability of forskolin to reproduce the natural response of epidermis to UV rays, even in subjects with loss-of-function MC1R variants, increasing skin pigmentation and ability to repair DNA photodamage, without needing UV exposure. 5.3.2.2 Oligonucleotides and p53 Activation The small DNA fragment thymidine dinucleotide pTpT induces photoprotective responses in cultured cells and intact skin via melanogenesis, enhanced DNA repair, and induction of TNF-α. The induction and activation of the p53 tumor suppressor and transcription factor is the key signaling pathway. Larger oligonucleotides induce the pigmentation response even more efficiently than pTpT. The ability of these oligonucleotides to stimulate pigmentation is highly dependent on the presence of a 5′-phosphate group on the molecule [297]. 5.3.2.3 Piperin The water extract (0.1 mg/ml) of black pepper (Piper nigrum), and its alkaloid piperine, has been demonstrated to have in vitro proliferative activity, probably mediated by activation of PKC, on melanocytes. Some mouse observations have confirmed the in vitro data [298]. 5.3.2.4 Lipids (Sphingolipids and Prostaglandins) The occasional report of prostaglandin E2-induced hyperpigmentation after eyedrop usage in ophthalmology suggested its possible utilization for the topical treatment of vitiligo lesions. In vitro, prostaglandin E2 has been reported to induce the expression of mRNA for bFGF and the oxidative stress-mediated glutathione depletion may decrease the prostaglandin E2 level in vitiligo epidermis. A recent trial indicates that the topical application of a gel containing prostaglandin E2 gives rise to repigmentation, mainly on the face, after 6 months of therapy. Segmental

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and focal vitiligo have been reported to be characterized by the highest percentage of repigmentation [299]. 5.3.2.5 Phospholipase A2 Phospholipase A2, a component of bee venom, has been considered for vitiligo repigmentation [300]. When tested in vitro, bee venom was able to promote melanocyte proliferation, dendriticity, and migration; the tyrosinase activity was also positively affected. The molecular mechanism has been supposed to be mediated by the activation of intracellular signal transduction mechanisms, in particular of PKA, ERK, and PI3K/Akt. 5.3.2.6 PPAR Activators PPARs play an important role in cellular responses. It was reported that three subtypes of PPAR are expressed in human melanocytes. The PPAR-γ agonist, ciglitazone, stimulates the melanin content of cells and cultured skin, in a tyrosinase/MITF-dependent pathway. Migration of melanocytes was increased after ciglitazone treatment [301]. 5.3.2.7 Psoralens and Photosensitizing Agents PUVA (psoralens plus UV-A) induces pigmentation that may develop without clinically evident erythema. PUVA is used for the treatment of vitiligo and for the preventive treatment of photodermatoses. In normal skin, PUVA pigmentation peaks about 7 days after exposure and may last from several weeks to months. A few PUVA exposures result in a much deeper tan than that produced by multiple exposures to solar irradiation. Psoralens belong to the furocoumarin group of compounds that are derived from the fusion of the furan ring with coumarin. These are found in a large number of plants and there are also several synthetic psoralen compounds. 8-Methoxypsoralen (8-MOP) is of plant origin, but it is also available as a synthetic drug. It is used primarily for oral and topical PUVA. The synthetic compound 4,5′,8-trimethylpsoralen (TMP, trioxsalen) is less phototoxic after oral administration, but more phototoxic when bath-water delivered. 5-MOP (bergapten) is only used in some European countries, and is less erythemogenic and not associated with gastrointestinal intolerance. Without UV radiation, psoralens intercalate in DNA double strands. After the absorption of UV-A photons the formation of 3,4- or 4′,5′-cyclobutane monoadducts with pyrimidine bases of native DNA occurs. Some psoralens, such as 8-MOP, 5-MOP, and TMP, can absorb a second photon, and this reaction leads to the formation of a bifunctional adduct that inhibits DNA replication and causes cell cycle arrest. Psoralens also stimulate melanogenesis. This involves the photoconjugation of psoralens to DNA in melanocytes followed by mitosis and subsequent proliferation of melanocytes, increased formation and melanization of melanosomes, increased transfer of melanosomes to keratinocytes, and activation and increased synthesis of tyrosinase via stimulation of cAMP activity [302].

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Nishiyama, T. (2002) Antimelanogenic activity of hydrocumarins in cultured normal human melanocytes by stimulating intracellular glutathione synthesis. Arch. Dermatol. Res., 294, 349–354. Fujimori, H., Hisama, M., Shibayama, H., Kawase, A., and Iwaki, M. (2010) Inhibitory effects of phytoncide solution on melanin biosynthesis. Biosci. Biotechnol. Biochem., 74, 918–922. Yokozawa, T. and Kim, Y.J. (2007) Piceatannol inhibits melanogenesis by its antioxidative actions. Biol. Pharm. Bull., 30, 2007–2011. Kim, Y.J., Kang, K.S., and Yokozawa, T. (2008) The anti-melanogenic effect of pycnogenol by its anti-oxidative actions. Food Chem. Toxicol., 46, 2466–2471. Kim, Y.J. (2007) Antimelanogenic and antioxidant properties of gallic acid. Biol. Pharm. Bull., 30, 1052–1055. Roy, S. and Packer, L. (1998) Redox regulation of cell functions by alphalipoate: biochemical and molecular aspects. Biofactors, 8, 17–21. Podda, M., Zollner, T.M., GrundmannKollmann, M., Thiele, J.J., Packer, L., and Kaufmann, R. (2001) Activity of alpha-lipoic acid in the protection against oxidative stress in skin. Curr. Probl. Dermatol., 29, 43–51. Saliou, C., Kitazawa, M., McLaughlin, L., Yang, J.P., Lodge, J.K., Tetsuka, T., Iwasaki, K., Cillard, J., Okamoto, T., and Packer, L. (1999) Antioxidants modulate acute solar ultraviolet radiation-induced NF-kappa-B activation in a human keratinocyte cell line. Free Radic. Biol. Med., 26, 174–183. Kim, J.H., Sim, G.S., Bae, J.T., Oh, J.Y., Lee, G.S., Lee, D.H., Lee, B.C., and Pyo, H.B. (2008) Synthesis and antimelanogenic effects of lipoic acidpolyethylene glycol ester. J. Pharm. Pharmacol., 60, 863–870. Ito, S. and Prota, G. (1977) A facile one-step synthesis of cysteinyldopas using mushroom tyrosinase. Experentia, 33, 118–119. Benedetto, J.P., Ortonne, J.P., Voulot, C., Khatchadourian, C., Prota, G., and Thivolet, J. (1982) Role of thiol

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melanosome transfer and dendrite retraction. J. Dermatol. Sci., 42, 68–70. Lin, J., Chiang, H., Lin, Y., and Wen, K. (2008) Natural products with skinwhitening effects. J. Food Drug Anal., 16, 1–10. Greatens, A., Hakozaki, T., Koshoffer, A., Epstein, H., Schwemberger, S., Babcock, G., Bissett, D., Takiwaki, H., Arase, S., Wickett, R., and Boissy, R. (2005) Effective inhibition of melanosome transfer to keratinocytes by lectins and niacinamide is reversible. Exp. Dermatol., 14, 498–508. Bissett, D., Oblong, J., and Berge, C. (2005) Niacinamide: a B vitamin that improves aging facial skin appearance. Dermatol. Surg., 31, 860–865. Bissett, D., Miyamoto, K., Sun, P., Li, J., and Berge, C. (2004) Topical niacinamide reduces yellowing, wrinkling, red blotchiness, and hyperpigmented spots in aging facial skin. Int. J. Cosmet. Sci., 26, 231–238. Bissett, D., Oblong, J., Saud, A., Berge, C., Trejo, A., and Biedermann, K. (2003) Topical niacinamide provides skin aging appearance benefits while enhancing barrier function. J. Clin. Dermatol., 32S, 9–18. Van Den Bossche, K., Naeyaert, J., and Lambert, J. (2006) The quest for the mechanism of melanin transfer. Traffic, 7, 769–778. Seiberg, M., Paine, C., Sharlow, E., Andrade-Gordon, P., Costanzo, M., Eisinger, M., and Shapiro, S. (2000) The protease-activated receptor 2 regulates pigmentation via keratinocyte– melanocyte interactions. Exp. Cell Res., 254, 25–32. Lin, C., Chen, N., Scarpa, R., Guan, F., Babiarz-Magee, L., Liebel, F., Li, W., Kizoulis, M., Shapiro, S., and Seiberg, M. (2008) LIGR, a protease-activated receptor-2-derived peptide, enhances skin pigmentation without inducing inflammatory processes. Pigment Cell Melanoma Res., 21, 172–183. Derian, C., Eckardt, A., and AndradeGordon, P. (1997) Differential regulation of human keratinocyte growth and differentiation by a novel family of

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References In vivo evaluation of piperine and synthetic analogues as potential treatments for vitiligo using a sparsely pigmented mouse model. Br. J. Dermatol., 158, 941–950. 299 Kapoor, R., Phiske, M.M., and Jerajani, H.R. (2009) Evaluation of safety and efficacy of topical prostaglandin E2 in treatment of vitiligo. Br. J. Dermatol., 160, 861–863. 300 Jeon, S., Kim, N.H., Koo, B.S., Lee, H.J., and Lee, A.Y. (2007) Bee venom stimulates human melanocyte proliferation, melanogenesis, dendricity

and migration. Exp. Mol. Med., 39, 603–613. 301 Lee, J.S., Choi, Y.M., and Kang, H.Y. (2007) PPAR-gamma agonist, ciglitazone, increases pigmentation and migration of human melanocytes. Exp. Dermatol., 16, 118–123. 302 Burchill, S.A., Marks, J.M., and Thody, A.J. (1990) Tyrosinase synthesis in different skin types and the effects of alpha-melanocyte-stimulating hormone and cyclic AMP. J. Invest. Dermatol., 95, 558–561.

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6 Structure of Melanins Shosuke Ito, Kazumasa Wakamatsu, Marco d’Ischia, Alessandra Napolitano, and Alessandro Pezzella

6.1 Introduction

Melanins (Greek μελαηoς = black) is a descriptive term relating to a variety of black, brown, or even yellowish and reddish natural biopolymers of diverse nature and chemical composition that arise biogenetically from the oxidation of phenolic metabolites [1, 2]. The term melanins refers to two main groups of intracellular nitrogenous pigments, the black/dark brown eumelanins (εν = good) and the lighter, yellowish/brown, sulfur-containing pheomelanins (φαεoς = dusky), which arise from a bifurcation of a common biosynthetic pathway involving the tyrosinasecatalyzed oxidation of tyrosine. All too often, however, the term melanins is used synonymously with eumelanins or, more in general, to denote any black insoluble pigment of phenolic origin. In this broader usage, melanins include the pigments found in higher plants, fungi, and bacteria. However, in this chapter we focus exclusively on those pigments derived biogenetically from tyrosine and produced intracellularly within specialized cells. Working on melanins has usually been regarded as an intriguing, although sometimes frustrating, experience. This is due to the extreme heterogeneity of their molecular systems and several adverse properties, including for eumelanins an almost complete insolubility in all solvents, an amorphous particulate character, and the lack of well-defined spectral features. Additional complexities stem from the biological matrix (melanosome) in which the pigments are usually found. Thus, it may not be surprising that melanins’ fundamental structure (if indeed the term “structure” can be applied to such heterogeneous materials) is still uncertain despite extensive studies. Apart from extensive physicochemical investigations, traditional approaches to melanin structure have been based on biosynthetic studies modeling the process of melanogenesis in vitro coupled with chemical degradation leading to fragments of variable diagnostic significance. These approaches form the core of this chapter, which is aimed at illustrating current views about melanin structural properties as derived from the biosynthetic and degradative approach. In addition, the main analytical Melanins and Melanosomes: Biosynthesis, Biogenesis, Physiological, and Pathological Functions, First Edition. Edited by Jan Borovanský and Patrick A. Riley. © 2011 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2011 by Wiley-VCH Verlag GmbH & Co. KGaA.

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6 Structure of Melanins

methods applicable for melanin determination in pigmented tissues are surveyed.

6.2 Classification and General Properties of Melanins

Several reviews of melanin structure and chemistry are available to which the interested reader is referred for a more in-depth description of these pigments [2–4]. Although eumelanins and pheomelanins have the primary biogenetic precursor tyrosine in common, they differ significantly in their chemical composition and physical properties. Typical eumelanins include the pigments that can be extracted from human and mammalian black/brown hair and irides, as well as those found in the inner ear and melanomas. Eumelanin pigments are also found in various vertebrates, like birds, reptiles, amphibians, and fish. The most remarkable example of eumelanin from lower animals is represented by the ink of cephalopods, including Sepia officinalis, Octopus vulgaris, and Loligo vulgaris. Studies of natural eumelanins have been carried out mainly on pigments isolated from Sepia ink, black human hair, and melanoma specimens. Isolation procedures are basically aimed at removing the protein matrix of the tissue while limiting oxidative damage to the pigment. A typical method for isolating eumelanin from black human hair involves sequential digestion with protease, proteinase K, and papaine in the presence of dithiothreitol, leading to a pigment with a 6–14% (w/w) protein content [5, 6]. Eumelanins display a peculiar set of physicochemical properties that have been considered an operational definition of eumelanins [7]. In the condensed phase or finely dispersed in an aqueous medium most eumelanins exhibit a featureless broadband monotonic absorption accounting for their black appearance. The origin of the broad spectrum of eumelanin is attributed to different contributions, including absorption and scattering. Electrical conductivity and photoconductivity are other peculiar properties that distinguish eumelanins from other bio-organic materials. Despite intensive investigations, the origin of these properties and the mechanism by which a charge is generated and transported are still poorly understood. Eumelanins are capable of reducing and oxidizing other molecules – a property that stems from the redox behavior of its monomer units – and display a persistent electron paramagnetic resonance (EPR) signal indicating free radical centers. The ability to bind various metal ions is still another characteristic feature of eumelanins (e.g., [8]). Whereas eumelanins are relatively widespread in nature, pheomelanins are found only in mammalian skin, hair, and eyes, and in bird feathers [9, 10]. In mice and other mammals, pheomelanin in the typical agouti-banding pattern provides camouflage. Natural pheomelanins are difficult to isolate from their biological matrix and are usually obtained with a high protein content [6]. The most charac-

6.3 Biosynthetic Studies

teristic feature of pheomelanins is their sulfur content, which accounts for up to around 10% in pigment samples from red feathers and is due to the incorporation of cysteine in the biosynthetic pathway [4, 11]. Most of the studies of pheomelanins have been done on partially purified samples obtained from hen feathers (gallopheomelanins), red human hair, and mammalian fur. Purification protocols should avoid harsh treatments causing degradative changes to the pheomelanin and should preferably involve repeated enzymatic digestions [6, 12]. Unlike eumelanins, which are completely insoluble in most solvents, pheomelanins exhibit a limited solubility in aqueous alkali. Natural and synthetic pheomelanins display remarkably similar absorption spectra featuring a flat maximum around 305 nm, and inflections at around 260 and 360 nm, with limited absorption above 450– 500 nm [13]. Apparently different molecular constituents in pheomelanin are responsible for the chromophoric features. The photoreactive chromophores of pheomelanins are of low molecular weight and exhibit similar photophysics in the aggregated state. They may include benzothiazine structural motifs [14], but there appears to be also a significant participation of benzothiazoles and other structural units of the trichochrome type [13, 15]. Pheomelanins exhibit an EPR signal with distinct immobilized nitroxide-like features attributed to a partial localization of the unpaired electron on the nitrogen atom of the o-semiquinone imine form of 1,4-benzothiazine subunits [16]. Like eumelanins, pheomelanins also bind metal cations, with a Fe(III) content that is 4 times higher in the red pheomelanosomes as compared with the black eumelanosomes [6].

6.3 Biosynthetic Studies 6.3.1 Early Stages of Melanogenesis

Eumelanin and pheomelanin are both derived from the common precursor dopaquinone formed by oxidation of the common amino acid l-tyrosine by tyrosinase (Scheme 6.1; see also Chapters 3 and 4). Dopaquinone is the first intermediate during the initial stage of melanogenesis [17]. As an o-quinone, dopaquinone is highly reactive and in the absence of sulfydryl compounds it undergoes the intramolecular addition of the amino group to produce cyclodopa (also called leucodopachrome). The chemistry of o-quinones is described in detail in Chapter 3, and Ito and Wakamatsu [18] also summarize pivotal roles of dopaquinone in controlling melanogenesis. A redox exchange between cyclodopa and dopaquinone then gives rise to dopachrome, an orange/red intermediate, and 3,4-dihydroxyphenylalanine (dopa). This latter reaction is considered the source of dopa formed during melanogenesis (Scheme 6.1). Dopachrome then gradually rearranges to generate mostly 5,6-dihydroxyindole (DHI) and to a minor extent DHI-2-carboxylic acid (DHICA) [19]. Under spontaneous reaction at neutral pH,

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6 Structure of Melanins

COOH NH2

HO

tyrosine Tyrosinase

HO

O2 Tyrosinase

DOPA

COOH Cys NH2

O

COOH NH2

HO

O2

O

COOH

HO

NH2

HO S

dopaquinone

H2N COOH cysteinyldopa O O

HO

N H cyclodopa

N H DHI [O]

HO

EUMELANIN

N

OH NH2

S

HO

HO

COOH

(HOOC)

tyrp-2

–CO2

HO

HO

COOH N H dopachrome

N H DHICA [O]

COOH

COOH

[O] PHEOMELANIN

OH N

(COOH)H N 2 COOH S

S HOOC

NH2

O

N H

OH

TRICHOCHROMES Schematic outline of the melanogenetic pathway showing the switch from eumelanin to pheomelanin by intervention of cysteine.

Scheme 6.1

DHI and DHICA are formed in a ratio of 70 : 1 [20]. These two DHIs are then further oxidized and polymerized to produce eumelanin. In addition to tyrosinase, two tyrosinase-related proteins have been shown to modulate/accelerate eumelanogenesis (see Chapter 4 for more details). Dopachrome tautomerase (Dct; also known as tyrosinase-related protein 2 (TRP-2/ Tyrp2)) catalyzes the tautomerization of dopachrome to DHICA [21, 22]. Certain divalent metal ions, especially Cu2+, can also promote tautomerization of dopachrome to DHICA, but Dct seems to be more effective in catalyzing the tautomerization [20, 23]. The oxidative polymerization of DHI is catalyzed by mammalian tyrosinase [24]. A recent pulse radiolysis study, however, indicates that DHI can be effectively oxidized by dopaquinone through redox exchange [25]. On the other hand, oxidative polymerization of DHICA appears to be catalyzed by tyrosinase in humans [26] or by Tyrp1 in mice [27, 28]. Thus, the activities of these tyrosinase-related

6.3 Biosynthetic Studies

proteins greatly affect the quantity and quality (the ratio of DHI to DHICA and the degree of polymerization) of eumelanins produced. Regarding the production of pheomelanin, the intervention of sulfydryl compounds such as cysteine gives rise exclusively to thiol adducts of dopa, cysteinyldopas. Tyrosinase oxidation of dopa in the presence of excess cysteine produces a high yield of 5-S-cysteinyldopa (74%) and 2-S-cysteinyldopa (14%) together with minor amounts of 6-S-cysteinyldopa (1%) and a di-adduct, 2,5-S,S′-dicysteinyldopa (5%) [29]. Further oxidation of the cysteine adducts leads to the formation of pheomelanin via benzothiazine intermediates [14]. 6.3.2 Late Stages of Eumelanogenesis

Based on elemental analysis, chemical degradation, and isotope labeling experiments [19, 30, 31], it has been concluded that the proportion of DHI- and DHICAderived units in eumelanins varies significantly depending on the origin of the pigment (natural or synthetic); in particular, intact natural eumelanins contain a higher proportion of DHICA (up to 75%) than enzymatically prepared synthetic eumelanins (less than 10%). Investigation of the oxidative polymerization of DHIs has been used as a strategy for inquiring into the mode of formation of eumelanins as well as for developing a consistent structural model [32]. The role of DHI and DHICA as the basic eumelanin building blocks is indicated by their rapid conversion to black insoluble pigments following exposure to oxidizing enzymes, UV radiation, chemical oxidants, or even on standing at neutral physiologic pH. Oxidation of DHI leads to a collection of dimers and trimers that reveals the prevalent mode of coupling of the indole monomer through 2,4′- and 2,7′bondings (Scheme 6.2). A unified synthetic strategy for the preparation of 2,7′-, 2,2′-, and 2,3′-dimers has recently been developed [33]. The mechanism of indole dimerization is uncertain, but a catechol–quinone interaction seems the most likely route based on product structures, theoretical calculations [34], and quinone trapping experiments with sulfur nucleophiles [35]. Pulse radiolysis experiments suggest that one-electron oxidation of DHI in aqueous solution at pH 7.4 leads to a semiquinone, which decays by a second-order process [25, 36]. Oxidative coupling of dimers proceeds likewise via semiquinone and quinone intermediates [37], and leads to tetramers in which other types of interring bonds are formed (e.g., 2,3′-, 4,4′-, and 7,7′-bonds), depending on the dimer substrate [38, 39]. These model studies point to a mechanism of polymerization of DHI in which monomer units couple through the 2- and 4- or 7-positions. However, as oligomer coupling steps become significant, other modes of bonding may become important, with consequent bending of the growing oligomer chain. As a result, a high degree of structural diversity is expectedly generated during eumelanin biosynthesis, accounting for the marked heterogeneity of the pigment. It may be relevant in this regard to mention the formation of a cyclic pentamer by co-oxidation of a dimer and a trimer from 5,6-dihydroxy-1-methylindole [40],

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6 Structure of Melanins

HO HO [O] HO

HO

OH

HO

NH +

N H HN

HO

[O] HO metal cations N HO H

HO

DHI N HHO

HO

OH

HO OH N H HN

HO HO HO

HO HO

H HN N

HO

+ HO HO NH

N H HO

OH HO N NH H

OH

OH OH

+

OH

NH H N

N H HN

HO

OH

OH

OH H N

HN

N H

OH

OH

HO

HO

HO

+ HO

N H

HO OH

N H NH

HO OH HO

NH

OHHO HO

HO

Scheme 6.2

HO

N H

OH

[O]

[O] HO

OH

N H HO

OH

NH

N H

OH N H

OH

OH

Main oligomers formed by oxidation of DHI.

raising the possibility that cyclic structural motifs are involved in the process of eumelanin formation [41]. Oxidation of DHICA is a process of considerable biological importance considering not only the prevalent involvement of this indole in the biosynthesis of natural eumelanins [19] and ocular pigments (e.g., the brown tapetum lucidum of the sea catfish (Arius felis L.) [42] ), but also its putative role as an endogenous antioxidant [43, 44]. Since the carboxylic group at C2 limits the range of options available for indole coupling, a lower number of positional isomers are expected to populate the various oligomer stages. Main oligomers formed by oxidative coupling of DHICA include the 4,4′-biindolyl, the 4,7′-biindolyl, and other minor dimers, a series of trimers, and tetramers [32, 45, 46] (Scheme 6.3). Notably, oligomers of DHICA exhibit hindered rotation around inter-ring bonds and the negatively charged carboxylate functions contribute to maintain significant twist angles between units. It is therefore suggested that the effective conjugation length in oligomeric/polymeric eumelanin components from DHICA may be controlled by such hindered rotation around interring bonds preventing planarization of the continuous array of indole units [47]. This may provide an explanation for the difference in the absorption properties of polymers from the two key eumelanin monomers.

6.3 Biosynthetic Studies

HO CO2H HO

N H [O]

HO

HO

CO2H

N H CO2H

HO HO

HO

N H

HO

N H

N H

HO

HO OH HN

OH

HO

HO2C HO HO

N H

CO2H

H N

CO2H

HO N H

HO HO

N H

CO2H

CO2H

CO2H

HO CO2H

CO2H N H [O]

CO2H

H N

HO N H N H

HO N H

CO2H

N H H N

CO2H

CO2H

HO HO

CO2H

CO2H

HO

HO

HO

HO

CO2H

HO

HO

HO

H N

HO HO

[O]

HO

HO HO

HO

N H H N

HO HO

HO DHICA

HO

HO

CO2H

HO

CO2H N H

CO2H

Scheme 6.3 Main oligomers formed by oxidation of DHICA.

Despite the extensive investigations described above, the detailed structural features of DHI polymers are largely unknown. To what extent DHI and DHICA oligomers model eumelanin fundamental building blocks is a most critical issue, and attention is also called to the possibility that DHI and DHICA can form copolymeric structures on oxidation [48, 49]. Early structural models envisaged a high-molecular-weight heteropolymeric system formed via random bonding between different DHI units. Later, an alternative view of eumelanin structure was proposed, based on scanning tunneling microscopy and X-ray studies [50–52], which suggested that eumelanins were not large extended heteropolymers, but rather mixtures of π-stacked oligomeric “protomolecules” consisting of three to four planar layers of no more than five or six indolic units and 15–20 Å in size. It should be emphasized, however, that this

173

174

6 Structure of Melanins

model would apply only to planar oligomer sheets made up of DHI units and not to the twisted DHICA oligomers, and is based on hypothetical structures devoid of experimental support. In this regard, small-angle X-ray scattering (SAXS) data [53] and atomic force microscopy (AFM) investigations of sepia eumelanin [54] would be compatible with eumelanins being mixtures of oligomers. Matrixassisted laser desorption/ionization (MALDI) mass spectroscopic studies have also identified several oligomeric and partially degraded species both in sepia melanin and synthetic eumelanins [55]. The structural and electronic properties of putative monomer and oligomer components of eumelanin architecture have also been investigated by theoretical methods, showing that different oxidation states and tautomeric forms can have dramatically different highest occupied molecular orbital to lowest unoccupied molecular orbital (HOMO–LUMO) gaps, and that an increase in oligomer size and π-stacking induce a significant compression of the gap, with bathochromic shift of the absorption maximum [56, 57]. The concepts emerging from theoretical studies seem to lend support to oligomer-based structures in which a broad range of variations is possible in terms of molecular size and redox states. However, further work is needed before the structural problem is definitively settled. As mentioned earlier, a fundamental issue concerning eumelanins is why they are black. The experimental difficulty lies in the virtual insolubility of these pigments, and the associated scattering effects and hindering characterization of the intrinsic absorption properties of the heterogeneous species produced by oxidative polymerization of monomer precursors. Important insights into this issue have derived from studies of soluble eumelanin-like materials aimed at disentangling intrinsic absorption properties of basic components by circumventing scattering effects. This goal was recently achieved, for example, by polymerizing DHI in the presence of polyvinylalcohol [58] and by oxidation of a glycated derivative of DHI (5,6-dihydroxy-3-indolyl-1-thio-β-d-galactopyranoside) [59]. The resulting dark brown soluble polymer exhibited a distinct UV band around 315 nm and a broad visible absorption, resembling that of natural eumelanins. By a systematic series of reduction and dilution experiments it was established that eumelanin black color is not only intrinsically defined by the overlap of π-electron conjugated chromophores within the individual polymer components, as commonly believed, but also by oxidation state- and aggregation-dependent interchromophoric interactions causing perturbations of the heterogeneous ensemble of π-electron systems and overall spectral broadening. These findings may overall set the basis for an improved interpretative model of eumelanin structure and properties that is based on a dynamic molecular disorder with extensive intermolecular and intramolecular redox and charge transfer interactions. 6.3.3 Late Stages of Pheomelanogenesis

Oxidation of dopa in the presence of cysteine is undoubtedly the process that mimics at best pheomelanin synthesis within pigment cells. However, oxidation

6.3 Biosynthetic Studies

of 5-S-cysteinyldopa apparently leads to less heterogeneous pigments that may be more useful for structural investigations. The reaction involves the formation of an o-quinone, which rapidly cyclizes to give an unstable o-quinonimine intermediate. This may undergo redox exchange with cysteinyldopa leading to a dihydrobenzothiazine intermediate, or may be converted to 2H-1,4-benzothiazine species by rearrangement with (85%) or without (15%) decarboxylation [11, 60]. These may be oxidized to pheomelanins by way of different processes, including phenolic dimerizations [61] and imine–enamine reactions with coupling at the 2-position to give trichochrome-like dimers [62, 63]. Notably, small amounts of trichochromelike pigments are slowly formed by oxidation of cysteinyldopas, a process speeded up by acidification of the reaction mixtures [64], but whether this behavior has some bearing on the identification of trichochromes in pigmented tissues remains to be assessed. Oxygenated systems related to 3-oxo-2H-1,4-benzothiazine units and benzothiazole moieties are also likely to be involved in pigment formation (Scheme 6.4). A recent biosynthetic study starting from dopa and cysteine [14] has shown that dihydro-1,4-benzothiazine species are the last major, isolable intermediates in pheomelanogenesis, and are then oxidized and polymerized to give pheomelanin with a gradual conversion of the benzothiazine units to benzothiazole units in polymer backbone. Beyond this level, knowledge of the molecular mechanism of pheomelanogenesis is virtually lacking. Nevertheless, it should be stressed that dopaquinone undergoes redox exchange reactions with cysteinyldopas and dihydro-1,4-benzothiazines to generate the corresponding o-quinone or o-quinonimine, thus spontaneously promoting pheomelanogenesis [14]. It stresses again the pivotal roles of dopaquinone in promoting the whole process of (mixed) melanogenesis. 6.3.4 Concept of Mixed Melanogenesis

Kinetic studies using the pulse radiolysis technique have provided a number of useful insights into the dynamic process of mixed melanogenesis. Thus, melanogenesis proceeds in three distinct stages (see [18] for more detail). The initial stage is the production of cysteinyldopas, as long as cysteine concentration is above 0.13 μM. The second stage is the oxidation of cysteinyldopas by dopaquinone to produce pheomelanins, which continues to proceed as long as cysteinyldopas are present at concentrations above 9 μM. The last stage is the production of eumelanins, which begins only after most cysteinyldopas (and cysteine) are depleted. Therefore, the ratio of eumelanin to pheomelanin is determined by tyrosinase activity, and the availability of tyrosine and cysteine in melanosomes [65]. This time course of melanogenesis further suggests the “casing model” for mixed melanogenesis. Thus, in the process of mixed melanogenesis, pheomelanic pigment is produced first making a core, followed by the deposit of eumelanic pigment to make an outer layer [18]. This model was originally proposed by Agrup et al. [66], based on biochemical findings, and then Bush et al. [67] provided direct, biophysical evidence that supports this model, by showing that neuromelanin

175

176

6 Structure of Melanins

HOOC

OH

HOOC NH2

NH2

OH

5-S-cysteinyldopa HOOC

O O S

S HOOC

NH2

NH2

O

HOOC OH NH2 HOOC

NH2

H N

N

(COOH)

S

COOH

S OH NH2 HOOC OH

NH2 HOOC

H N S

N

(COOH) OH

S

N

(COOH) H N 2

S

O HOOC OH

3-oxo-3,4-dihydrobenzothiazine N NH2 (λ max 299 nm) S HOOC benzothiazoles

NH2 (HOOC)

COOH

S N

OH 2,2'-bi(2H-1,4-benzothiazine) (COOH) (λ max 346 nm )

PHEOMELANIN Oxidation routes of 5-S-cysteinyldopa to pheomelanin through benzothiazine intermediates. Scheme 6.4

granules have a eumelanic surface with a pheomelanic core. The casing model was further supported on mixed melanins produced in iridal melanocytes [4, 68]. Although those melanins have varying ratios of eumelanin to pheomelanin, they have the surface oxidation potentials characteristic for eumelanin.

6.4 Degradative Studies 6.4.1 Eumelanins

Owing to their high chemical heterogeneity, elemental analyses of eumelanins lack reproducibility. The data vary significantly with the nature of the sample, isola-

6.4 Degradative Studies

HO HO

HO

HO COOH

N H

N H

HO

2-substituted DHI units

DHICA units

[O]

2-unsubstituted DHI units [O]

[O] HOOC

HOOC HOOC

N H

HO

N H

COOH

pyrrole-2,3,5-tricarboxylic acid (PTCA)

HOOC

N H

pyrrole-2,3-dicarboxylic acid (PDCA)

Scheme 6.5 Origin of pyrrole carboxylic acids by oxidative degradation of eumelanins.

tion procedure, storage method, and other experimental parameters, including hydration [1]. However, the C/N ratio provides an indication of the proportion of DHICA versus DHI units [19], with the caveat that it may provide limited information in the case of extensive degradation of the benzene moieties of the indole units and is applicable mainly for synthetic eumelanins. The carboxyl group attached to the indole or pyrrole ring is labile and may easily be split off as CO2 by heating a melanin suspension in 6 M HCl [2, 19]. This methodology has been applied successfully to estimate the carboxyl content in natural and synthetic eumelanins [5, 19, 31, 69]. Chemical analysis of eumelanins is based on oxidative degradation with potassium permanganate [70, 71] or alkaline hydrogen peroxide [72, 73] followed by high-performance liquid chromatography (HPLC) detection of pyrrole-2,3,5tricarboxylic acid (PTCA) and pyrrole-2,3-dicarboxylic acid (PDCA) (Scheme 6.5). In addition to these major degradation products, related pyrrole carboxylic acids such as 2-carboxymethylpyrrole-3,5-dicarboxylic acid are produced [74]. Although usually formed in rather small amounts (below 6–7%), pyrrole carboxylic acids are diagnostic markers for eumelanins and provide perhaps the most cogent evidence for the presence of indolic building blocks in the eumelanin backbone. DHICA-rich polymers give higher yields of PTCA relative to DHI polymers, which in turn furnish more PDCA [71]. DHI units with 2- and 3-positions unsubstituted in eumelanins afford PDCA [75]. Thus, the ratio of PTCA to PDCA may reflect the ratio of DHICA-derived units to DHI-derived units in eumelanins. Upon oxidation, pyrrole carboxylic acids arise not only from indolic building blocks but also from degraded pyrrolic building blocks. The (dihydroxy)indolic

177

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6 Structure of Melanins

OH

OH N

NH2 HOOC

(COOH) HI

S

NH2

NH2 HOOC

pheomelanin structural unit

aminohydroxyphenylalanine (AHP)

oxidative degradation OH NH2 HOOC

N COOH S

benzothiazolecarboxylic acid (BTCA) Scheme 6.6

HOOC

N

HOOC

S

COOH

+

thiazole-2,4,5-tricarboxylic acid (TTCA)

Main degradation products of pheomelanins.

units can provide the redox property of eumelanins while the pyrrolic units cannot. Thus, the ratio of indolic units to pyrrolic units is important in determining the degree of the redox property of eumelanins. In this connection, sepia melanin is highly degraded to a pyrrolic structure, thus implying little redox property compared with the high redox property of synthetic DHI or DHICA melanin [31]. Pyrrole carboxylic acids PTCA and PDCA are present also in the free, extractable form in melanosomes obtained from Sepia, human black hair, and bovine eyes [76]. The free form consists of as much as 20% of the total amounts obtained after hydrogen peroxide oxidation and demonstrates extensive oxidative breakdown of indolic units in natural eumelanins. This degradation is likely to proceed through peroxidative cleavage as occurs in the chemical degradation. In this connection, it should be noted that UV-A irradiation induces fluorescence of melanin accompanied by pigment bleaching [77]. As PTCA and PDCA are fluorescent compounds this provides evidence of degradation by photo-oxidation. 6.4.2 Pheomelanins

Pheomelanin treated with HI leads to the formation of aminohydroxyphenylalanine (AHP) isomers by reductive hydrolysis of benzothiazine moieties (Scheme 6.6). An advantage of hydriodic acid (HI) hydrolysis is the rather high yield (around 20%) of AHP isomers [70, 71, 78]. AHP isomers, 4-amino-3-hydroxyphenylalanine (4-AHP) and 3-amino-4-hydroxyphenylalanine (3-AHP), derive from 5-S-cysteinyldopa- and 2-S-cysteinyldopa-derived benzothiazine units in pheomelanins, respectively [78]. It should be noted that AHP isomers are not derived from benzothiazole

6.4 Degradative Studies

units in the pheomelanin structure [14]. 3-AHP may also arise from 3-nitrotyrosine produced in vivo by nitric oxide-dependent nitration of tyrosine. An alternative approach involves oxidative degradation with alkaline hydrogen peroxide leading to a number of diagnostic structural fragments including thiazole-2,4,5-tricarboxylic acid (TTCA) and thiazole-4,5-dicarboxylic acid (TDCA [79]), while alkaline permanganate produces a series of pyridine-containing polycarboxylic acids, including pyridine-2,3,4,6-tetracarboxylic acid and 2-(2′-(4′,5′-dicarboxythiazolyl))-3,4,6-pyridinetricarboxylic acid [80]. More recent work on human red hair pheomelanin has led to the identification of two additional degradation products: the isomeric benzothiazolecarboxylic acids BTCA (from 5-S-cysteinyldopa) and BTCA-2 (from 2-S-cysteinyldopa) [81]. Analysis of different segments of red hair locks (4–20 cm) with the improved dual-marker methodology revealed a remarkable degradation of the 5-Scysteinyldopa-derived constituents on passing from the root region near the scalp (or very short hair) to the tip, whereas 2-S-cysteinyldopa-related units were little modified throughout the entire hair length. Lethal yellow and recessive yellow mouse hair and red chicken feathers gave values of BTCA and BTCA-2 similar to those of red hair regions near the root, and better reflective of the typical 5-Scysteinyldopa/2-S-cysteinyldopa formation ratio. Prolonged exposure of red hair locks to intense sunlight caused a modest decrease in the yields of BTCA, but not BTCA-2. It appears that red hair pheomelanin consists of stable structural components made up of 2-S-cysteinyldopa-derived units, associated to a degradable 5-S-cysteinyldopa component. Degradation occurs during hair growth probably as a result of oxidative processes related in part, but not exclusively, to sun exposure. The structural origin of the isomeric benzothiazole carboxylic acids BTCA and BTCA-2 appears to be benzothiazine units despite of the benzothiazole structure of BTCA and BTCA-2 [80]. Overall, these data have led to the hypothesis that pheomelanins consist of benzothiazine, benzothiazole, and isoquinoline units linked at various levels and through different bondings. However, while benzothiazine and benzothiazole units have been identified in all natural and synthetic pheomelanins, and have been substantiated by chemical isolation and degradation studies, and a straightforward synthetic access is available [82], the actual presence of isoquinoline units still needs to be demonstrated. The origin of benzothiazole fragments is clearly via contraction of benzothiazine ring precursors, but whether this occurs exclusively during biosynthesis, as has been shown recently [14], or is also in part associated with postsynthetic oxidative degradation processes is still uncertain and requires further elaboration. Specifically, it should be clarified what type of chemical modification takes place during the selective (photo)degradation of 5-S-cysteinyldopa-derived benzothiazine units in pheomelanin, whether it is the ring contraction of benzothiazine to benzothiazole structure or a more complex modification of the whole pheomelanin skeleton. A hint to the possible occurrence of the ring contraction to benzothiazole units is the facile conversion of 1,4-dihydrobenzothiazine-3carboxylic acid to 2-methylbenzothiazole upon UV-A radiation [83]. The ring contraction is also markedly promoted by the presence of Fe3+ ions [84].

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6 Structure of Melanins

6.5 Analysis of Eumelanins and Pheomelanins

Regulation of melanogenesis has been a subject of extensive studies. In most such studies, quantification of melanins in pigmented tissues such as hair, skin, melanocytes, and melanomas is essential. However, previous methods for characterizing melanins in pigmented tissues required the isolation of melanin pigments. Moreover, none of those methods was suitable for distinguishing eumelanins and pheomelanins [2]. Only one exception is the EPR spectrum in which eumelanins give a single peak while pheomelanins afford two characteristic peaks [16]. Under these circumstances, Ito and Fujita [70] introduced a rapid and sensitive method for quantitatively analyzing eumelanin and pheomelanin in tissue samples by taking advantage of degradative studies. The method is based on the formation of PTCA (Scheme 6.5) by acidic permanganate oxidation of eumelanins and of AHP isomers by HI hydrolysis of pheomelanins. These specific degradation products are determined by HPLC with UV or electrochemical detection, respectively. An advantage of PTCA by permanganate oxidation as a marker for eumelanins is that it is highly specific for eumelanins [70, 85]. However, the permanganate oxidation method is rather tedious and not suitable as routine assays, and is now being replaced by alkaline hydrogen peroxide oxidation that is much easier to perform (because of the exclusion of ether extraction and the direct injection into HPLC), and gives higher yields of PTCA and PDCA [72, 73, 86]. The ratio of PTCA to PDCA can be used to assess the ratio of DHICA-derived units to DHI-derived units in eumelanins (unpublished results by Ito and Wakamatsu). The ratios are much higher in mouse hairs than in human hairs, indicating a large difference in the ratio of DHICA to DHI between the two species. Previously, the relative content of DHICA in eumelanins was assessed indirectly from the ratio of PTCA to total melanin [85, 87]. Regarding differential roles of DHICA and DHI as eumelanin building blocks, it should be stressed that DHICA melanin has a high ability to scavenge hydroxyl radicals while DHI melanin does not [44]. However, whether this advantageous property of DHICA unit in eumelanins plays a physiological role in vivo remains to be clarified.

6.6 Conclusions

Melanin pigments, eumelanins and pheomelanins, can be described today as collections of molecular species with different chemical structures and properties, thus encompassing diverse degrees of chemical heterogeneity. This molecular heterogeneity can be ascribed to a number of factors, including: the range of different monomer units (several precursor compounds can take part in the polymerization process); the different modes of inter-unit coupling; differences in molecular weight (arising from the variable degree of polymerization); the redox

References

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Pavel, S., C.A. Ramsden, Riley, P.A., and Smit, N.P. (1997) Evidence of the indirect formation of the catecholic intermediate substrate responsible for the autoactivation kinetics of tyrosinase. J. Biol. Chem., 272, 26226–26235. Ito, S. and Wakamatsu, K. (2008) Chemistry of mixed melanogenesis – pivotal roles of dopaquinone. Photochem. Photobiol., 84, 582–592. Ito, S. (1986) Reexamination of the structure of eumelanin. Biochim. Biophys. Acta, 883, 155–161. Palumbo, A., d’Ischia, M., Misuraca, G., and Prota, G. (1987) Effect of metal ions on the rearrangement of dopachrome. Biochim. Biophys. Acta, 925, 203–209. Pawelek, J., Körner, A., Bergstrom, A., and Bologna, J. (1980) New regulators of melanin biosynthesis and the autodestruction of melanoma cells. Nature, 286, 617–619. Tsukamoto, K., Jackson, I.J., Urabe, K., Montague, P.M., and Hearing, V.J. (1992) A second tyrosinase-related protein, TRP-2, is a melanogenic enzyme termed DOPAchrome tautomerase. EMBO J., 11, 519–526. Palumbo, A., Solano, F., Misuraca, G., Aroca, P., García-Borròn, J.C., Lozano, J.A., and Prota, G. (1991) Comparative action of dopachrome tautomerase and metal ions on the rearrangement of dopachrome. Biochim. Biophys. Acta, 1115, 1–5. Tripathi, R.K., Hearing, V.J., Urabe, K., Aroca, P., and Spritz, R.A. (1992) Mutational mapping of the catalytic activities of human tyrosinase. J. Biol. Chem., 267, 23707–23712. Edge, R., d’Ischia, M., Land, E.J., Napolitano, A., Navaratnam, S., Panzella, L., Pezzella, A., Ramsden, C.A., and Riley, P.A. (2006) Dopaquinone redox exchange with dihydroxyindole and dihydroxyindole carboxylic acid. Pigment Cell Res., 19, 443–450. Olivares, C., Jiménez-Cervantes, C., Lozano, J.A., Solano, F., and GarcíaBorrón, J.C. (2001) The 5,6-dihydroxyindole-2-carboxylic acid

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(DHICA) oxidase activity of human tyrosinase. Biochem. J., 354, 131–139. Jiménez-Cervantes, C., Solano, F., Kobayashi, T., Urabe, K., Hearing, V.J., Lozano, J.A., and García-Borrón, J.C. (1994) A new enzymatic function in the melanogenic pathway. The 5,6-dihydroxyindole-2-carboxylic acid oxidase activity of tyrosinase-related protein-1. J. Biol. Chem., 269, 17993–18000. Kobayashi, T., Urabe, K., Winder, A.J., Jiménez-Cervantes, C., Imokawa, G., Brewington, T., Solano, F., GarcíaBorrón, J.C., and Hearing, V.J. (1994) Tyrosinase related protein 1 (TRP1) functions as a DHICA oxidase in melanin biosynthesis. EMBO J., 13, 5818–5825. Ito, S. and Prota, G. (1977) A facile one-step synthesis of cysteinyldopas using mushroom tyrosinase. Experientia, 33, 1118–1119. Tsukamoto, K., Palumbo, A., d’Ischia, M., Hearing, V.J., and Prota, G. (1992) 5,6-Dihydroxyindole-2-carboxylic acid is incorporated in mammalian melanin. Biochem. J., 286, 491–495. Pezzella, A., d’Ischia, M., Napolitano, A., Palumbo, A., and Prota, G. (1997) An integrated approach to the structure of Sepia melanin. Evidence for a high proportion of degraded 5,6-dihydroxyindole-2-carboxylic acid units in the pigment backbone. Tetrahedron, 53, 8281–8286. d’Ischia, M., Napolitano, A., Pezzella, A., Land, E.J., Ramsden, C.A., and Riley, P.A. (2005) 5,6-Dihydroxyindoles and indole-5,6-diones. Adv. Heterocycl. Chem., 89, 1–63. Capelli, L., Manini, P., Pezzella, A., Napolitano, A., and d’Ischia, M. (2009) Efficient synthesis of 5,6-dihydroxyindole dimers, key eumelanin building blocks, by a unified o-ethynylaniline-based strategy for the construction of 2-linked biindolyl scaffolds. J. Org. Chem., 74, 7191–7194. Okuda, H., Wakamatsu, K., Ito, S., and Sota, T. (2008) Possible oxidative polymerization mechanism of 5,6-dihydroxyindole from ab initio calculations. J. Phys. Chem. A, 112, 11213–11222.

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Misuraca, G., Palumbo, A., and Prota, G. (1997) Diffusible melanin-related metabolites are potent inhibitors of lipid peroxidation. Biochim. Biophys. Acta, 1346, 61–68. Jiang, S., Liu, X.M., Dai, X., Zhou, Q., Lei, T.C., Beermann, F., Wakamatsu, K., and Xu, S.Z. (2010) Regulation of DHICA-mediated antioxidation by dopachrome tautomerase: implication for skin photoprotection against UVA radiation. Free Radic. Biol. Med., 48, 1144–1151. Pezzella, A., Vogna, D., and Prota, G. (2002) Atropoisomeric melanin intermediates by oxidation of the melanogenic precursor 5,6-dihydroxyindole-2-carboxylic acid under biomimetic conditions. Tetrahedron, 58, 3681–3687. Pezzella, A., Vogna, D., and Prota, G. (2003) Synthesis of optically active tetrameric melanin intermediates by oxidation of the melanogenic precursor 5,6-dihydroxyindole-2-carboxylic acid under biomimetic conditions. Tetrahedron Asymmetry, 14, 1133–1140. Pezzella, A., Panzella, L., Crescenzi, O., Napolitano, A., Navaratnam, S., Edge, R., Land, E.J., Barone, V., and d’Ischia, M. (2009) Lack of visible chromophore development in the pulse radiolysis oxidation of 5,6-dihydroxyindole-2carboxylic acid oligomers: DFT investigation and implications for eumelanin absorption properties. J. Org. Chem., 74, 3727–3734. Napolitano, A., Crescenzi, O., and Prota, G. (1993) Copolymerization of 5,6-dihydroxyindole and 5,6-dihydroxyindole-2-carboxylic acid in melanogenesis: isolation of a crosscoupling product. Tetrahedron Lett., 34, 885–888. Okuda, H., Wakamatsu, K., Ito, S., and Sota, T. (2010) Regioselectivity on the cooxidation of 5,6-dihydroxyindole and its 2-carboxy derivative from the quantum chemical calculations. Chem. Phys. Lett., 490, 226–229. Zajac, G.W., Gallas, J.M., Cheng, J., Eisner, M., Moss, S.C., and AlvaradoSwaisgood, A.E. (1994) The fundamental

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Nemanich, R.J., and Simon, J.D. (2009) Human iridal stroma melanosomes of varying pheomelanin content possess a common eumelanic outer surface. J. Phys. Chem. B, 113, 11346–11351. Palumbo, A., d’Ischia, M., Misuraca, G., Prota, G., and Schultz, T. (1988) Structural modifications in biosynthetic melanins induced by metal ions. Biochim. Biophys. Acta, 964, 193–199. Ito, S. and Fujita, K. (1985) Microanalysis of eumelanin and pheomelanin in hair and melanomas by chemical degradation and liquid chromatography. Anal. Biochem., 144, 527–536. Wakamatsu, K. and Ito, S. (2002) Advanced chemical methods in melanin determination. Pigment Cell Res., 15, 174–183. Ito, S. and Wakamatsu, K. (1998) Chemical degradation of melanins: application to identification of dopaminemelanin. Pigment Cell Res., 11, 120–126. Napolitano, A., Vincensi, M.R., Di Donato, P., Monfrecola, G., and Prota, G. (2000) Microanalysis of melanins in mammalian hair by alkaline hydrogen peroxide degradation: identification of a new structural marker of pheomelanins. J. Invest. Dermatol., 114, 1141–1147. Napolitano, A., Pezzella, A., d’Ischia, M., and Prota, G. (1996) New pyrrole acids by oxidative degradation of eumelanins with hydrogen peroxide. Further hints to the mechanism of pigment breakdown. Tetrahedron, 52, 8775–8780. Napolitano, A., Pezzella, A., Vincensi, M.R., and Prota, G. (1995) Oxidative degradation of melanins to pyrrole acids: a model study. Tetrahedron, 51, 5913–5920. Ward, W.C., Lamb, E.C., Cooden, D., Chen, X., Burinsky, D.J., and Simon, J.D. (2008) Quantification of naturally occurring pyrrole acids in melanosomes. Photochem. Photobiol., 84, 700–705. Borovansky, J. and Elleder, M. (2003) Melanosome degradation: fact and fiction. Pigment Cell Res., 16, 280–286. Wakamatsu, K., Ito, S., and Rees, J.L. (2002) The usefulness of 4-amino-3hydroxyphenylalanine as a specific marker of pheomelanin. Pigment Cell Res., 3, 225–232.

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M., and Prota, G. (1996) A new benzothiazole derivative by degradation of pheomelanins with alkaline hydrogen peroxide. Tetrahedron Lett., 37, 6799–6802. Fattorusso, E., Minale, L., Cimino, G., Stefano, S.D., and Nicolaous, R.A. (1969) Struttura e biogenesi delle feomelanine. Nota VI. Sulla struttura della gallofeomelanina-1. Gazz. Chim. Ital., 99, 29–45. Greco, G., Wakamatsu, K., Panzella, L., Ito, S., Napolitano, A., and d’Ischia, M. (2009) Isomeric cysteinyldopas provide a (photo)degradable bulk component and a robust structural element in red human hair pheomelanin. Pigment Cell Melanoma Res., 22, 319–327. Greco, G., Panzella, L., Napolitano, A., and d’Ischia, M. (2009) Biologically inspired one-pot access routes to 4-hydroxybenzothiazole amino acids, red hair-specific markers of UV susceptibility and skin cancer risk. Tetrahedron Lett., 50, 3095–3097. Costantini, C., Testa, G., Crescenzi, O., and d’Ischia, M. (1994) Photochemical ring contraction of dihydro-1,4benzothiazines. Tetrahedron Lett., 35, 3365–3366. Di Donato, P., Napolitano, A., and Prota, G. (2002) Metal ions as potential regulatory factors in the biosynthesis of red hair pigments: a new benzothiazole intermediate in the iron or copper assisted oxidation of 5-S-cysteinyldopa. Biochim. Biophys. Acta, 1571, 157–166. Ozeki, H., Ito, S., Wakamatsu, K., and Hirobe, T. (1995) Chemical characterization of hair melanins in various coat-color mutants of mice. J. Invest. Dermatol., 105, 361–366. Wakamatsu, K., Fujikawa, K., Zucca, F., Zecca, L., and Ito, S. (2003) The structure of neuromelanin as studied by chemical degradative methods. J. Neurochem., 86, 1015–1023. Lamoreux, M.L., Wakamatsu, K., and Ito, S. (2001) Interaction of major coat color gene functions in mice as studied by chemical analysis of eumelanin and pheomelanin. Pigment Cell Res., 14, 23–31.

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7 Properties and Functions of Ocular Melanins and Melanosomes Małgorzata Rózᠨanowska 7.1 Introduction

The color of human eyes is largely dependent on the amount, type, and distribution of melanin present in the iris [1, 2]. However, the iris is not the only part of the uveal tract that contains melanin (Figure 7.1). Melanin is in great abundance also in the remaining parts of the highly vascularized uvea – the choroid and the ciliary body. In the choroid, melanin is present in melanocytes, whereas in the iris and ciliary body melanin is present in two cell types: melanocytes in the stroma and in epithelial cells. Another melanin-containing epithelium in the eye is the retinal pigment epithelium (RPE), an essential monolayer of cells that separate the neurosensory retina from the choroidal blood supply. This chapter is focused on our current understanding of the properties and functions of melanin in the mammalian eye, particularly the human eye.

7.2 Biogenesis of Ocular Melanosomes and Melanogenesis

Ocular melanins, like most other natural melanins, are a mixture of brown/black eumelanin and red/brown pheomelanin. Melanin is synthesized in intracellular organelles, the melanosomes, which are present in uveal melanocytes or in specialized pigment epithelial cells of the uvea and retina. In the RPE the biogenesis of melanosomes and melanogenesis was followed and divided in several stages [4–6]. The first stage involves budding off from the smooth endoplasmic reticulum (ER) of ovoid, membrane-bound organelles, called premelanosomes. Formation of premelanosomes occurs in the RPE early in fetal development, then ceases within a few weeks. Premelanosomes develop into stage I melanosomes, where the protein matrix is regimented, filaments stretch from one pole to the other, and the granule becomes more osmiophilic. Melanogenesis in the eye can occur only in the presence of functional tyrosinase [7], although a number of other proteins are likely to be involved such as tyrosinase-related protein (TRP)-1 and TRP-2 [8, Melanins and Melanosomes: Biosynthesis, Biogenesis, Physiological, and Pathological Functions, First Edition. Edited by Jan Borovanský and Patrick A. Riley. © 2011 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2011 by Wiley-VCH Verlag GmbH & Co. KGaA.

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Figure 7.1 Diagram of a cross-section of the human eye depicting melanin-containing structures: RPE and the uveal tract composed of the choroid, ciliary body and iris (left). A photograph of the human fundus

showing the characteristic areas: macula and fovea with the highest density of photoreceptors responsible for acute vision (right). (Modified from [3].)

9], p locus protein [10], and lysosomal membrane surface proteins [11]. Tyrosinase encapsulated in coated vesicles is delivered to premelanosomes and stage I melanosomes. Melanogenesis in the eye starts very early during fetal development. Pigmentation in the RPE cells is present already in the fourth week of gestation. Polymerization of melanin within the RPE melanosomes continues until approximately 2 years of age in humans after which pigment turnover is believed to be quite low [5, 6]. Once full melanization has occurred, melanosome reaches the final stage IV with no tyrosinase activity. Melanosomes in the melanocytes of the iris stroma in Caucasian eyes start developing pigmentation only after birth [4]. This can be observed easily in Caucasian babies born with blue irides, which may change color to brown within a few months after birth. Current understanding of pathways leading to biosynthesis of eumelanin and pheomelanin has been recently reviewed by Simon, Ito and colleagues [12, 13]. In short, both types of melanin derive from enzymatic oxidation of l-tyrosine catalyzed by tyrosinase to the common precursor dopaquinone (o-quinone of 3,4-dihydroxyphenylalanine (dopa)). In the absence of sulfhydryl compounds, dopaquinone undergoes intramolecular cyclization to form cyclodopa, which is then rapidly oxidized by a redox reaction with dopaquinone to give dopachrome (and dopa). Dopachrome then gradually rearranges to form 5,6-dihydroxyindole (DHI) and to a lesser extent DHI-2-carboxylic acid (DHICA). The DHI/DHICA ratio is determined by a dopachrome tautomerase (Dct; also known as TRP-2). Oxidation and subsequent polymerization of the dihydroxyindoles leads to the production of eumelanin. In the presence of cysteine, dopaquinone gives rise to cysteinyldopa isomers. Cysteinyldopas subsequently react with dopaquinone to

7.2 Biogenesis of Ocular Melanosomes and Melanogenesis

form cysteinyldopaquinones, eventually leading to the production of pheomelanin. The total amount of melanin produced is proportional to tyrosinase activity. It has been proposed that synthesis of natural melanins, which are a mixture of eumelanin and pheomelanin, proceeds in three distinct stages [12, 13]. In the first stage, when the concentration of cysteine is above 0.13 μM, cysteinyldopa isomers are produced. When the concentration of cysteine is decreased but is still greater than 9 μM, cysteinyldopas are oxidized to produce pheomelanin. Only after most cysteinyldopas and cysteine are depleted is eumelanin produced in the third stage of melanogenesis. The ratio of eumelanin to pheomelanin is determined by tyrosinase activity, and the availability of tyrosine and cysteine in melanosomes. This theory of three-stage mixed melanogenesis is supported by studies on melanogenesis in cultures of human uveal and epidermal melanocytes [12, 13]. In these cells pheomelanin is produced at constant levels regardless of the degree of pigmentation, whereas eumelanin production is proportional to pigmentation. Another factor that affects melanin synthesis is melanosomal pH [12, 13]. Tyrosinase activity peaks at about pH 7.3 and is almost completely inactive under acidic conditions. It has been shown that melanosomes isolated from Caucasian skin exhibit acidic pH, while pH of melanosomes isolated from black skin is more neutral, and that difference corresponds to tyrosinase activities in these melanosomes and melanin production [14, 15]. By employing drugs to neutralize pH in melanosomes, the tyrosinase activity can be strongly upregulated and melanin content increased [14–16]. Considering that the cyclization of dopaquinone leading to eumelanin production proceeds slower at lower pH, while the cysteinyldopa– quinone cyclization leading to pheomelanin is faster, it has been suggested that neutral melanosomes will favor synthesis of eumelanin, while pheomelanin is more likely to be produced in acidic melanosomes [12]. Adult human RPE contains only mature melanosomes [6]. Consistently, adult bovine RPE cells exhibit very little (about 1/20 th of that in the choroid) activity of tyrosinase [17, 18]. In contrast, the adult bovine uvea exhibits considerable tyrosinase activity, particularly in the ciliary body where it is 2 times higher than in the choroid or iris [18]. Tyrosinase activity has been also detected in the ciliary body of the adult human eye [19]. The melanin content of the human choroid remains approximately constant with age, whereas melanin content in the human RPE decreases significantly in aging eyes [20–23], supporting the view that melanin production is either absent in the adult human RPE or occurs only at a very slow rate. Interestingly, it has been demonstrated in the cultured RPE cell line ARPE-19 transfected with tyrosinase that melanogenesis can also occur in the absence of premelanosomes [24]. In the transfected cells, tyrosinase was associated with multivesicular and multilamellar organelles in which melanogenesis took place despite the fact that the organelles did not contain detectable amounts of proteins typical of premelanosomes, such as structural protein PMEL17 or TRP-1. These findings indicate that the classical pathway of melanogenesis, which occurs in four stages in melanosomes, is not essential for melanin synthesis and there is a possibility that melanin can be synthesized in the adult RPE via an alternative pathway also in vivo.

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7.3 Melanin Content in Pigmented Structures of the Eye

The total melanin content as well as the relative content of pheomelanin to eumelanin varies in different melanin-containing parts of the eye. The following sections will discuss melanin composition in the RPE, choroid, and iris, and how it is affected by race and age. 7.3.1 Melanin Content in the RPE

RPE melanin is composed mostly of eumelanin [2]. Melanin content in the RPE in human eyes from 19 white and 16 black donors of different ages was studied by Weiter et al. by measurements of optical density of the RPE and their morphometric analysis [21]. To calculate optical density of the RPE, Weiter et al. measured intensities of 500- to 600-nm spectral light delivered from a rectangular aperture of 30 × 3 μm and transmitted through 8-μm thick sections of either the apical or the basal parts of the RPE. From these measurements the absorption coefficients of the basal and apical portions of the RPE were calculated. The total optical density of the RPE monolayer was calculated as the product of the RPE height and the mean of the apical and basal absorption coefficients. The optical densities were ascribed exclusively to melanin even though RPE cells contained lipofuscin likely to contribute to absorption and scatter of the incident 500- to 600-nm light. Nevertheless, the optical density measurements exhibited a positive statistical correlation with the counts of melanin granules. The measurements of the optical densities have demonstrated that the RPE exhibits a broad and flat minimum in the paramacula, and increases almost 2-fold in both directions – towards the center of the macula and towards the equator, reaching in those locations values of about 0.3 ± 0.1 (Figure 7.2) [21, 25]. The highest melanin density in the RPE appears as a ring 1 mm proximal of the ora serrata, where the RPE terminates and is continuous with the ciliary body [5]. Morphological measurements have demonstrated that RPE melanin exhibits a highly polarized intracellular distribution in the RPE, particularly in young donors where most melanin is present in the apical portion of the cell [21]. It has been shown that there is no statistically significant difference in melanin content in the RPE between whites and blacks [21]. In the RPE of both whites and blacks, melanin concentrations decrease with age [20, 21]. Feeney-Burns et al. studied the effect of donor age on RPE granule morphology and content in a 50 Caucasian cadaver eyes with five eyes per each decade of age [20]. The measurements demonstrated that there is a statistically significant decrease with age in “pure” melanosomes accompanied by an increase of lipofuscin and complex granules: melanolysosomes and melanolipofuscin. The percentage area of the macular RPE cell occupied by melanosomes of about 8% in the first two decades of life declines gradually to 3.5% of cell area in the 41–90 age group. The number of melanolipofuscin and melanolysosomes per RPE cell

7.3 Melanin Content in Pigmented Structures of the Eye a)

b)

Melanin (a) and lipofuscin (b) distribution in the RPE and choroid based on measurements of optical density (a) and fluorescence (b). (Based on data from [21].)

Figure 7.2

section is considerable already in the first decade of life (6.9 granules per cell section) and continues to rise to almost 15 complex granules per cell section in donors older than 61 years (Figure 7.3). As a percentage of cell area, the complex granules occupy 3.3% in the first two decades and this rises to 8–10% of the RPE cell area in donors over 61 years old. In the 90- to 101-year-old maculas, most RPE melanin is present as melanolipofuscin [26]. At the equator, the effect of age on the percentage of RPE cell areas occupied by melanosomes and melanosome number is less pronounced than for the macular RPE (Figure 7.3) [20]. The area occupied by melanosomes decreases from 8% in the first two decades to 5–6% in the elderly. Complex granules are rather sparse in the first decade (0.8 granules per cell section). As a percentage of cell

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Figure 7.3 Age-related changes in the content of pigment granules: melanosomes (Ms), melanolipofuscin (MLf), and lipofuscin (Lf) in human RPE. Mean number of

pigment granules per RPE cell section in the macular, equatorial and peripheral area of the retina from donors aged 1–20, 21–60, and 61–100 years. (Based on data from [20].)

area, the complex granules increase from 0.7% in the first two decades to 4% in donors over 61 years old. In the farthest periphery (the last 4 mm before the ora serrata) the concentration of melanosomes is the highest in the youngest RPE with a mean of 34 granules per cell section (Figure 7.3) [20]. The percentage of RPE area occupied here by melanosomes decreases from 15% in the first two decades to 4% in donors over 61 years old. The number of complex granules is the lowest here, but also significantly increases with age. The percentage area occupied by melanolipofuscin and melanolysosomes increases from 0.5% in the 0- to 20-year group to 3.6% in the 61- to 100-year group. These morphological measurements of age-related decrease in melanin content in human RPE are overall consistent with results obtained by two other methods [22, 23]. Solubilization of RPE cells by sodium borohydride followed by centrifugation to separate the soluble part from the insoluble cellular debris provides a solution with the absorption spectrum identical to the absorption spectrum of synthetic eumelanin model [22]. As noted by the authors, the procedure leads to lipofuscin, melanolysosomes, and melanolipofuscin being pelleted during centrifugation, and, therefore, melanin present in the complex granules is excluded from the measurements. Based on absorption maximum at 260 nm of the solubilized fraction, melanin content has been calculated in human RPE from 61 eyes from donors ranging in age between 14 and 97 years. Fitting data to a linear regression shows that content of solubilized melanin decreases within eight decades of life about 3-fold. In another method of melanin quantification, measurements of melanin free radicals by electron spin resonance (ESR) at pH 1 at liquid nitrogen temperature have been employed as a probe of melanin content in RPE cells scraped postmortem from the entire eye-cups of 105 donors [23]. The data obtained can be fitted rea-

7.3 Melanin Content in Pigmented Structures of the Eye

sonably well using linear regression demonstrating that melanin content normalized to total cellular protein decreases 1.8-fold between the ages of 10 and 90 years. 7.3.2 Melanin Content in the Choroid

The optical density of choroid is maximal at the fovea and gradually decreases almost 2-fold towards the equator (Figure 7.2) [21]. The concentration of melanin in the choroid is on average 2.4 ± 1.0 times higher in the outer half of the choroid (adjacent to the sclera) than in the inner half (adjacent to the RPE). There is a highly significant difference between whites and blacks in the optical density of the choroid [21]. The optical densities of the choroid for 500- to 600-nm light are 0.7 ± 0.5 and 1.4 ± 0.7 in the fovea, and 0.4 ± 0.3 and 0.7 ± 0.4 in the equator for whites and blacks, respectively. There is no statistically significant change with age in the total choroidal optical density; however, there is a trend for the ratio of the outer/inner choroidal melanin density to increase with age [21]. These measurements of melanin density in postmortem RPE and choroid are consistent with measurements of optical density in vivo based on reflectance spectroscopy. From measurements of 675 nm light reflected from an area of 7 ° × 7 ° of the fundus incorporating the macula, the optical density of the RPE–choroid complex has been calculated as 0.79 ± 0.17 for 16 Caucasians, 1.36 for a black, and 0.06 for an albino [27]. Measurements of reflectance at 500 nm from smaller areas of the fundi in 10 subjects have shown that the optical densities of the RPE-choroid complex vary within the range of 0.79–8.5 in the fovea and 0.72–6.90 in the perifovea [28–30]. 7.3.3 Melanin Content in the Iris

The iris is the most anterior portion of the uveal tract and is the most visible. It is composed of three parts: an anterior limiting membrane, stroma, and the posterior pigment epithelium. As mentioned earlier, iridial melanosomes are present in stromal melanocytes and iris pigment epithelial (IPE) cells. Melanin from the IPE is similar in eyes of different color and it is mostly eumelanin [31]. The composition of iris stroma varies with respect to total melanin content and eumelanin/ pheomelanin ratio [31, 32]. Blue irides exhibit very small melanin content in the stroma, with no detectable eumelanin and pheomelanin content of only 0.03 μg/ iris. The stroma of green irides exhibits a total melanin content of 4.7 μg/iris and a eumelanin/pheomelanin ratio of 0.88. The stroma of green/blue mixed-color irides contains mostly eumelanin (17.8 μg/iris) and a very high eumelanin/ pheomelanin ratio of 44.5. Interestingly, the pheomelanin/eumelanin ratio of 3.7 in stromas of green/brown mixed-color and brown irides falls in between green and green/blue irides. The total melanin content is highest in stromas of brown irides (32.2 μg/iris).

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While the color of the iris is largely determined by the variation in pigmentation in the iridial stroma, it is important to note that the iris color visible through the cornea results from different optical phenomena, including light absorption by different chromophores and multiple light scattering on melanosomes as well as other components of the connective tissue forming the stroma [2]. In albinism, where the melanin content in the pigment epithelium is markedly decreased or even absent, the color of irides may vary from yellow to a pink color. Studies of normal human donor eyes using light and electron microscopy have revealed that there is no significant difference in the content and distribution of melanocytes in irides of different colors. The difference in iris color is mainly determined by the variation of the melanosome structure and distribution within the iridial melanocytes. Darker irides have larger melanin granules and greater granule density. Another study on quantification of iridial melanin has been done on homogenates of whole human irides from donors of different age [33]. ESR measurements of melanin free radicals in the light blue irides showed no significant age-related changes in the ESR signal parameters or melanin content. Considering that in these irides most melanin is expected to be localized to the IPE [31], it can be suggested that the content of IPE melanin does not change with age. Darkening of the iris can occur in the adult eye as a side effect of topical application of prostaglandin analogs, such as latanoprost, used to lower intraocular pressure in the treatment of glaucoma [34]. Morphometric studies have demonstrated that the only change induced by latanoprost occurs in the iris stroma where the existing melanosomes undergo a small enlargement and this is sufficient to account for the darkening effect. Analysis of melanin composition of the iris stroma of cynomolgus monkeys subjected to 25–38 weeks of treatment with latanoprost has demonstrated that in the latanoprost-treated irides the amount of eumelanin increases 3- to 7-fold, while the pheomelanin content remains almost the same as in untreated irides [35]. As a result, latanoprost leads to the 3- to 5-fold increase in eumelanin/pheomelanin ratio in comparison with the control irides. Interestingly, latanoprost does not affect pigmentation of the iris epithelium.

7.4 Structure of Ocular Melanosomes

The structure of ocular melanosomes has been a subject of investigation for many years. The melanin composition of melanosomes from different ocular tissues has already been discussed. Early studies using transmission electron microscopy have shown that in ocular melanosomes, in similarity to other melanosomes, melanin is synthesized on a protein matrix and the whole granule is surrounded by a lipid bilayer. This section reviews the morphology of ocular melanosomes and their composition with regard to lipids and proteins.

7.4 Structure of Ocular Melanosomes

7.4.1 Morphology of Ocular Melanosomes

Melanosomes from different parts of the eye vary in shape, size, and age. Iridial stroma melanosomes in the young Rhesus monkey are cylindrical with the long axis often exceeding 2 μm and the short axis of about 0.2 μm [36]. With monkey aging there is progressive fusion of melanosomes forming aggregates composed initially of from two to four melanosomes and reaching numbers of 15–20 in the old monkeys; initially abundant long melanosomes seem to be replaced by shorter ovoid-shaped granules. Melanosomes from the human iridial stroma isolated from donors aged 30–67 years are rather uniform in size; small ovoid-shaped organelles with width and length dimensions of 0.25 ± 0.05 and 0.64 ± 0.10 μm, respectively [37]. In contrast, melanosomes from the human iris epithelium are spherical, with an average diameter of 1.02 μm [38]. Adult bovine melanosomes isolated from the whole iris–ciliary body complex seem to fall between these two with a long axis ranging from about 0.68 to 0.96 μm and a short axis ranging from 0.59 to 0.71 μm. Adult bovine melanosomes from the choroid also tend to have an ovoid shape with a long axis ranging from about 0.74 to 0.98 μm [39]. The choroidal melanosomes of the rat appear smaller and spherical with a diameter close to 0.4 μm [40]. Studies using transmission electron microscopy of age-related changes in choroidal melanosomes of Rhesus monkeys demonstrated that melanosomes in young animals are uniform in size and electron density, but with age there is an increasing number of melanosomes with altered morphology [41]. In aged monkeys almost all melanosomes appear as having an electron-dense “core” surrounded by material with very low electron density. No changes like these have been reported for humans. Choroidal melanosomes from human adults up to 67 years old appear similar to melanosomes from the human iridial stroma with width and length dimensions of 0.25 and 0.64 μm, respectively [37]. In the RPE of many species, including human, bovine, and rat, there are many elliptical or cigar-like melanosomes [20, 39, 40]. The long axis of the rat RPE melanosomes is close to 0.8 μm and the short axis is close to 0.4 μm [40]. Adult bovine RPE melanosomes tend to be larger than in the rat with a long axis within a range of about 0.6–2.9 μm and a short axis within a range of about 0.5–0.8 μm [39]. In the newborn bovine RPE there are considerably more cylindrical melanosomes than in the adult RPE. As discussed already, the most dramatic age-related changes in morphology of melanin-containing granules occur in the human RPE: the cylindrical melanosomes predominant in the newborn RPE seem to be disappear with aging so that in the adult RPE the melanosomes are more spherical and gradually seem to be replaced by complex granules – melanolysosomes and melanolipofuscin (Figure 7.3) [20, 26, 42]. Interestingly, in scanning electron microscopy, the morphology and size of the bovine ocular melanosomes appear unaffected by the procedure of chloroform– methanol extraction of melanosomal lipids [43]. It would be of interest to see the morphology of the aged human melanosomes, and melanolipofuscin/ melanolysosomes granules in particular, after such a procedure.

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Imaging by atomic force microscopy of ocular melanosomes isolated from human and bovine iridial stroma and RPE reveals that these melanosomes, in a manner similar to the melanosomes isolated from the ink sac of the cuttlefish Sepia officinalis, or from human black and red hair, appear to be comprised of smaller substructures [44]. The substructures appear as nonporous spheres with lateral dimensions of a few tens of nanometers consistent with the structure of premelanosomes where zigzagging longitudinal strands form cross-links every 20 nm and melanin deposition occurs around these strands [45]. Additional information about melanosomal substructure has been elegantly deduced based on melanin composition, models of mixed melanogenesis, and comparisons of the surface photoionization thresholds of melanosomes isolated from dark brown and blue/green irides and from black and red hair using photoemission electron microscopy (PEEM) [44]. PEEM analysis of melanosomes isolated from black and red hair with a predominant content of eumelanin and pheomelanin, respectively, has demonstrated that they substantially differ in their ionization thresholds [44]. The ionization threshold for black hair eumelanosomes is 4.6 ± 0.2 eV (corresponding to an oxidation potential of −0.2 V versus normal hydrogen electrode (HNE)) [46, 47]. Red hair pheomelanosomes exhibit two ionization thresholds, one of which is the same as for eumelanosomes of 4.6 ± 0.2 eV and the other which is lower, 3.9 ± 0.2 eV (corresponding to an oxidation potential of +0.5 V versus HNE). Eumelanosomes of other origin, namely Sepia, bovine, and human RPE, also exhibit a single ionization threshold within the range of 4.4–4.8 (±0.2) eV. Melanosomes isolated from blue/green and dark brown irides are expected to have eumelanin/pheomelanin ratios of 1.3 and 14.8, respectively [32]. However, melanosomes from both dark brown and blue/green irides exhibited the same single ionization threshold of 4.9 ± 0.2 eV [44]. These results have been explained by a mixed melanogenesis model in which pheomelanin is initially synthesized and the pheomelanin core is covered by eumelanin exterior. Considering melanosomal substructure as a sphere of 30 nm diameter, the thickness of the eumelanin exterior would be ~9 and 3.6 nm for the dark brown and blue/green iridial melanosome, respectively. 7.4.2 Molecular Composition of Ocular Melanosomes

In addition to eumelanin and pheomelanin content, which has been discussed above, melanosomes also include a considerable fraction of proteins and lipids, which will be discussed below. 7.4.2.1 Melanosomal Proteins The proteomics of mature melanosomes is challenging because many melanosomal proteins are insoluble, presumably due to covalent binding to melanin [48]. Nevertheless, proteomic analyses have been applied to mature melanosomes and melanolipofuscin from porcine and human RPE, respectively, yielding a considerable number of proteins [48, 49]. To date, more than 100 proteins have been

7.4 Structure of Ocular Melanosomes

identified in RPE melanosomes. These include proteins involved in melanogenesis, organelle acidification, organelle motility, cytoskeleton as well as proteolytic enzymes, channels, and transporters. Out of 102 proteins identified in the RPE melanosome by proteomics, only 12 proteins are common with the proteome of early stage melanosomes isolated from the human melanoma cell line MNT1. The majority of proteins in the RPE melanosome proteome are common with proteomes of other organelles, such as ER microsomes, mitochondria, Golgi apparatus, phagosomes, lysosomes, peroxisomes, the cytoskeleton, and the nucleus. Due to the small contamination of melanosomal preparations by mitochondria and other organelles, extramelanosomal origin of some of these proteins cannot be excluded. Unequivocal evidence for the presence of one of the lysosomal enzymes, cathepsin D, has been obtained by immunolabeling of cathepsin D in fixed RPE and its detection by transmission electron microscopy. Cathepsin D was localized close to the surface of RPE melanosomes. As mentioned earlier, aging of the human RPE is accompanied by accumulation of complex granules: melanolysosomes and melanolipofuscin. Proteomics of human RPE melanolipofuscin has identified 110 proteins, 23 of which are common with the porcine RPE melanosomes and 18 with macrophage phagosomes [49]. Interestingly, only 14 proteins were identified as common for melanolipofuscin and lipofuscin studied by the same group. In another study of lipofuscin proteomics, 38 and 17 proteins common with human melanolipofuscin and porcine RPE melanosomes, respectively, have been identified [50]. Treatment of lipofuscin granules with sodium dodecyl sulfate (SDS) and/or proteinase K results in removal of all identifiable proteins and a 5-fold decrease in their amino acid content [50]. It would be interesting to see which proteins remain after the SDS/proteinase K treatment is applied to melanolipofuscin or melanosomes. 7.4.2.2 Melanosomal Lipids The melanin-rich center of the melanosome is surrounded by a lipid membrane. This membrane tends to disappear after melanosome isolation and purification in a density gradient [48], imposing the requirement for a large number of melanosomes for lipid extraction. A thorough tandem mass spectrometry analysis of phospholipids was performed on lipids extracted from bovine ocular melanosomes isolated from three structures: iris–ciliary body complex, choroid, and RPE [43]. The analysis has revealed that the choroidal and iris–ciliary body melanosomes exhibit a similar composition of lipids with sphingomyelin, accounting for 54 and 41% of phospholipids in the choroid and iris, respectively. This is followed by glycerophosphocholine, lyso-glycerophosphocholine, lyso-glycerophosphoethanolamine, and phosphatidylglycerol. The fatty acid composition of these phospholipids is also rather similar in the choroid and iris. RPE melanosomes contain all of the lipids listed above; however, they exhibit striking differences in fatty acid composition in these phospholipids in comparison to the uveal melanosomes [43]. In the RPE, melanosomal phospholipids appear to contain more polyunsaturated fatty acyl chains than in the uvea. For example, in the RPE melanosomes, 93% of glycerophosphoethanolamine and 66%

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of glycerophosphocholine contain long-chain polyunsaturated fatty acid species. In the iridial and choroidal melanosomes glycerophosphocholine contains at most 43 and 31% polyunsaturated lipids, respectively. The most unsaturated fatty acid of the mammalian body, docosahexaenoic acid, is esterified to 26.7% of glycerophosphoethanolamine and 9.6% of glycerophosphocholine in the RPE melanosomes. In contrast to uveal melanosomes, RPE melanosomes contain detectable quantities of glycerophosphoserine and glycerophosphate.

7.5 Role of Ocular Melanin as a Broadband Optical Filter

Absorption of light is considered as one of the most important functions of melanin in the eye [4, 6]. Melanins exhibit broadband absorption spectra increasing monotonically from the IR through visible to UV light. These spectra result from combined effects of light absorption and scatter. Direct measurements of single intact melanosomes with PEEM have allowed the determination of the product of the absorption coefficient ε and the concentration of chromophores c absorbing 244 nm light [51]. For bovine RPE melanosomes isolated from a 1-week-old animal the product εc = 3895 ± 880 cm−1, whereas for melanosomes from an adult animal εc = 3515 ± 900 cm−1. The decrease of εc by a factor of 1.1 in the adult melanosomes compared to the newborn is consistent with an increase in the ratio of the eumelanin precursors DHI and DHICA. The [DHI]/[DHICA] ratio increases from 0.77 in newborn melanosomes to 1.25 in adult melanosomes. The absorption coefficient of DHICA at 244 nm is about 3.8 times higher than that for DHI, so it can be calculated that, as a result of the increase of the [DHI]/[DHICA] ratio, the ratio of εc decreases by the same factor of about 1.1, provided the DHICA content and melanosome size remain the same. In addition to melanin, melanosomal proteins and their oxidation products are likely to contribute to the absorption of 244 nm light. Most of the light energy absorbed by melanin, particularly eumelanin, is dissipated as heat with only a tiny fraction released as fluorescence, phosphorescence, or utilized for chemical changes such as formation of melanin free radicals and electron transfer to molecular oxygen [52]. 7.5.1 Role of the Iris as a Filter of Light

The human cornea absorbs UV-C and part of the UV-B radiation up to 295 nm [53], so only light above 295 nm reaches the iris and lens. The iris partly absorbs the remaining UV-B, UV-A, visible, and IR radiation so less light passes to the lens and the posterior segment comprised of the vitreous and retina. The iris regulates the size of the aperture through which light enters the more posterior parts of the eye. The iridial stroma is connected to the iris sphincter and dilator muscles. In response to light reaching the retina, either the sphincter or

7.5 Role of Ocular Melanin as a Broadband Optical Filter

the dilator contracts, in this was regulating the diameter of the circular opening in the center of iris – the pupil. The human pupil diameter depends on age and varies from 2–7 mm, when it is fully constricted in bright light, up to around 9 mm when it is fully dilated in the dark [54]. This pupillary light reflex plays an important role in adaptation of the eye to different levels of light, which normally vary within 11 orders of magnitude. By changing the size of the pupil, the pigmented iris can regulate the amount of light passing through the pupil up to 20-fold. While light enters the posterior segment of the eye mostly through the pupil, some light can be transmitted through the iris itself [55]. There are substantial differences in the transmittance through the iris itself depending on iris color [55]. In the rabbit, the average transmittance of freshly extracted blue irides is around 4.2 ± 1.7% and is greater by at least a factor of 4.2 than for brown irides. Blue irides transmit 7 times less light than albino irides. The difference in transmission properties of blue and brown irides seem to be less pronounced in humans where the ratio of transmittance for blue and brown irides isolated from human cadavers is only about 2.5 [55]. Transmission values of flattened irides are quite considerable: 5.5 ± 2.8% for brown irides and 14 ± 6.3% for blue irides. Considering the surface of the iris and fully constricted pupil the amount of light passing through the iris in addition to that passing through the pupil can be estimated and compared for blue and brown irides. Calculation, based on a diameter of 11.7 mm of the opening through which light enters the iris [56] and a diameter of the constricted pupil of 2 mm [54], shows that the surface of the iris can be 34-fold greater than the pupil area. Considering the transmittances of human blue and brown irides cited above [55], it can be estimated that the light intensity entering the eye through the iris can be 1.9 and 4.8 times greater than the light entering the eye through the pupil for brown and blue irides, respectively. It should be noted that the human irides used for these measurements were used 2–3 days after enucleation and the IPE might have been dislodged due to extended storage [55]. Nevertheless, the study demonstrated that blue irides transmit 2.5–4.2 times more light than brown irides, and that this can account for a substantial part of light reaching the lens and posterior segment. These measurements of transmission of light through extracted irides are consistent with more recent psychophysical measurements of stray light in healthy human volunteers [57, 58]. The photoprotective role of iridial melanocytes is suggested by epidemiological studies of opacities in the lens known as cataract [59]. It has been shown that nuclear cataract and posterior subcapsular cataract are significantly correlated with iris color, with higher a risk of cataract among medium brown, light brown, and yellow/green, in comparison with dark brown, irides. While there is no doubt pigmentation of the iris partly blocks light from reaching the posterior segment, it can also be suggested that absorption of light by iridial melanosomes can play a role in screening from light, and thus protection from oxidative and nitrative stress induced by UV and visible light, of different cell types present within the iris, including endothelial cells and erythrocytes as well as pigmented cells themselves [60–64]. Epidemiological studies of iridial melanoma support this protective function of melanosomes of the iridial stroma. Iridial

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melanocytes are exposed to UV light above 295 nm transmitted through the cornea (Figure 7.1). Melanomas of the iris tend to originate from the inferior part of the iris where the exposure to sunlight is the greatest [2, 65]. The risk factors for the development of uveal melanoma include light iris color (blue, hazel, or gray), fair skin, inability to tan, and arc welding. It has been suggested that the high abundance of eumelanosomes in dark irides may act as an effective filter of light, thus providing protection from photo-oxidative stress and mutagenic effects. Light irides, on the other hand, do not exhibit a high density of stromal pigmentation. Moreover, blue/green and hazel irides contain a high proportion of pheomelanin to eumelanin, and therefore a thinner coating of eumelanin over the reactive pheomelanin core. It has also been suggested that exposure to sunlight may cause photodegradation of eumelanin and expose the more reactive pheomelanin, thus increasing the oxidative stress in the iridial melanocyte. 7.5.2 Role of Melanin in Light Transmission Through the RPE and Choroid

Light reaching the back of the adult human eye is mostly visible and IR covering the range from 390 to 1400 nm (Figure 7.1) [3]. UV light in the range 295–390 nm that passes through the cornea is almost completely absorbed by the lens. In young children there is a transmission window centered at about 320 nm with a transmission maximum of about 8%. As the lens ages, its transmission in the visible spectral range gradually decreases, with the effect being more pronounced the shorter the wavelength. Thus, the spectral characteristics of radiation incident on RPE and choroid are strongly dependent on the age and the presence of the crystalline lens. In the case of cataract surgery, the crystalline lens is replaced with an artificial lens, which could be either clear, transmitting all visible light above 390 nm, or yellow, mimicking the absorption of light by an old human lens. Melanosomes in the young RPE are localized in the apical portion of the RPE including its microvilli. The microvilli extend into the retinal layer containing photoreceptor outer segments with high concentrations of visual pigments. Here, in the apical portion of the RPE melanin can absorb light passing through photoreceptors thus minimizing light reflections and improving image quality. Based on optical densities measured by Weiter et al. of the RPE from cadaver eyes [21], it can be estimated that the RPE absorbs about 34–60% of incident light in the fovea, 21–40% in the paramacula, and 26–57% at the equator. As mentioned earlier, optical densities of choroidal melanin vary considerably between individuals, and there is a significant difference between whites and blacks [21]. In the area underlying the fovea the choroid absorbs 37–94 and 80–99% of incident light in whites and blacks, respectively. At the equator, where the melanin density is the smallest, the choroid absorbs 21–80 and 50–92% of incident light in whites and blacks, respectively. Altogether, in the area of the fovea, the RPE and choroid absorb 58–97 and 87–100% of incident light for whites and blacks, respectively. At the equator, RPE

7.6 Antioxidant Properties of Ocular Melanin

and choroid absorb together 41–91 and 62–97% of incident light for whites and blacks, respectively. In addition to light entering the eye globe through the cornea, some light can be transmitted through the exposed part of the sclera adjacent to the cornea. Psychophysical measurements of volunteers with different degrees of ocular pigmentation have demonstrated that transmission of light through the anterior sclera–RPE–choroid complex is two orders of magnitude greater in people with blue irides than dark brown irides [58].

7.6 Antioxidant Properties of Ocular Melanin

Ocular melanin is present in structures exposed to light with high levels of oxygen and photosensitizers – an ideal environment for the production of reactive oxygen species (ROS). As discussed above, melanin can act as an efficient natural sunscreen, thus protecting ocular tissues from adverse reactions induced by light. In addition, properties of synthetic melanin studied in suspension indicate that it can act as an efficient scavenger of free radicals and quencher of electronically excited states of photosensitizers and singlet oxygen. Studies of both synthetic melanin and ocular melanosomes indicate that both can bind redox-active metal ions, such as iron or copper, and inhibit Fenton-type reactions catalyzed by these ions. These properties will be discussed below with regard to the potential functions of melanin in the eye. 7.6.1 Scavenging of Free Radicals

The ability of melanin to scavenge free radicals as well as reduce and oxidize other molecules is related to its redox properties. The key role in electron transfer is attributed to the functional subunits present in melanin: DHI, DHICA, and their fully oxidized (quinone) and semioxidized (semiquinone) forms in eumelanin, and o-aminophenols, such as 1,4-benzothiazine and their corresponding fully oxidized o-quinonimine and semioxidized o-semiquinonimine in pheomelanin [52, 66]. However, these subunits present in the melanin polymer exhibit much smaller reactivity in comparison to the very reactive free o-quinones and o-semiquinones. The relatively small reactivity of melanin subunits may be ascribed to their restricted accessibility and/or modified ionization potentials and electronic affinity. From pulse radiolysis measurements of interaction of synthetic melanins with a number of oxidizing and reducing radicals it appears that the synthetic model of eumelanin requires stronger reducing radicals than the synthetic model of pheomelanin [67]. The one-electron reduction potential of the eumelanin model is about 0.52 V versus NHE; whereas for the pheomelanin model, the reduction potential is close to 0.35 V versus NHE. Assuming that all monomer units of the

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eumelanin are available for redox reactions, it can be estimated that about 25% of the eumelanin sites are available for reduction, while 75% of the sites are available for oxidation. Synthetic models of eumelanin have also been investigated by electrochemistry yielding potentials of +460 and +525 mV versus saturated calomel electrode (SCE) for a two-electron oxidation and +20 and −355 mV versus SCE for a two-electron reduction [68]. Synthetic melanins can scavenge effectively a number of free radicals that can be formed in the eye, such as the hydroxyl radical, superoxide radical, nitrogen dioxide radical, or carotenoid cation radical [67, 69]. Hydroxyl radicals can be formed as a result of decomposition of hydrogen peroxide by redox active metal ions such as iron or copper [70]. In the cellular environment there is a high abundance of molecules with which •OH can react with bimolecular rate constants approaching the diffusion-controlled limits. Due to this extremely high reactivity of the hydroxyl radical in the cell, the ability of melanin to scavenge •OH is likely to play a role only if •OH is generated within the melanosomes or close proximity. Superoxide radicals are formed in all cells as a byproduct of mitochondrial respiration. In addition, in the RPE, superoxide is formed during phagocytosis of photoreceptor outer segments, or by photoexcited melanosomes or lipofuscin [71, 72]. Oxidation of melanin-bound metal ions such as Fe(II) or Cu(I) by molecular oxygen also leads to generation of superoxide [66]. Interaction of melanin with the superoxide radical leads either to its reduction to hydrogen peroxide or oxidation to molecular oxygen. Thus melanin can be viewed as a pseudo-dismutase. NO2ᠨ is formed as a result of interaction of long-lived nitric oxide with oxygen or as a result of oxidation of nitrite [73]. Nitric oxide, which is generated both in the retina and choroid, has a relatively long lifetime in tissues and therefore it may diffuse near melanosomes. It can be suggested that in proximity to melanosomes it may encounter superoxide, forming another reactive nitrogen species (RNS), peroxynitrite. Even though peroxynitrite is not a free radical, it is highly reactive with proteins and lipids, leading to their nitration. Scavenging of peroxynitrite by a synthetic model of neuromelanin has been demonstrated [74, 75]. Considering that protein nitration occurs in the retina and is substantially increased as a result of exposure to light or in retinal diseases [76], it is of high importance to investigate interactions of ocular melanins and melanosomes with RNS. The ability of melanin to scavenge the carotenoid radical cation may be important in human RPE where lutein, zeaxanthin, and β-carotene are present [77]. These carotenoids are believed to play a protective role in the RPE; however, they may become pro-oxidant and cytotoxic upon oxidation. Semioxidized forms of all these carotenoids are scavenged by synthetic models of eumelanin and pheomelanin [69], presumably by electron/hydrogen transfer, thus regenerating the parent compound. It remains to be shown whether or not RPE melanosomes can also protect from carotenoid degradation. An important question is whether melanins can scavenge peroxyl radicals propagating lipid peroxidation. While the interaction of melanins with lipid-derived

7.6 Antioxidant Properties of Ocular Melanin

peroxyl radicals has not been studied directly, the efficient scavenging of peroxyl radicals derived from methanol by synthetic models of eumelanin and pheomelanin, suggests this possibility [78]. As mentioned earlier, RPE melanosomes contain polyunsaturated lipids. Oxidation of these lipids can lead to the formation of a number of noxious species, including some small polar aldehydes that can diffuse outside the granule. Thus, the ability of melanin to inhibit lipid peroxidation within RPE melanosomes would be a highly desirable function. While melanin structure appears very complex and resistant to elucidation, the structure of melanosomes is even more complex. As already mentioned, redox properties of whole melanosomes have been studied using PEEM [44]. PEEM is inherently a surface technique with electrons originating from no further than within a few nanometers of the melanosomal surface. This can be viewed as an advantage as interaction of melanosomes with many extragranular molecules is likely to proceed at the surface. As mentioned earlier, PEEM results have revealed that all ocular melanosomes studied so far, bovine and human RPE melanosomes, and human iridial stroma melanosomes isolated from dark brown and blue/green irides, exhibit similar single photoionization thresholds ranging from 4.4 ± 0.2 to 4.9 ± 0.2 eV (corresponding to 253–282 nm wavelength of light and oxidation potential of about −0.2 V versus NHE). This single photoionization threshold seems to be typical for eumelanosomes. Red hair pheomelanosomes exhibit an additional photoionization threshold of 3.8 ± 0.2 eV corresponding to 326 nm wavelength of light and oxidation potential of +0.5 V versus NHE) [44, 46, 47]. 7.6.2 Quenching of Electronically Excited States of Photosensitizers and Singlet Oxygen

Interaction of synthetic melanins with electronically excited states of photosensitizers or with the excited state of molecular oxygen, known as singlet oxygen (1Δg), can lead to transfer of the excess energy to melanin [66]. This effect suggests that melanin can offer photoprotection not only as a passive absorber of light, but also by preventing photosensitized oxidation. The energy transfer from electronically excited states is particularly efficient for positively charged photosensitizers, such as cationic derivatives of porphyrins, which bind to melanins. When a melanin-bound photosensitizer absorbs light, the energy transfer occurs within femtoseconds from the excited singlet state of the photosensitizer to melanin [79]. As a result, the photosensitizer molecule returns to the ground state, and is unable to undergo intersystem crossing and form the excited triplet state – the usual culprit responsible for subsequent interactions with oxygen and formation of damaging ROS [3]. It has been demonstrated that human RPE melanosomes exhibit similar ability to synthetic melanins to bind cationic photosensitizers and strongly inhibit their photosensitizing properties (Figure 7.4) [80, 81]. Quenching of excited states of photosensitizers by melanosomes may be important in vivo in the presence of photosensitizing molecules of both exogenous and

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Effect of native human RPE melanosomes isolated from donors 60–90 years old (HMs) and photodegraded human RPE melanosomes (dHMs) on photosensitized oxidation of histidine mediated by cationic derivative of porphyrin. Samples

Figure 7.4

contained melanosomes at indicated concentrations, 88 μM porphyrin, 2 mM histidine, and 0.1 mM ESR oximetry spin probe. Human RPE melanosomes were photodegraded by exposure to 5.54 kJ/cm2 of blue light (390–490 nm). (Modified from [80].)

endogenous origin. Xenobiotics with photosensitizing properties and high affinity for binding to melanin include chloroquinone derivatives used as antimalarial drugs [82–84]. These antimalarial drugs cause phototoxic effects in the skin and the eye, and the ocular effects of their binding to melanin and accumulation in the eye are discussed below. Other drugs that deserve consideration with respect to interactions with melanin are drugs used for phototherapy where their photosensitizing properties are highly desired. When phototherapeutic drugs are targeted to affect melanin-free tissues, their binding to melanin and quenching of their excited states may be seen as protective for pigmented cells. For example, photodynamic therapy of choroidal neovascularization is meant to destroy pathological blood vessels growing into the retina in the course of the “wet” form of age-related macular degeneration (AMD). However, the photosensitizing drug used for that therapy, verteporfin (Visudyne®) tends to enter the RPE causing, in some cases, particularly after multiple treatments, collateral damage to this extremely important monolayer of postmitotic cells essential for vision [85]. It would be of interest to investigate the possibility that RPE melanosomes/melanolipofuscin bind to verteporfin (or other newly developed photosensitizers) and moderate their photosensitizing action. It could be suggested that during initial treatments, this binding could indeed play a protective role. However, if the photosensitizer remains bound to melanin, the

7.6 Antioxidant Properties of Ocular Melanin

binding capacity of melanin could be exceeded during subsequent treatments. Moreover, while binding of photosensitizers to melanosomes may be protective, it is unknown what the effects are of binding of photosensitizers to melanolipofuscin. Understanding the interactions between melanin-containing granules and photosensitizing drugs could allow better design of photodynamic treatment. Melanolipofuscin is expected to contain similar photosensitizer(s) as those present in lipofuscin granules [86]. It can be suggested that due to the proximity of melanin to these photosensitizers in melanolipofuscin granules, melanin can quench their photoexcited states, thus reducing the photoreactivity of melanolipofuscin. Whether or not this energy transfer occurs in melanolipofuscin still awaits experimental confirmation. Another molecule present in lipofuscin is a pyridinium bisretinoid (so-called A2E), which, because of its positive charge, can be expected to bind to melanin. Although A2E provides an almost negligible contribution to photosensitizing properties of whole lipofuscin [3, 86], quenching by melanin of its excited state may be beneficial by preventing A2E photodegradation that leads to formation of deleterious degradation products. Determination of whether or not photodegradation of A2E is slower in melanolipofuscin than in lipofuscin also needs experimental testing. Interestingly, it has been demonstrated that the inhibitory effect of human RPE melanosomes on photosensitized oxidation is substantially enhanced in the case of photodegraded melanosomes (Figure 7.4) [80]. Photodegradation was induced by a dose of blue light corresponding to 4.4 years of daily exposure of the human eye to ambient light. Under conditions where nondegraded melanosomes inhibited photosensitized oxidation of histidine mediated by cationic porphyrin by about 42%, the inhibition in suspension of degraded melanosomes was greater than 90%. It needs to be stressed that in this experiment melanosome photodegradation induced by blue light was rather extensive leading to a 2.3-fold decrease in the intrinsic free radical signal of melanin. Thus, it is intriguing why, despite the substantial degradation of melanin polymer, melanosome ability to inhibit photosensitized oxidation is enhanced. One possibility, which requires further investigation, is that access to melanin binding sites becomes facilitated upon melanosome photodegradation. It has been demonstrated that synthetic melanins and RPE melanosomes solubilized at pH 12 (and thus most likely oxidized) can quench the excited state of molecular oxygen that can be formed as a result of energy transfer from photoexcited photosensitizers to molecular oxygen [3, 66, 87]. In contrast to ground state oxygen, singlet oxygen can readily oxidize proteins, nucleic acids and unsaturated lipids. Yet, the cellular defenses against singlet oxygen seem to be rather limited. In contrast to other ROS, such as superoxide radical anion, hydrogen peroxide, or lipid hydroperoxides, no enzymes have evolved to deactivate singlet oxygen. Thus, melanin as a singlet oxygen quencher is potentially important for all pigmented tissues exposed to light. However, it can be suggested that due to the relatively short lifetime of singlet oxygen, which in water is only about 3–4 μs, and the abundance of substrates of oxidation in the cellular environment, the quenching

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of singlet oxygen by melanin is likely to occur only if singlet oxygen is generated in proximity or within melanin-containing granules. These conditions are probably met in melanolipofuscin; however, experimental proof is still lacking. 7.6.3 Sequestration of Redox-Active Metal Ions

Melanin has the capacity to bind and accumulate many metal ions including redoxactive metals such as iron and copper [4, 52, 88]. Iron and copper can be damaging by catalyzing the decomposition of hydrogen peroxide and lipid hydroperoxide that result in the formation of the highly reactive free hydroxyl radical and/or initiation of a chain of lipid peroxidation [70]. It has been demonstrated that binding of iron and copper ions to melanin diminishes their ability to catalyze the generation of free hydroxyl radicals and lipid peroxidation [4, 52]. Even though melanin complexes with Fe(II) ions are readily oxidized by hydrogen peroxide and oxygen, the hydroxyl radicals produced are mostly scavenged by melanin and few leak out of the melanin polymer. Once iron ions are oxidized, melanin-bound Fe(III) becomes substantially more difficult to reduce by mild biological reductants such as NAD(P)H or ascorbate in comparison with free Fe(III) ions or Fe(III) ions bound to ADP or citrate. Thus, melanin can prevent redox cycling of iron ions and their action as catalysts in Fenton-type reactions. It has been shown that ocular melanosomes isolated from human, porcine, and bovine RPE can also bind iron ions, and exhibit similar protective effects as synthetic melanins [80, 81, 89, 90]. A very convincing example of protective action of melanin against iron-catalyzed oxidation has been obtained by comparison of ironcatalyzed oxidation in a suspension of bovine RPE homogenate isolated from pigmented and nonpigmented parts of bovine fundus (Figure 7.5) [80]. To test whether or not the presence of melanin in pigmented cells was responsible for the inhibitory effect, the nonpigmented RPE cells were supplemented with either bovine melanosomes or synthetic eumelanin. As a result of addition of bovine melanosomes, the rate of oxidation in suspension of nonpigmented cells was slowed down to a similar level as in pigmented cells. Human RPE melanosomes isolated from 60- to 90-year-old donors are also effective in inhibiting iron-catalyzed oxidation (Figure 7.5). These findings are of particular importance considering that RPE cells provide the blood–retina barrier, and therefore are heavily involved in continual trafficking of iron between the choroidal blood supply and photoreceptors [91, 92]. Iron delivered from blood to the RPE is partly used there as a cofactor for mitochondrial and other enzymes, such as enzymes essential for the regeneration of visual pigments. A portion of iron is transported through the RPE to photoreceptors and incorporated in their outer segments. The distal tips of photoreceptor outer segments are phagocytosed daily by the RPE, providing another source of iron. Due to their pro-oxidant properties, the free iron ion concentrations in the cell are meant to be maintained at very low levels by efficient homeostatic mechanisms,

7.6 Antioxidant Properties of Ocular Melanin a)

Effect of bovine (a) and human (b) RPE melanosomes on oxidation catalyzed by iron ions. Oxidation was induced by Fe-ADP/ascorbate (0.05 mM/0.20 mM) in a suspension of bovine RPE homogenate isolated from pigmented (BP) and nonpigmented (BNP) parts of bovine fundus (a). To test whether the presence of melanin in pigmented bovine fundus was responsible for the inhibitory effect, either bovine

Figure 7.5

b)

melanosomes (BMs) or synthetic eumelanin (DM) were added to nonpigmented bovine RPE at concentrations corresponding to concentration of melanin of 30 or 60 μg/ml. Inhibitory effect of human RPE melanosomes (HMs) was tested on iron ion-catalyzed lipid oxidation in a suspension of 10 mM linoleate (LA), 0.2 mM ascorbate, and 0.05 mM Fe-ADP (b). (Modified from [80].)

controlling its compartmentalization and regulating its release and trafficking. However, it can be suggested that due to its physiological functions RPE is at continual risk of exposure to iron ions that escape from iron-binding proteins. With aging there is a substantial increase in iron content in the RPE and choroid [93]. Iron content is further increased in several retinal degenerations. For example, there is a 5-fold increased iron in the RPE affected by AMD than in age-matched healthy RPE [94]. Another factor likely to contribute to increasing the risk of Fenton-type processes in the aged RPE is age-related decrease of activity of catalase – the major enzyme responsible for decomposition of hydrogen peroxide [95]. It remains to be investigated in which intracellular compartment(s) of the RPE the accumulation of iron occurs and whether melanosomes can protect RPE cells from deleterious effects of iron. 7.6.4 Testing Protective Effects of Ocular Melanin in Cultured Cells

All the antioxidant properties discussed above indicate that melanosomes may play a protective role in the eye in vivo. However, providing experimental evidence on animals with different degrees of ocular pigmentation to support this notion has

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proven to be unexpectedly difficult and conflicting results have been obtained [96]. Also, attempts to demonstrate the protective effect of RPE melanosomes in cultured cells have proven so far unsuccessful [97, 98]. Susceptibility to apoptosis of primary cultures of fetal human RPE cells with different degrees of pigmentation has been studied by Seagle et al. [97]. Melanin content was assessed by spectrophotometry. The intensity of melanin free radicals in the dark and upon exposure to increasing intensity of 355-nm laser light was monitored by ESR. To assess potential photoprotective effects of melanin, cells were exposed for 7 days to continuous blue light irradiation (440 nm, 4.55 mW/ cm2) while cells kept in the dark served as controls. Staining of exposed phosphatidylserine with Annexin V was used as a measure of apoptosis. It has been stated in the results that the RPE cells less susceptible to apoptosis have relatively low optical absorption spectra even though the intensities of the ESR signals of melanin are relatively high [97]. Plotting the data points shows that the cell cultures employed exhibited considerable variability and linear regression analysis of the data provide low correlation coefficients (Figure 7.6). It can be argued that the higher amplitudes of melanin free radicals measured for some RPE cells could reflect a lower content of melanin-bound iron and/or higher concentrations of reductants such as ascorbate [98, 99], each of which could also confer protection from light-induced apoptosis. In conclusion, the role of endogenous melanosomes in protection of the RPE against light damage requires further investigation. Zareba et al. have undertaken a thorough investigation of effects of exogenous melanosomes on the human RPE cell line, ARPE-19 [98]. The cells were fed with bovine or porcine melanosomes to accumulate intracellular granule concentrations of more than 20 melanosomes/cell and then exposed to visible light or oxidants. Several endpoints were evaluated including integrity of the plasma membrane, mitochondrial activity, cell adhesion, and lysosomal integrity, and compared with cells fed with melanosomes, charcoal, silica particles, and control cells. The results have demonstrated that the presence of intracellular melanosomes neither prevented nor exacerbated the cytotoxic effects of hydrogen peroxide, tert-butyl hydroperoxide, and visible light. However, it can be argued that the lack of effect of melanosomes could be due to the majority of oxidants being formed in the extracellular culture medium. The culture medium contained iron ions, and therefore it is likely that the decomposition of tert-butyl hydroperoxide and hydrogen peroxide occurred mainly extracellularly. The riboflavin present in the culture medium used could also be a source of extracellular phototoxic effects as observed in cultured cells exposed to UV-A or blue light [100]. Furthermore, phagocytosed melanosomes may be surrounded by an additional lipid membrane thereby hindering their interaction with ROS. Under physiological conditions RPE cells are likely to be exposed to oxidative stress due to accumulation of lipofuscin [3, 86]. It would be of interest to test the effects of melanosomes on oxidative stress induced inside the RPE cell by photoexcited lipofuscin.

7.7 Pro-Oxidant Properties of Ocular Melanosomes a)

b)

d)

Susceptibility to apoptosis in primary cultures of fetal human RPE cells after 7 days of exposure to blue light (circles) or kept in the dark (triangles), and arithmetical differences between apoptosis values of cells exposed to light and kept in the dark (rectangles) as functions of optical density of RPE cells measured at 500 nm (a), amplitude of melanin radical in the dark (b) or melanin

Figure 7.6

c)

e)

radical photoinduced by exposure to laser light (250 mW; 355 nm) (c). (d and e) Amplitudes of melanin radical in the dark (d) or during exposure to laser light (e) as a function of optical density of RPE cells. Linear regression lines and their corresponding correlation coefficients are included in diagrams. (Based on data from [97].)

7.7 Pro-Oxidant Properties of Ocular Melanosomes

Ocular melanosomes exhibit several pro-oxidant properties. They can be a source of ROS, induce depletion of important cellular reductants, and facilitate Fentontype reactions. This can be another reason why it is so difficult to observe protective effects of melanosomes in cultured cells or in vivo, where the antioxidant action can be counterbalanced by the pro-oxidant action of melanosomes. Next, the prooxidant properties of ocular melanosomes and their age-related changes will be discussed.

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7.7.1 Generation of ROS and Oxidation of Cellular Reductants

Photoexcitation with UV or visible light of ocular melanosomes results in similar effects as those observed for synthetic melanins: generation of photoinduced melanin free radicals and formation of ROS, such as superoxide radical and hydrogen peroxide [66, 71]. While for synthetic eumelanin most oxygen consumed during irradiation with visible light is converted to hydrogen peroxide, only around 35% of consumed oxygen is converted to hydrogen peroxide for RPE melanosomes isolated from donors younger than 33 years. The ratio of hydrogen peroxide produced to total oxygen consumed decreases with donor age. For donors older than 78 only 13% of oxygen consumed during irradiation of RPE melanosomes accumulates as hydrogen peroxide. This indicates that RPE melanosomes are susceptible to photo-oxidation of intragranular components. Consistently, upon irradiation of human RPE melanosomes with visible light accumulation of lipid hydroperoxides can be detected. Light-induced oxidation is strongly dependent on the irradiation wavelength. The rate of photoinduced oxygen uptake in suspension of human or bovine RPE melanosomes increases with decreasing wavelength measured in the range of 300 to 600 nm (Figure 7.7) [101]. While for UV and short-wavelength blue light the rate of photo-oxidation of human RPE melanosomes is slower than for lipofuscin by almost an order of magnitude, the difference becomes smaller at longer wave-

Figure 7.7 Wavelength dependence of susceptibility of bovine (BMs) and human (HMs) RPE melanosomes to photo-oxidation and their comparison with human RPE lipofuscin (HLf). Human granules were

isolated from donors above 60 years old. The initial rates of oxygen uptake have been normalized to equal number of incident photons. (Modified from [102].)

7.7 Pro-Oxidant Properties of Ocular Melanosomes a)

c)

b)

Figure 7.8 Photogeneration of superoxide by human RPE melanosomes isolated from donors less than 40 (MS < 40) or more than 80 years old (MS > 80). ESR spectra recorded in samples containing melanosomes (0.9 × 109 granules/ml) and 0.2 M spin trap 5,5-dimethylpyrroline-N-oxide (DMPO) in dimethyl sulfoxide before (a, b), and after (d, e) 22 min of irradiation with broadband blue light. (c) Kinetics of changes

d)

e)

in amplitude of DMPO-OOH spin adduct formed during irradiation. Arrow: beginning of irradiation. Incubation of melanosomes with DMPO in the dark did not result in the appearance of an ESR signal of spin adducts. Instrument settings: time constant 328 ms; sweep time 160 s; microwave power 10 mW; modulation amplitude 1.0 G. (Modified from [71].)

lengths. At wavelengths longer than 550 nm human RPE melanosomes are more photoreactive than lipofuscin. Interestingly, the susceptibility to photo-oxidation and the light-induced generation of superoxide radical by human RPE melanosomes substantially increases with donor age (Figure 7.8) [71]. Irradiation with blue light of suspension of the RPE melanosomes from donors more than 80 years old induces 2.4-fold faster oxygen uptake than for melanosomes from donors less than 40 years old. Despite the 40% increase in production of superoxide by older melanosomes in comparison to young melanosomes, there is no significant difference in accumulation of the product of superoxide dismutation, hydrogen peroxide. This indicates that with age melanosomes may lose their ability to reduce superoxide and/or gain an ability to decompose H2O2. It has been shown that photoexcited human RPE melanosomes can oxidize extragranular unsaturated lipids (Figure 7.9) [71, 103]. Generation of hydrogen peroxide by photoexcited melanosomes from the RPE or iris strongly increases in the presence of physiological reductants, such as ascorbate or NADH, whereas the reductants are depleted (Figures 7.9 and 7.10) [71, 99, 104–107]. Altogether, the properties of isolated melanosomes indicate that they may contribute to oxidative stress in the RPE by generation of reactive species and depleting cellular reductants.

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Figure 7.9 The effect of the addition of liposomes with polyunsatuarated lipids (PUFA), bovine serum albumin (BSA), NADH, or mM ascorbate (AscH) on the rate of broadband, blue-light-induced oxygen uptake in suspensions of human RPE melanosomes (MS) or lipofuscin (LF).

Control samples were with melanosomes or lipofuscin without additional substrate of oxidation (CTRL). The rates of oxygen uptake in the dark were negligible for all samples studied. Significant differences are relative to the controls. *P < 0.05, **P < 0.01. (Modified from [71].)

a)

b)

Depletion of ascorbate (a) and formation of hydrogen peroxide (b) during aerobic irradiation with blue light of bovine RPE melanosomes (BMs) in the presence of ascorbate (Asc). Controls show levels of ascorbate during incubation with bovine RPE

melanosomes in the dark (a) and formation of hydrogen peroxide during incubation of bovine RPE melanosomes with ascorbate in the dark and during irradiation of bovine RPE melanosomes in the absence of ascorbate (b). (Modified from [99].)

Figure 7.10

7.7 Pro-Oxidant Properties of Ocular Melanosomes

7.7.2 Pro-Oxidant Effects of Interactions of Melanosomes with Metal Ions

While melanin can effectively inhibit redox-cycling of metal ions by cellular reductants and decomposition of hydrogen peroxide and lipid hydroperoxides catalyzed by metal ions, melanin itself can reduce Fe(III) or Cu(II) ions when they are complexed with a strong chelator and/or when they exceed the number of melanin binding sites [108]. It remains to be investigated how close to saturation with iron melanosomes in the aged RPE are, especially if affected by AMD. Superoxide radical, which can be generated by photoexcited melanin/ melanosomes, can also reduce iron and copper ions. Oxidation of melanin-bound Fe(II) or Cu(I) by hydrogen peroxide or molecular oxygen leads to the formation of hydroxyl radical and superoxide radical, respectively. As both radicals are generated within the melanin granule, they are likely to be scavenged by melanin. However, in cells such as RPE, life-long exposure to visible light and scavenging of radicals may lead to melanin degradation. Degradation of isolated RPE melanosomes can be easily achieved by their exposure to visible light [80, 89, 90]. For human RPE melanosomes isolated from donors 60–90 years old, the dose of blue light leading to 2.3-fold decrease in melanin free radical was equivalent to 4.4 years of daily exposures to ambient light [80]. It is still an open question whether melanosome degradation occurs in vivo or whether cellular antioxidants are able to prevent or minimize it. With aging, RPE melanosomes undergo substantial changes with regard to their morphology, melanin content, and photophysical and photochemical properties. Previous studies have shown that photodegradation of melanosomes mimics several changes observed in the aging of human RPE melanosomes, including (i) an increase in fluorescence [23, 109], (ii) a decrease in intrinsic melanin free radicals [80, 90, 110], and (iii) an increase in photoinduced generation of superoxide [71, 89, 90]. Initially, melanosome degradation is accompanied by reduction in their capacity to bind iron ions, and to offer protection against iron ion-mediated decomposition of hydrogen peroxide and lipid peroxidation (Figure 7.11). Further degradation of melanosomes leads to a complete loss of inhibitory effect on oxidation catalyzed by metal ions. Instead, degraded melanosomes become pro-oxidant and mediate metal ion-induced oxidation. It remains to be investigated what changes in melanin structure are responsible for the switch from an anti- to pro-oxidant. It is tempting to suggest that RPE melanosomes may undergo degradation in vivo due to life-long exposure to light and oxidants and, as a result, lose their ability to protect the cells from oxidation catalyzed by metal ions or even become prooxidant and mediate metal ion-induced oxidation. This could have profound consequences for the retina where the impairment in iron transport and metabolism, and accumulation of excessive amounts of iron, is associated with several retinal degenerations [91, 92]. In the aging RPE, oxidative changes in melanin may increase due to formation of complex granules with lipofuscin, which can generate ROS upon excitation with

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Effect of photodegradation of human RPE melanosomes on oxygen uptake induced by Fe-ADP/ascorbate (0.05 mM/0.2 mM) in the absence (a) and the presence (b) of 10 mM linoleate (LA). To induce degradation, melanosomes were

Figure 7.11

b)

exposed to 0.95 or 5.54 kJ/cm2 of blue light delivered from a compact arc high-pressure mercury lamp equipped with broadband filter transmitting 390- to 490-nm light. (Modified from [80].)

blue light [3, 86]. Moreover, the complex granules of melanolipofuscin are 2.8- and 2.1-fold more susceptible to oxidation induced by iron ions than melanosomes or lipofuscin, respectively, isolated from the same donors (Figure 7.12). 7.7.3 Cytotoxic Properties of Aged RPE Melanin Granules and Their Potential Consequences for Retinal Aging and AMD

Deleterious effects of aged human melanosomes and melanolipofuscin have been demonstrated in the cultured RPE cell line ARPE-19 exposed to blue/green light (Figure 7.13) [49, 110]. The cells were enriched with bovine and human RPE melanosomes and human melanolipofuscin by feeding. Due to differences in the experimental conditions used in these two studies, the comparison of the effects of melanosomes and melanolipofuscin on susceptibility to light-induced toxicity can only be made indirectly. Exposure to light of cells enriched with bovine melanosomes led to a small decrease of 11% of mitochondrial activity. Human melanosomes isolated from donors aged 60–90 years induced death of about 44% of cells, whereas exposure of lipofuscin-laden cells under the same conditions led to death of about 70% cells. Exposure of melanolipofuscin-laden cells to blue/ green light induced death of about 58% of cells, whereas exposure of lipofuscinladen cells under the same conditions led to death of 80% cells. These data demonstrate that aged melanosomes and melanolipofuscin exhibit substantial phototoxicity. Considering that in elderly people melanolipofuscin is the predomi-

7.7 Pro-Oxidant Properties of Ocular Melanosomes

Figure 7.12 Comparison of the rates of oxygen uptake induced by Fe-ADP/ascorbate (0.05 mM/0.2 mM) in suspensions of bovine (BMs) and human (HMs) RPE melanosomes, melanolipofuscin (HMLf), and lipofuscin (HLf). (Modified from [80].)

Figure 7.13 Mitochondrial activity of ARPE-19 cells without (Control) and with internalized human RPE melanosomes (OHMs) or bovine melanosomes (BMs) as a

function of time of exposure to blue/green light (390–550 nm; 2.8 mW/cm2). Human RPE melanosomes were isolated from 60- to 90-year-old donors. (Modified from [110].)

nant pigment granule in the RPE, its properties with regard to interaction with light and redox-active metal ions require further investigation. The properties of RPE melanosomes and their changes that occur with age suggest that RPE melanosomes may play a role in retinal aging and diseases, such as AMD. Aging of the RPE is accompanied by partial loss of melanin, and accumulation of melanolipofuscin and iron. It can be suggested that with aging the antioxidant properties of RPE melanosomes become gradually outbalanced by their pro-oxidant properties. Due to these pro-oxidant properties RPE melanosomes may play a causal role in the impairment of iron homeostasis and the increase of oxidative stress in the RPE. There is a growing body of evidence

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pointing to the RPE as the primary site of damage leading to AMD. The characteristic features of AMD include increased accumulation of iron in the RPE and several markers of oxidative damage. Several products resulting from oxidative damage to constituents that accumulate in the retina can trigger a vicious cycle involving proinflammatory and proangiogenic pathways, which results in more oxidative stress in the retina and further damage. Thus, understanding the agerelated changes in structure and properties of RPE melanosomes may help to understand the initial pathways leading to RPE dysfunction followed by development of AMD.

7.8 Other Properties of Ocular Melanosomes and Their Implications

Apart from the anti- and pro-oxidant properties discussed above, ocular melanosomes exhibit several other characteristics that are important for the eye, starting from early embryogenesis. Proper maturation of melanosomes is required for normal development of the visual system [6]. Due to the congenital reduction or absence of melanin synthesis in the eye, not only does hypopigmentation of ocular structures occur, but the retina remains underdeveloped with an absent fovea and there are misguided neurons in the optic chiasma. The defects associated with ocular albinism manifest clinically as lack of acute vision, nystagmus, and strabismus. In addition to antimalarial drugs mentioned previously, there are a number of other xenobiotics with high affinity for binding to melanin, including drugs for rheumatoid arthritis, local anesthetics, aminoglycoside antibiotics, and phenothiazine-based drugs such as chlorpromazine used as antipsychotics [84, 111]. Moreover, some environmental pollutants including herbicides, dyes, and alkaloids also bind to melanin. Some types of bonds between melanin and xenobiotics are ionic and easily reversible, but irreversible binding was observed with chloroquine and chlorpromazine where autoradiographic study of [14C]chloroquine and [35S]chlorpromazine revealed that radioactivity in pigmented structures persisted for up to 90 days and 1 year for [14C]chloroquine and [35S]chlorpromazine, respectively. On one hand, binding of xenobiotics to melanin may be protective when the noxious chemicals are rapidly bound to melanin and slowly released at small nontoxic concentrations [112]. It may be particularly important for the retina where the RPE provides the blood–retina barrier so potentially toxic substances may become bound to RPE melanin and therefore disabled from reaching photoreceptors. Adverse effects of binding of drugs to melanin include accumulation of drugs in pigmented tissues, leading eventually to degenerative changes and toxicity such as those observed in the RPE. Binding of drugs to melanin can also affect ocular drug delivery [113]. Ocular melanosomes accumulate considerable amounts of metals such as calcium and zinc [84, 114, 115]. It has been suggested that binding of calcium to

7.9 Conclusions

melanin may play a role in calcium regulation and buffering, which seem to be particularly important for cell adhesion and for retinal photoreceptors with tightly regulated calcium levels. Interestingly, the concentration of calcium in ocular pigmented tissues is very high, exceeding even that of the bone where calcium is deposited in a mineral form [116–118]. X-ray microanalyses of elemental compositions of choroidal and RPE melanosomes from an 8-month-old rat, 2-year-old Cynomolgus monkey and 68-year-old human show that calcium accounts for 0.04 to 0.78% mol fraction. X-ray microanalyses of fixed sections of ocular tissues of various species have demonstrated that melanosomes of the ciliary body, iris, RPE, and choroid contain up to 2–10 times more calcium than adjacent nonpigmented cellular organelles [40, 118]. Choroidal melanosomes of the rat, rabbit, pig, and monkey contain up to 7-fold greater concentrations of calcium than RPE melanosomes [40]. RPE melanosomes contain cathepsin D, which is the main hydrolase responsible for the proteolysis of opsin from phagocytosed photoreceptor outer segments [48]. The presence of cathepsin D at the surface of RPE melanosomes and observations of fusion between RPE melanosomes and phagosomes suggest that melanosomes may be involved in the degradation of photoreceptor outer segments. Support for this hypothesis comes also from experiments on cultured RPE cells fed daily for up to 4 weeks with photoreceptor outer segments [119]. Nonpigmented RPE cells isolated from the albino rabbit and nonpigmented tapetal part of bovine fundus showed greater accumulation of nondigested photoreceptor outer segments than pigmented rabbit and bovine RPE cells. Absorption of light by the RPE and choroidal melanosomes and their IR fluorescence have been employed in the development of diagnostic tools for imaging of the retina–choroid complex [120]. Absorption of light together with an efficient thermal deactivation pathway in melanosomes have also been employed in therapeutic photocoagulation of ocular tissues [121].

7.9 Conclusions

Undoubtedly, ocular melanosomes play multiple roles in the eye. The most obvious and well documented is their role as a broadband optical filter that, in the case of the iris, can play a protective role by limiting light intensity reaching the retina, crystalline lens, as well as the nuclei of iridial melanocytes. A potential photoprotective role of RPE and choroidal melanosomes still awaits experimental evidence. Aging of the RPE melanosomes leads to an increase in their pro-oxidant properties which may counterbalance or even exceed their antioxidant properties. This may increase the oxidative stress in the aged RPE and contribute to agerelated retinal dysfunction and development of AMD. It remains to be determined at the molecular level what structural changes of the RPE melanin/melanosomes are responsible for the observed changes in their morphology and physicochemical properties.

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8 Biological Role of Neuromelanin in the Human Brain and Its Importance in Parkinson’s Disease Kay L. Double, Wakako Maruyama, Makoko Naoi, Manfred Gerlach, and Peter Riederer

8.1 What are Neuromelanins?

Neuromelanins are one of the two primary pigment molecules in the human brain, the other being the so-called “age pigment” lipofuscin. No formal criteria to define a neuromelanin have been agreed upon, but a “neuromelanin” is generally considered to consist of a dark-colored, highly insoluble intracellular granular pigment found in the brain. In the human brain, neuromelanin granules have an amorphic shape, ranging in size from 0.5 to 2.5 μm and, because of their characteristic dark brown color, can readily be seen both macroscopically and at the light microscope level without the need for histochemical staining or immunohistochemical visualization (Figures 8.1a and 8.5a). Unlike lipofuscin, which is readily fluorescent, neuromelanin does not immediate fluoresce on exposure to UV light, although the pigment may develop this characteristic upon extended UV exposure [1]. In the healthy human brain the pigment is abundant in the cytoplasm of neurons [2] (Figure 8.1a), although it may also extend into cellular processes. Neuromelanin does not form in glial cells, but may sometimes be found in these cell types in the postmortem human brain in neurological disorders where pigmented neurons degenerate, as a result of the phagocytosis of the pigment released from dying cells. Unlike other melanins, at the electron microscope level neuromelanin granules exhibit a unique structure consisting of three components of different electron density (Figure 8.1b): an electron-dense melanin polymer, a component of intermediate electron density, and an electron-lucent lipid component not found in peripheral melanins or other nonpigmented central nervous system cells [3]. Individual pigment granules are heterogeneous in size and also in the proportion of each of the three components that they contain (Figure 8.1b). Further, we have observed large bodies of the electron-dense component of neuromelanin within neurons of the substantia nigra, although the function of these bodies is unknown [3]. Although a double membrane has been reported to surround granules of a synthetic neuromelanin formed in an in vitro system [4], there is no clear evidence for a membrane surrounding native neuromelanin granules in vivo in the human Melanins and Melanosomes: Biosynthesis, Biogenesis, Physiological, and Pathological Functions, First Edition. Edited by Jan Borovanský and Patrick A. Riley. © 2011 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2011 by Wiley-VCH Verlag GmbH & Co. KGaA.

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8 Biological Role of Neuromelanin in the Human Brain and Its Importance in Parkinson’s Disease a)

b)

Figure 8.1 (a) Neuromelanin-pigmented neuron from the human substantia nigra. The pigment appears dark under the light microscope and fills much of the cytoplasm. N, nucleus. (b) Appearance of neuromelanin granules under the electron microscope. The

pigment consists of three components of differing electron density: ED, electron-dense component; EI, electron intermediate component; EL, electron-lucent component. (Reproduced from [3].)

8.3 Development and Metabolism of Neuromelanin

brain [3, 5], suggesting that, unlike most other melanins, neuromelanins may interact directly with other molecules present in the cytoplasm.

8.2 Phylogenetic Development of Neuromelanin

Although there is a lack of studies specifically addressing this question, the presence of neuromelanin in the brain appears to be phylogenetically related to brain development. Studies of the midbrains of common laboratory and agricultural animals demonstrate an absence of the pigment in the substantia nigrae of lower species, such as mice, rats, and sheep. The pigment begins to first appear in the brains of higher primate species, with small amounts of the pigment reported in the tyrosine hydroxylase (TH+)-positive neurons of the substantia nigra of primates, such as the baboon (Papio papio, Figure 8.2), although there have been no comprehensive comparative studies on the melanization of primate brains. The greatest quantify of the pigment, however, is clearly found in the human brain where the pigment fills 50% of the cytoplasm of TH+ neurons of the substantia nigra [2] (Figures 8.1 and 8.2), giving this brain region the characteristic dark appearance for which it is named (Figure 8.5a). Neuromelanin is also abundant in the neurons of the locus coeruleus, and is further found in the ventrolateral reticular formation and the nucleus of the solitary tract of the medulla oblongata (Figure 8.3). Neuromelanins appear to form from two of the three catecholamines – dopamine and norepinephrine, but not adrenalin [6, 7]. More recently, neuromelanins have been reported from other regions of the human brain such as the putamen, the premotor cortex, and the cerebellum [8]; however, as these brain regions do not exhibit dark pigments when viewed macro- or microscopically the nature of these putative neuromelanins is unknown.

8.3 Development and Metabolism of Neuromelanin

While the adult human midbrain contains the largest amounts of neuromelanin it is interesting that the development and subsequent appearance of the pigment is age-related. In fact, a dark neuromelanin is not present at all in the prenatal or infant human substantia nigra. Within the human substantia nigra, neuromelanin develops in three distinct stages [9]. The pigment first appears in these neurons at approximately 3 years of age as small, pale granules, and the size and number of the granules increases until age 20, when the neuronal cytoplasm contains the adult complement of neuromelanin granules [9] (Figure 8.4). After the age of 20 the proportion of the neuron occupied by neuromelanin remains unaltered [9], but the amount of pigment, quantified biochemically, increases with increasing age [10], suggesting pigment granules are more compact in the aging brain,

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8 Biological Role of Neuromelanin in the Human Brain and Its Importance in Parkinson’s Disease a)

b)

c)

8.3 Development and Metabolism of Neuromelanin Cresyl violet-stained sections from the human (a), baboon (b), and rat (c) substantia nigrae. Human dopamine neurons contain the greatest quantify of neuromela-

Figure 8.2

nin pigment, while those of the baboon contain a relatively small amount of pigment and these neurons are unpigmented in the rat. (Reproduced from [18].)

a)

b)

c) Within the human brain, three main regions contain neuromelaninproducing cells: the substantia nigra of the midbrain (a), the locus coeruleus within the pons (b), and the ventrolateral reticular formation and the nucleus of the solitary

Figure 8.3

tract in the medulla oblongata (c). Of these regions, only the substantia nigra (a) and the locus coeruleus (b) contain a large cluster of pigmented neurons that can be seen macroscopically as a darkened area. (Reproduced from [18].)

possibly accounting for the observed darkening of the pigment with age [9]. Interestingly, the second common pigment in the human brain, lipofuscin, is sometimes referred to as the “age pigment,” yet the characteristic changes observed for neuromelanin with aging suggests this term could equally be applied to neuromelanin [3]. While the visual features of neuromelanin development in the human substantia nigra have now been described in detail, the pathways regulating the biosynthesis of the pigment remain largely unknown. The majority of melanins in the body are regulated via an enzymatic process, yet an enzyme that regulates neuromelanin synthesis is yet to be identified. The enzyme tyrosinase that controls melaninogenesis from the substrate l-tyrosine in other human tissues is absent in the substantia nigra and, despite a number of studies investigating this question, no other enzymatic regulatory pathway has been identified to date [9, 11]. The lack of a known regulatory system has resulted in a generalized belief that neuromelanin forms from an uncontrolled process of autoxidation of the

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8 Biological Role of Neuromelanin in the Human Brain and Its Importance in Parkinson’s Disease a)

3 years

d)

31 years

b)

8 years

e)

74 years

c)

19 years

f)

94 years

100µm Figure 8.4 Intermediate and high (inset) magnification photomicrographs of representative unstained neuromelaninpigmented neurons from the ventral region of the substantia nigra of humans at various ages. Scale bars in (f) are equivalent for all micrographs. The first appearance of neuromelanin pigment is at 3 years of age

50µm

(a). The optical density of the neuromelanin pigment increases with age (a–f) while the average cellular volume occupied by neuromelanin pigment increases up to 20 years of age (a–c). After 20 the average cellular volume occupied by neuromelanin does not increase significantly (d–f). (Reproduced from [9].)

8.4 Structure of Neuromelanin

catecholamines on which they are based. If this were true, the pigment would be expected to be present in approximately equal quantities in all catecholaminergic neurons of a given type. This is not the case, however, as, for example, 95% of the TH+ neurons of the substantia nigra are pigmented, while in the neighboring ventral tegmental area only 50% of TH+ neurons are pigmented [2]. Further, individuals medically treated with large quantities of l-3,4-dihydroxyphenylalanine (l-dopa), the precursor to dopamine, would be expected to exhibit increased levels of pigment within dopaminergic neurons as a result of increased autoxidation, but this is not the case [2]. The idea that neuromelanogenesis is nevertheless a controlled process is supported by the observation that nonpigmented fetal dopaminergic cells implanted into the striatal region of the brain as a treatment for Parkinson’s disease exhibit adult levels of pigmentation in just 3 years [12], suggesting that some yet to be identified factor or factors present in the adult brain support this process. Similarly, little is known about possible catabolism pathways for neuromelanin. Unlike pigmented cells in the periphery that may have a high rate of division and replacement, pigmented neurons in the brain do not divide. There is no known catabolism pathway for neuromelanin and, given the highly insoluble nature of the pigment, it is generally assumed that catabolism of the pigment, once formed, does not occur. Peripheral melanins are suggested to be catabolized via oxidative degradation and, interestingly, neuromelanin can be degraded under highly oxidative conditions in vitro. Recent biophysical data obtained from the pigment ex vivo demonstrates age-associated changes in highly oxidized forms of sulfur-based compounds in the pigment, suggesting that oxidative degradation may modify the mature pigment in the healthy human brain [13]. In disorders characterized by the death of the pigmented neurons of the substantia nigra, such as Parkinson’s disease and related disorders and toxin-induced substantia nigra cell death, the pigment is released by the dying neurons and can be seen within glial cells, which are assumed to remove and degrade the pigment from the brain [14, 15]. Changes in the appearance of neuromelanin in the human substantia nigra have been reported with aging, but carefully designed morphological studies suggest that the number of pigmented dopaminergic neurons in this brain region does not decrease with increasing age [16]. Nevertheless neuromelanin pigment is reported to be more commonly observed inside glial cells in the aged, compared with the young, brain [17].

8.4 Structure of Neuromelanin

As described above, neuromelanins form in some, but not all, catecholaminesynthesizing neurons and these neurotransmitters are considered to be primary substrates for the synthesis of the pigment. Thus, the neuromelanin of the substantia nigra is considered to be a dopamine-based melanin, while those of the locus coeruleus, reticular formation, and medulla oblongata are considered

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norepinephrine-based melanins. To date, the chemical structures of the norepinephrine-based neuromelanins are unknown, primarily because of the small amount of these melanins present in the human brain. The dopamine-based melanin of the human substantia nigra is relatively more abundant, but is very limited in availability because it occurs almost exclusively in the human brain. Nevertheless, studies on both synthetic melanin pigments and on native human neuromelanin have illuminated some of the structural features of this pigment. Neuromelanin is considered to be a copolymer of black insoluble eumelanin and the brown alkali-soluble pheomelanin, and shares structural features of both of these melanin types [18], with a polymer backbone based primarily upon l-dopa with reduced dopamine and dopamine metabolites also incorporated into the polymer [19], as well as a variety of reduced and oxidized sulfur species [13]. Interestingly, in contrast to the rat brain, which contains only one isoform of TH, the rate-limiting enzyme for dopamine production, the human brain involves four differentially regulated isoforms of this enzyme [20, 21]. This suggests that cellular levels of dopamine, and its metabolites, may be more variable in the human midbrain. Unlike other melanins, neuromelanin contains a significant amount of associated lipids which make up 30% of the granule mass [22] (Figure 8.1b). The primary lipid associated with the melanin is the little-known isoprenoid dolichol with small amounts of other hydrophobic compounds such as cholesterol, ubiquinone, and ω-tocopherol also present [22, 23]. Amino acid analyses have demonstrated that a proteinaceous component comprises 5–15% of the pigment [19, 24]. In addition, proteomic analysis identified 72 proteins, some of which are associated with lysosomes and lysosome-related organelles [5, 25]. The apparent relative complexity of the structure of the pigment argues against the idea that the pigment is produced purely via a simple autoxidation pathway.

8.5 Biological Role of Neuromelanin in the Human Brain

For many years neuromelanin was considered to be an inert molecule with little or no physiological relevance, but more recent thinking suggests this view may not be correct. In tissues outside the central nervous system melanins are considered to have protective functions, the most obvious of which are the photoprotective functions of varying levels of melanin in the human skin, eye, and hair [18]. Neuromelanin may represent a cellular mechanism that evolved within catecholaminergic neurons to protect these cells from the potentially toxic metabolic products of these neurotransmitters. The enzymatic and autoxidation of dopamine, for example, produces highly oxidative quinone and semiquinone species that are suggested to be incorporated into the melanin polymer, effectively resulting in their inactivation [26–28]. Peripheral melanins have also been posited to be protective by virtue of their radical scavenging [29] and metal-binding properties [18, 30], and the free radical scavenging abilities of neuromelanins may represent an addi-

8.6 Is Neuromelanin Involved in Neurological Disease?

tional capability by which these molecules might benefit the cell. We have demonstrated reduced cell death in the presence of neuromelanin in primary rat mesencephalic cultures challenged with an oxidative stimulus [31], suggesting that the polymer can reduce oxidative damage. Interestingly, this protective effect is not seen for synthetic model dopamine pigment where the polymerization process may be incomplete [31]. A further role for neuromelanin appears to be binding a variety of potentially harmful exogenous compounds, such as pesticides and toxins [32–34]. In particular, its role as a metal binder has received attention. Neuromelanin binds a range of metals, including a range of potentially toxic cations [13, 35] and the concentrations of metals associated with the polymer increases with aging [13]. We have demonstrated high- and low-affinity binding sites for iron in neuromelanin [36], suggesting that the interaction of this physiologically important metal with neuromelanin is not random, but highly regulated. The substantia nigra is naturally rich in iron and, given the absence of the iron-binding protein ferritin in the dopaminergic neurons of the human substantia nigra, it is feasible that neuromelanin plays this role as an iron binder within these pigmented cells [37]. Supporting this notion, Mössbauer studies demonstrate many similarities between the physical properties of the iron core in ferritin and the iron clusters found in neuromelanin [38, 39]. Like the iron core in ferritin, neuromelanin binds ferric iron in oxyhydroxy clusters, although these clusters are smaller and less regular than that in ferritin [36]. Proteomic analysis of neuromelanin isolated from the human substantia nigra recently identified ferritin localized within these granules [40]. It has been suggested that the iron-binding capacity of neuromelanin in vivo is unsaturated [27], thus the pigment may maintain the capacity to bind iron. It is therefore feasible that the pigmented neurons employ this capacity to prevent iron-mediated free radical production. Experimental data using synthetic dopamine melanin (DA-M) supports this hypothesis in that under conditions of low iron, most iron ions are bound to the polymer and free radical production is low [41]. Such data support a protective role for neuromelanin in the healthy human brain. A further biological role of neuromelanin in the degradation pathway of proteins has been suggested from proteomic studies of neuromelanin granules isolated from human substantia nigra [5, 25]. These studies identified the lysosome integral membrane protein II, cathepsin B and D, and tripeptidyl-peptidase, proteins associated with lysosomes and lysosome-related organelles. It can therefore be hypothesized that, like lysosomes, neuromelanin granules may also play a role in degrading aggregated or misfolded proteins.

8.6 Is Neuromelanin Involved in Neurological Disease?

Despite some evidence for an active and protective role for neuromelanin in the normal brain, the pigment has also been linked with the etiology of the common

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8 Biological Role of Neuromelanin in the Human Brain and Its Importance in Parkinson’s Disease a)

b)

2cm c)

d)

1mm Figure 8.5 Neuromelanin pigmentation of the healthy human substantia nigra (a) and the characteristic pallor of this region in the Parkinson’s disease brain seen macroscopically (b). Loss of pigmented cells in the Parkinson’s disease substantia nigra at the microscopic level can be seen in (d),

compared with the healthy substantia nigra in (c). Associated with the death of many of the pigmented neurons in this region is the formation of Lewy bodies (insert in c) in some surviving neurons. (Figure provided with kind permission from Professor Glenda Halliday, Neuroscience Research Australia.)

neurodegenerative disorder Parkinson’s disease. Parkinson’s disease is characterized pathologically by the relatively selective and massive death of the pigmented neurons of the substantia nigra, giving this region a characteristic pale appearance, although the volume of this region is unchanged (Figure 8.5). Correlative analyses of the relative vulnerability of substantia nigra neurons in Parkinson’s disease compared with other pigmented regions of the human brain demonstrated a positive association between regional degree of pigmentation and cell loss in this disorder [42, 43]. Quantitative analyses of pigment volume and the relative vulnerability of pigmented neurons within the various nuclei that make up the substantia nigra, however, does not support the hypothesis that cell vulnerability in Parkinson’s disease is directly related to degree of pigmentation as the differentially vulnerable tiers of this nuclei contain that same amount of pigment [2]. This does not exclude the possibility of a role for the pigment in Parkinson’s

8.7 Effects of Neuromelanin In Vitro and In Vivo

disease. Given the lack of neuromelanin in most laboratory species and the small amount of the pigment in the human brain, particularly in Parkinson’s disease where the amount of pigment is dramatically reduced due to the death of these cells [10, 43], experimental investigations into this question in the human brain are limited. The small amount of available data, however, suggests that changes in the pigment occur in Parkinson’s disease. Pigmented neurons in the parkinsonian brain are reported to contain less pigment than those in the healthy brain [43, 44], but the optical density of the pigment is increased [2]. Biophysical analyzes of neuromelanin isolated from the Parkinson’s disease brain appears to contain a protease-resistant, lipoproteic material not seen in the healthy brain [30, 44] and pigment-associated cholesterol is also reduced [2]. α-Synuclein, a synaptic protein which forms Lewy bodies, abnormal inclusions found in the parkinsonian brain, is cross-linked to neuromelanin pigment from the parkinsonian brain [45]. Further, this protein aggregates specifically on the vulnerable pigmented cells of the substantia nigra in early disease, suggesting the pigment plays a role in neurodegenerative cascades in this disorder [2]. It has also been reported that the ability of neuromelanin to bind iron is reduced in Parkinson’s disease [46, 47]. This may be significant as iron levels have been reported to be increased in the substantia nigra in this disorder [48]. If the iron chelation ability of the pigment is reduced this could potentially result in increased levels of intraneuronal free iron and oxidative stress via the stimulation of free radical production, contributing to perhaps many factors that increase the vulnerability of these pigmented neurons [16].

8.7 Effects of Neuromelanin In Vitro and In Vivo

While factors other than melanization influence the vulnerability of an individual nigral neuron in Parkinson’s disease [16], pigmentation of these neurons nevertheless appears to play a significant role in their eventual fate. Thus, an understanding of the influence of this pigment is important in understanding the pathogenesis and progression of this disorder. In the remaining sections we review in vitro and in vivo studies investigating the functional effects of neuromelanin. 8.7.1 Mechanisms of Neuromelanin Cytotoxicity

In vitro studies suggest that neuromelanin can induce mitochondria-initiated cell death and inhibit the ubiquitin–proteasome system (UPS) [49–52]. In contrast, however, neuromelanin may also protect neurons by scavenging toxic free radicals, reactive ions, and toxic dopamine quinone, a metabolite of dopamine autoxidation, thus functioning as an antioxidant [53, 54]. These contrasting data suggest that neuromelanin can exert either a neuroprotective or neurotoxic influence, and that data from cellular and animal experiments with purified neuromelanin must be carefully interpreted with respect to the human brain.

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Table 8.1 Neurotoxicity of isolated neuromelanin in in vivo and in vitro models.

Melanin

Model system

In vivo experiments

Administered in:

Neuromelanin

Cytotoxicity

Mechanism of cytotoxicity

References

rat substantia nigra

cell loss of dopamine neurons

inflammation

[55]

rat substantia nigra (iron-laden neuromelanin)

loss of dopamine

iron-induced oxidative stress

[56]

rat substantia nigra

cell loss of dopamine neurons

glia-mediated inflammation

[57]

SH-SY5Y cells

cell death

activation of apoptotic signal

[52, 58]

SH-SY5Y cells

iron-dependent cell death

oxidative stress, UPS inhibition

[49–51]

SK-N-SH, rat mesencephalic cells

no toxicity

proinflammatory signaling

[31]

rat microglia

activation

oxidative stress

[59]

mesencephalic neurons

cell death

[60]

rat mesencephalic neurons

no toxicity

[61]

In vitro experiments Neuromelanin

A limited number of in vivo and in vitro experiments investigating the possible cytotoxicity of neuromelanin have been reported, and are summarized in Table 8.1. The data are contradictory, dependent on the model system, amount and type of neuromelanin used, and the measured endpoints. Studies injecting ironsaturated neuromelanin into the rat substantia nigra report a loss of 50% of dopamine neurons compared with controls – a smaller decrease in neuronal number than the 95% loss following injection of free Fe(III) alone [56, 62, 63]. In another study, injection of human neuromelanin into the rat substantia nigra and cerebral cortex induced a significant reduction of dopamine cells in the substantia nigra – an effect attributed to strong microglia activation [57]. Activation of microglia by neuromelanin has also been demonstrated in rat primary cultured microglial cells – an effect suggested to be mediated by p38-activated protein kinases [59]. These data may be pertinent for the marked microglial reaction associated with extraneuronal neuromelanin pigment deposits in the human parkinsonian

8.7 Effects of Neuromelanin In Vitro and In Vivo

substantia nigra. In in vitro systems, neuromelanin-mediated cell death appears to be dependent on cell type, as phagocytosis of both iron-saturated neuromelanin and synthetic DA-M stimulates significant cell death of neuron-like SK-N-SH cells, but not of glial-like U373 cells [31]. In contrast, phagocytosis of iron-depleted neuromelanin into SK-N-SH cells was not associated with cell death; indeed cell survival following an oxidative stimulus was increased [31]. These data suggest a neuroprotective function of neuromelanin via scavenging cytotoxic iron and reactive oxygen species (ROS) and reactive nitrogen species (RNS) under certain cellular conditions. In contrast, in human dopaminergic SH-SY5Y cells neuromelanin induces mitochondria-dependent apoptosis [52, 64]. Neuromelanin causes mitochondrial membrane permeabilization, permitting transmembrane movement of molecules up to around 1.5 kDa, release of cytochrome c into the cytosol, and activation of an apoptotic cascade, as shown in Figure 8.6. Interestingly, protease K treatment completely prevents induction of this apoptosis, and the cytotoxicity of synthetic DA-M and l-cysteinyl-DA-M (CysDA-M) is much lower than that of human neuromelanin, suggesting the protein component of neuromelanin may play a role in induction of apoptosis. 8.7.2 Neuromelanin Effects on Mitochondrial Function

In Parkinson’s disease, a defect of complex I (NADH : ubiquinone oxidoreductase, EC 1.6.5.3) in the mitochondrial respiratory chain has been reported [66–68]. Complex I is a macromolecule composed of 46 subunits, the activity of which is reduced by impaired subunit assembly. Neuromelanin disturbs the assembly of complex I, but protease K-treated neuromelanin or DA-M does not [52, 58]. Neuromelanin has accessible sulfhydryl groups within its proteinaceous component, the number of which is reduced by protease K treatment to one quarter, suggesting that peptide-bound sulfhydryl residues account for neuromelanin-induced mitochondrial dysfunction and apoptosis. Under physiological conditions or mild oxidative stress, ROS and RNS reversibly modify thiols in neuromelanin, protein, and reduced glutathione (GSH) into active intermediates, thiol radicals, sulfenic (RSOH), and sulfinic acid (RSO2H). Activated sulfhydryl groups in protein form mixed disulfide (S–S) bonds with those of GSH and other compounds in a reaction called “S-glutathionylation” (Pr-S-SG). Studies using synchrotron X-ray microscopy of human brain tissues have detect these oxidized reactive sulfur compounds in neuromelanin ex vivo [13]. Prolonged or intense exposure to ROS/RNS irreversibly oxidizes protein thiols into cysteine sulfonic acid. Sulfonate has been reported in neuromelanin in the healthy brain [69, 70], and also in the brains of patients with Parkinson’s disease and Alzheimer’s disease [4]. S-Glutathionylation is reversed by glutaredoxin, other thioredoxin, and protein disulfide isomerase (EC 5.3.4.1), yielding free protein sulfhydryl and GSH from Pr-S-SG. This reaction is recycled by thioredoxin reductase (EC 1.6.4.5) or GSH reductase (EC 1.6.4.2) using NADPH as a cofactor.

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8 Biological Role of Neuromelanin in the Human Brain and Its Importance in Parkinson’s Disease Activation by ROS-RNA NM-SH

a) Deglutathionylation of Complex I

Pr-SSG GSH

Pr-S-SG

Complex I

I II III IV V

I II III IV V

Pr-SH

Dissociation of Complex I, III

Inhibition of ATP synthesis

b) Inhibition of the UPS

Increased ROS-RNS Accumulation of modified proteins a-Synuclein fibril formation

Mitochondrial membrane permeabilization

Lewy body formation Caspase activation c) Induction of apoptosis Control

Figure 8.6 Mechanisms underlying the neurotoxicity of neuromelanin. In cellular models, neuromelanin deglutathionylates mitochondrial proteins and impairs assembly of complex I (a). Mitochondria were incubated in the presence of 10 μg/ml neuromelanin (III), or protease-K-treated neuromelanin (IV) or DA-M (V), or without melanin (II, control). Lane I represents molecular weight markers. After nonreducing sodium dodecyl sulfate–polyacrylamide gel electrophoresis, protein-bound SH residues were detected according to Brennan et al [65]. Complex I was stained with polyclonal antibody specific for complex I. Increased ROS/RNS and deglutathionylation in mitochondria-induced methylthiazoltetrazolium, and cytochrome c was released as a

+ NM

+ P-K treated NM

function of reaction time: I, II, III and V prior to, and 1, 2, 4 and 6 h after neuromelanin treatment. Caspase-3 was activated in neuromelanin-treated SH-SY5Y cells, but not in control and protease K-treated cells. UPS activity was simultaneously inhibited directly by the oxidative modification and indirectly by mitochondrial dysfunction. Inhibition of the 26S proteasome was visualized by accumulation of Green Fluorescent Protein in SH-SY5Y cells transfected with a proteasome sensor vector, pZsProSensor-1 (BD Biosciences) (b). Neuromelanin, but not protease K-treated neuromelanin, induced apoptosis, as shown by Hoechst 33342 cell staining (c). NM, neuromelanin; P-K, protease K.

S-Glutathionylation regulates cellular functions related to energy metabolism, cytoskeletons, signaling-modifying proteins (kinases, phosphatases), Ca2+ homeostasis, protein folding, and apoptosis cascades [71, 72]. S-Glutathionylation stabilizes the assembly of complex I and protects the 4Fe–4S cluster of the 51-kDa subunit [72]. A component of the mitochondrial permeability transition pore,

8.7 Effects of Neuromelanin In Vitro and In Vivo

adenine nucleotide transporter, is S-glutathionylated at Cys57 and the oxidation of this sulfhydryl group with nitric oxide induces mitochondrial membrane permeabilization [73]. In conclusion, glutathionylation appears to be an important mechanism related to neuromelanin function [52, 58]. 8.7.3 Neuromelanin Effects on the UPS

Lewy bodies and Lewy neurites have been suggested to contribute mechanistically to neuronal degeneration in Parkinson’s disease and other synucleinopathy disorders [74, 75], although more recently these inclusions have been considered to be protective [76]. Oxidatively modified proteins are conjugated with multiple ubiquitin molecules and the tagged protein is degraded by the 26S proteasome complex [77]. UPS dysfunction has been implicated in both familial [78] and sporadic [79] Parkinson’s disease. Interestingly, neuromelanin inhibits the in situ activity of the 26S proteasome [49], increases ROS/RNS production in mitochondria, reduces mitochondrial ATP synthesis, and inhibits the ATP-dependent function of UPS [80, 81]. Increased ROS/RNS also facilitates modification of proteins, resulting in proteins that can also inhibit the activity of the UPS [49, 51]. 8.7.4 Comparison of the Cytotoxicity of Neuromelanin with Synthetic DA-M

Synthetic DA-M and CysDA-M have both been employed as models of native neuromelanin, but their composition and biological functions are quite different. Proteins and lipids form major components of the structure of neuromelanin [5, 19, 23, 24], while the melanin component of the pigment is a mixture of indolebased eumelanin and benzothiazine-based pheomelanin, with different oxidation potentials [82, 83]. As summarized in Table 8.2, DA-M consistently induces cell death in animal and cellular models [52, 58, 60, 84–86], primarily via induction of “apoptosis-like” cell death in which ROS/RNS appear to play a decisive role. As summarized in Table 8.3, we have recently studied the cellular mechanisms of apoptosis induced by DA-M compared with that reported for neuromelanin [58]. DA-M oxidatively decreases GSH and sulfhydryl content in mitochondria, whereas neuromelanin increases GSH by dissociation of mixed disulfide bonds in complex I. Neuromelanin and DA-M induce mitochondrial membrane permeabilization, and cytochrome c release, whereas Cys-DAM does not. DA-M does not induce significant apoptosis, but thiol-targeting reducing reagents, such as GSH, dithiothreitol, and N-acetyl-cysteine, markedly enhance the cytotoxicity. In contrast, neuromelanin directly activates a mitochondria-initiated apoptotic cascade, which is suppressed completely by GSH. In DA-M-treated cells, reducing sulfhydryl reagents activate caspase-3, as shown by detection of activated caspase-3 with a molecular mass of 18 kDa. These results suggest that the pathways leading to cell death initiated by neuromelanin and DA-M differ, depending on

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8 Biological Role of Neuromelanin in the Human Brain and Its Importance in Parkinson’s Disease Table 8.2 Neurotoxicity of synthetic DA-M in in vitro models.

Cell system

Cytotoxicity

Mechanism of cytotoxicity

References

SK-N-SH, rat mesencephalic cells

cell death

ROS-mediated apoptosis?

[31]

Rat mesencephalic neurons

cell death

oxidative stress

[52]

SH-SY5Y cells

GSH-dependent apoptosis

mitochondrial membrane permeabilization, GSH-dependent caspase activation

[64]

Rat substantia nigra coculture

cell death

iron-induced oxidative stress

[84]

PC12 cells

apoptosis-like cell death

iron?

[85]

Mouse mesencephalic dopamine cells

apoptosis

[86]

Table 8.3 Comparison of the effects of neuromelanin and DA-M in vitro and in vivo.

Function

Neuromelanin

DA-M

Induction of apoptosis

yes

no; requires reducing sulfhydryl

Activation of apoptotic cascade in mitochondria

Reference

[52, 58, 64]

Induction of mitochondrial membrane permeabilization

yes

yes

Release of cytochrome c

yes

yes

Reduction of membrane potential (DYm)

yes

yes

Reduced ATP synthesis

yes

yes

Increased ROS/RNS production

yes

yes

Caspase-3 activation

yes

no; requires GSH

Protection by Bcl-2

yes

no

S-Glutathionylation, S-deglutathionylation

in mitochondria

no

Redox state in mitochondria

to reduced state

to oxidized state

Redox state in cytoplasm

no significant effect

to oxidative state

inhibition of 26S

no significant effect

[52, 64]

Effects on redox state

Effects on the UPS

[52, 58]

[49–51]

References

the differential regulation of the redox state in the cytoplasm and mitochondria [58, 64]. In summary, neuromelanin primarily perturbs S-glutathionylation in mitochondria and inhibits this function as the initial event in the cell death pathway, whereas DA-M oxidatively modifies proteins in subcellular components, especially mitochondria and the UPS. These results strongly suggest cytotoxicity mediated by neuromelanin is attributable not only to its melanin composition, but also to the proteinaceous and other components of this molecule.

8.8 Conclusions

Despite the advances of recent years, neuromelanins represent an enigmatic member of the pigment family, about which much still remains to be discovered. Nevertheless, it is clear that these pigments are not simply an inert waste product of neuronal metabolism as previously believed, but play important roles in neuronal function in the human brain. Neuromelanin appears to protect neurons under physiological conditions, but it may also promote cellular toxicity in certain degenerative conditions. Specifically, the neuromelanin of the substantia nigra appears to be involved in a number of pathways accepted to be involved in neurodegenerative cascades in Parkinson’s disease and related disorders. Characterization of the role of neuromelanin in the healthy and diseased brain will improve our understanding of this interesting pigment and our understanding of the etiology of a number of common neurodegenerative disorders.

Acknowledgments

K.L.D. holds a Research Fellowship from the National Health and Medical Research Council of Australia. This work was supported by the National Health and Medical Research Council of Australia, and a Grant-in-Aid for Scientific Research (C) of the Japanese Ministry of Education, Culture and Science and a Grant-in-Aid for Clinical Research Promotion of the Japanese Ministry of Welfare and Health (W. M.). K.L.D. and W.M. contributed equally to this work.

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redistributes to neuromelanin lipid in the substantia nigra early in Parkinson’s disease. Brain, 128, 2654–2664. 3 Double, K.L., Dedov, V.N., Fedorow, H., Kettle, E., Halliday, G., Garner, B., and Brunk, U.T. (2008) The comparative biology of neuromelanin and lipofuscin in the human brain. Cell. Mol. Life Sci., 65, 1669–1682.

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9 Biogenesis of Melanosomes Cédric Delevoye, Francesca Giordano, Michael S. Marks, and Graça Raposo

9.1 Introduction

Melanin pigments are synthesized within epidermal and ocular melanocytes and the retinal pigment epithelium (RPE) in specialized organelles called melanosomes. Synthesis and sequestration of melanin in membrane-enclosed compartments within these cells prevents exposure of most cellular components to oxidative reactants that are produced during synthesis and that would otherwise be deleterious. Melanins produced in melanocytes in the skin are transferred to keratinocytes, providing photoprotection against ionizing UV radiation, but also resulting in the characteristic pigmentation of skin and hair, and the very different visual cues in the animal kingdom [1]. In the eye, melanins synthesized within melanosomes of RPE cells are not released, but rather are retained within these cells where they function in focusing light and in detoxifying free radicals liberated from phagocytosed photoreceptor outer membrane segments [2]. Melanosomes are also found in the eye in melanocytes within the choroid and in the pigmented epithelium of the iris and ciliary body. The timing of melanin synthesis and thereby also of melanosome production differs in skin melanocytes and in the choroid and RPE. Whereas in the skin melanosomes are produced throughout life, in the RPE the majority of melanin synthesis occurs during embryonic and early postnatal life [3]. To accomplish their role as melanin factories within the pigment cell, melanosomes have unique morphological and compositional features. They are membrane-bound organelles that bear melanocyte-specific proteins involved in melanosome structure, melanin synthesis, and in maintenance of the most favorable ionic, redox, and osmotic environment for pigment synthesis. Melanosomes share a number of properties associated with conventional lysosomes, the major degradative compartment of the cell, and are therefore classified as members of a family of so-called lysosome-related organelles (LROs). Melanosomes have a low luminal pH, and contain lysosomal hydrolases and lysosomal membrane proteins (reviewed in [4]). Although melanocytes can make both brown/black eumelanin

Melanins and Melanosomes: Biosynthesis, Biogenesis, Physiological, and Pathological Functions, First Edition. Edited by Jan Borovanský and Patrick A. Riley. © 2011 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2011 by Wiley-VCH Verlag GmbH & Co. KGaA.

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and yellow/red pheomelanin, melanosomes that make predominantly pheomelanins differ in structure and composition, and much less is known about their biogenesis. For this reason this chapter focuses on melanocytes that make predominantly eumelanosomes. The biogenesis of eumelanosomes is a multistep process in which an immature unpigmented precursor organelle forms and then subsequently matures to a fully pigmented structure. Such maturation requires morphogenetic and structural modifications of endosomal intermediates accompanied by interorganellar transport of protein cargos. These cargoes largely consist of melanocyte-specific components required for melanosome structure and pigment synthesis, including melanogenic enzymes, transporters, and structural components. Mutations in the genes encoding many of these components result in oculocutaneous albinism (OCA) or ocular albinism (OA) due to a failure in melanosome maturation/ function. Melanosome maturation is also accompanied by the recruitment of effector proteins necessary for the appropriate transport of melanosomes to the cell periphery (as discussed in Chapter 10). Over the past few years, studies on skin melanocytes and pigmented melanoma cells have started to unravel the major pathways and molecular mechanisms used by melanocytes to generate eumelanosomes. These studies have highlighted how eumelanosomes become segregated from the main “conventional” endolysosomal system [5–7]. Our emerging understanding of melanosome biogenesis serves as an excellent model that likely applies to the formation of other LROs in different cell types. Like all other membrane and lumenal proteins of the endocytic system, melanosomal proteins are synthesized in the endoplasmic reticulum (ER) and processed during secretory traffic through the Golgi complex. From the Golgi, these cargoes are targeted to melanosomes via specialized post-Golgi trafficking routes that converge upon the endosomal system. The endosomal pathways through which these proteins traffic are only beginning to be understood, and are regulated by both ubiquitous and cell-type-specific effectors. Ubiquitous trafficking effectors include the heterotetrameric adaptors that recognize specific cytoplasmic targeting signals on cargo proteins, small GTPases of the Rab superfamily that recruit effectors from the cytoplasm to endosomal intermediates, SNARE (soluble Nethylmaleimide-sensitive factor attachment protein receptor) proteins that specify membrane fusion, and cytoskeleton-associated molecular motors (as detailed in Section 9.4). Importantly, the genes encoding some components of these and other requisite molecular machineries bear mutations in syndromic genetic diseases that are characterized in part by hypopigmentation due to melanosome dysfunction. Among these diseases are the Chediak–Higashi syndrome (CHS) and several variants of the Hermansky–Pudlak syndrome (HPS) [8]. A number of findings over the last 10 years have begun to unravel how pigment cells integrate unique and ubiquitous molecular mechanisms in specializing the endosomal system to generate cell-type-specific structures and their associated functions. These studies have shed light not only on the biogenesis of melanosomes and other LROs, but also on general aspects of vesicular transport in the endocytic system and major dysfunctions in genetic diseases.

9.2 Melanosomes: Intracellular Organelles Specialized in Melanin Synthesis

9.2 Melanosomes: Intracellular Organelles Specialized in Melanin Synthesis 9.2.1 Melanosomes Are Unique Organelles That Develop through Different Stages

As melanins are highly electron-dense, melanosomes within epidermal melanocytes were among the first organelles to be well characterized by electron microscopy [9]. Eumelanosomes – containing black and brown melanins – can be divided into four stages, termed I–IV, based on their morphology [10] (Figure 9.1). The earliest stages – also often referred to as premelanosomes – lack pigment, but are characterized by the formation of a fibrillar matrix upon which melanins are deposited in later stages. Stage I melanosomes, which are accessible to endocytic tracers and correspond to an early endosomal compartment [5], contain a few

Ultrastructure of melanosomes. Electron microscopy of MNT-1 human melanoma cells fixed by high-pressure freezing before cryosubstitution and embedding in Epon. The four stages of melanosome development are shown in the lower panels. Note the dense bilayered coat (arrowhead) and intralumenal vesicles

Figure 9.1

(arrow) of stage I melanosomes, the proteinaceous fibrils (arrow) of stage II, and the melanin deposition (black) in stages III and IV. The main panel shows a typical field of MNT-1 cytoplasm near the nucleus, which contains all four stages of melanosomes. m, mitochondria; N, nucleus; GA, Golgi apparatus. Scale bars: 200 nm.

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irregular fibrils and intralumenal membrane vesicles. Interestingly, the fibrils emanate from the intralumenal membranes and elongate as the organelle matures [11]. Stage II melanosomes are characterized by fully matured parallel arrays of fibrils organized into concentric sheets [10, 11]. Upon initiation of melanin synthesis, melanin polymers deposit on these fibrillar sheets, resulting in their thickening and blackening as observed by electron microscopy [5, 10]. Continued melanin synthesis results in melanin deposition throughout the melanosome and masking of the underlying contents. Pigmented eumelanosomes in which fibrillar sheets are still apparent are referred to as stage III melanosomes, whereas those in which the underlying fibrillar matrix is masked are considered stage IV [10]. In cells that make predominantly pheomelanins, the melanin content of melanosomes is dark and the underlying fibrillar structure is absent [12]. The formation of melanosomes in eye pigment cells has been much less studied than in skin melanocytes. Genetic analyses in mice with coat-color dilution and in OCA patients (reviewed in [13]), as well as a few limited studies of melanogenesis in early eye development, suggest that the mechanisms underlying melanosome biogenesis in eye pigment cells is similar to that in skin melanocytes. In the RPE and choroid, melanosomes also appear to develop progressively from an immature unpigmented precursor to a mature pigmented melanosome [14]. However, melanogenesis in the RPE occurs predominantly before birth and is largely completed soon after birth, such that there is very little melanin synthesis and melanosome renewal in the adult [15, 16]. Melanogenesis in adult RPE can be induced under certain experimental conditions [17, 18] and can occur in structures without the characteristic striated precursors [19], but it is not clear whether this can occur physiologically. How are the different stage melanosomes structures generated? Both the formation of the underlying fibrillar matrix in stage I and II melanosomes and melanin synthesis/deposition in stage III and IV melanosomes are dependent upon pigment cell-specific proteins that are specifically targeted to newly forming organelles. While melanosomes were once considered modified lysosomes because of their content of lysosomal membrane proteins and enzymes [4, 20–23], they are now appreciated to coexist with lysosomes and other organelles of the late endocytic pathway [5]. As described in detail below, stage I and II melanosomes develop from vacuolar domains of early endosomal compartments that are enriched in the pigment cell-specific protein, Pmel17 (also known as gp100 or SILV). Current models suggest that stage II melanosomes must be fully “matured” – including fully formed fibrillar sheets – before they become competent to synthesize melanins. Melanin synthesis to generate stage III and IV melanosomes coincides with the delivery to the mature stage II compartments of melanogenic enzymes and transporters that shape the environment of the organelle to favor melanin synthesis (reviewed in [7]). Below we describe these components and our current understanding of these trafficking pathways.

9.2 Melanosomes: Intracellular Organelles Specialized in Melanin Synthesis

9.2.2 Melanosomal Components

Most known melanosomal components are integral membrane proteins that are uniquely expressed in pigment cells. Mutations in the genes that encode many of these proteins cause coat-color dilution in animals or albinism in humans – characterized by variable loss of pigmentation in the skin, hair, and/or eyes due to an impairment of the number, structure, and/or function of melanosomes [24] (Table 9.1). Table 9.1

Melanosome main components and their functions.

Melanosomal proteins

Melanosome stages

Function

Human disease/ mouse model

Tyrosinase

III/IV

melanin synthesis

OCA1 (h)/TyrC (albino)

oxidation of tyrosinase to dopa Tyrp1

III/IV

melanin synthesis oxidation of DHICA to eumelanin

Tyrp2/Dct

III/IV

melanin synthesis tautomerization of dopachrome to DHICA

OCA3 (h)/Tyrp1b (brown) unknown/Dctslt (slaty)

Pmel17/ gp100/ME20

I–II (epitopes masked by melanin in III and IV)

component of the amyloid fibrillar sheets

unknown/si (silver)

P/OCA2

III/IV

melanosome acidification

OCA2 (h)/p (pink-eyed dilute)

putative anion transporter MATP/ SLC45A2

unknown

putative membrane transporter

OCA4 (h)

SLC24A5

unknown

putative membrane transporter

unknown/OA

OA1/ GPR143

II–III–IV–lysosomes (bulk is in II)

GPCR

OA1/Oa1

MART-1

I–II

accessory protein for Pmel17 and OA1 function

control of melanosome composition and size unknown

human melanoma antigen OCA1, OCA2, OCA3, OCA4: oculocutaneous albinism type 1, 2, 3, and 4, respectively; OA1: ocular albinism type 1.

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Pmel17 serves as the structural foundation of the fibrils in stage I and II melanosomes. Pmel17 is synthesized as a transmembrane protein, but is proteolytically processed to smaller lumenal fragments that polymerize to form the intralumenal fibrillar sheets (reviewed in [25]). The mechanisms regulating Pmel17 maturation are discussed below. The accumulation of Pmel17 fibrils is the only currently known defining factor for stage II melanosomes, although one study has suggested that autophagy markers, such as lipidated LC3a, are also present within these organelles [26], suggesting that premelanogenesis may require the same molecular machinery used by the autophagy pathway. Consistent with the onset of melanin synthesis in the transition from stage II melanosomes, the melanin biosynthetic enzymes tyrosinase, tyrosinase-related protein 1 (Tyrp1) and dopachrome tautomerase (Dct; also known as Tyrp2) are enriched in stage III and IV melanosomes [5, 27, 28]. Tyrosinase is a copperdependent enzyme that catalyzes the limiting steps in melanin synthesis, ltyrosine hydroxylation to l-3,4-dihydroxyphenylalanine (l-dopa) and l-dopa oxidation to dopaquinone [29]; tyrosinase also catalyzes the downstream oxidation of 5,6-dihydroxyindole (DHI) to 5,6-indolequinone [30]. Patients with OCA type 1 have mutations in the gene encoding tyrosinase, and most often suffer from a complete loss of pigmentation in the skin and eyes [31]. As the name suggests, Dct catalyzes the tautomerization of dopachrome to DHI [32]. The murine Tyrp1 homolog has been suggested to have DHI-2-carboxylic acid (DHICA) oxidase activity [33], but the enzyme activity of the human Tyrp1 homolog has been controversial [34]; Tyrp1 has also been suggested to stabilize tyrosinase [35]. OCA type 3 results from inactivating mutations in the gene encoding TYRP1 in humans, and is characterized by accumulation of red and brown pigments [34]. Tyrosinase, Dct and Tyrp1 are type I membrane glycoproteins that share around 40% amino acid homology [36]. All three enzymes contain an N-terminal signal sequence that targets the nascent protein to the ER, a large intralumenal domain that bears the catalytic activity, a single membrane-spanning domain, and a short C-terminal cytoplasmic domain. The cytoplasmic domains of tyrosinase and Tyrp1 contain signals that are crucial for correct targeting to the melanosome [37–41]. In addition to the melanogenic enzymes, a number of other proteins required for melanogenesis are thought to be associated with melanosomes. Proteomic analyses of melanosome-enriched subcellular fractions from melanocytic cells have identified numerous proteins associated with the organelle [42, 43], but only a few of the “hits” have been verified by more rigorous analyzes. Among them are the putative channel protein, OCA2 [44], and the G protein-coupled receptor (GPCR) GPR143, commonly called OA1 [45]. These proteins are the products of genes that are targeted by mutation in patients with OCA type 2 and OA type 1, respectively. The molecular function of OCA2, also called the P protein because it is mutated in the pink-eyed dilute mouse [46], is unknown, but melanosomes in OCA2-deficient melanocytes are severely hypopigmented [47, 48] and are depleted of tyrosinase [49, 50]. OCA2 is also thought to regulate melanosomal pH [51], perhaps by facilitating counteranion transport [52–54]. OA1 belongs to

9.2 Melanosomes: Intracellular Organelles Specialized in Melanin Synthesis

the large family of GPCRs with seven-transmembrane spanning domains [45, 55]. Its ligand has been recently suggested to be the melanin intermediate l-dopa [56]. OA1 has been proposed to control fusion and fission events with transport intermediates [57] as detailed in Section 9.3.4. Additional melanocyte-specific components that are likely residents of melanosomes include the potassiumdependent sodium/calcium exchanger SLC24A5 [58] and the putative transporter SLC45A2 [59]. Genetic variants of SLC24A5 are associated with skin color variation in humans [60] and mutations in SLC45A2 (also called membraneassociated transporter protein (MATP) or underwhite in mice) result in OCA type 4 [61]. Both were identified in melanosome-enriched fractions by proteomics analysis, but their predominant localization and molecular function are not yet clear. Mutations in the calcium channels TRPM1 [62] and TRPM7 [63] result in defects in melanogenesis, and thus these are also likely associated with melanosomes. Finally, the MART-1 tumor-associated antigen has been shown to interact with other melanosomal proteins, such as Pmel17 [64] and OA1 [57], and may play an important role as a chaperone in their trafficking within the melanocyte. The identity and function of many of these components have been reviewed in detail elsewhere, and their deficiencies result in pigmentation disorders in humans (ocular or oculocutaneous albinism) and/or in model organisms (Table 9.1). Melanosomes also harbor proteins that are ubiquitously expressed and that likely play a role in melanogenesis. Most of these components, however, are not necessarily enriched only in melanosomes but may be present in other cellular compartments as well. Among these are lysosomal enzymes such as acid phosphatase and cathepsins [20, 23] and lysosomal membrane proteins such as lysosomal membrane proteins (LAMP) 1 and 2 (reviewed in [4]). Evidence from proteomics analyzes suggest that other lysosomal proteins and components of the ER might also be present in different stage melanosomes [27, 42, 43], but these findings warrant verification by other means. One component that plays an important role in melanosomes is the copper transporter, ATP7A [65]. ATP7A is a member of the ABC family transporters that functions as an ATP-dependent pump to load copper into secretory/endocytic organelles from the cytosol [66]. Whereas in all cells ATP7A is localized predominantly to the trans-Golgi network (TGN) and endosomes, a cohort of ATP7A in melanocytes is additionally present within melanosomes, where it provides the copper cofactor required for tyrosinase activity; a failure to localize ATP7A to melanosomes results in severe hypopigmentation due to a loss of tyrosinase activity [65]. Genetic deficiencies in ATP7A result in Menkes disease, which is associated with severe neurological and developmental abnormalities [66]; mouse models of Menkes disease with mutations in ATP7A include the mottled and brindled mice, which were first noticed for their effects on pigmentation [13, 67].

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9.3 Endocytic System and Formation of Melanosomes 9.3.1 Organelles of the Endocytic Pathway

In most cells the endocytic pathway serves housekeeping functions such as nutrient uptake, control of signaling pathways, and macromolecule degradation [68]. These functions are carried out by exploiting distinct membrane delimited compartments and organized subdomains that are highly dynamic [69, 70]. Proteins and other macromolecules that are internalized at the plasma membrane by receptor-mediated clathrin-dependent and -independent pathways or bulk-flow endocytosis [71] reach distinct compartments that are classified as “early” and “late” endosomes (Figure 9.2). This classification is based on the sequential accessibility of these different compartments to free endocytic tracers and to ligands bound to internalized cell surface receptors. Endocytic tracers access early endosomes within 5–15 min following internalization. They consist of different domains defined, morphologically, by the relative enrichment in specific effectors and by the kinetics of trafficking of ligands bound

Coated pit

Early recycling endosome

Early sorting endosome

Late endosome/MVB

Lysosome

TGN Golgi Apparatus Endoplasmic Reticulum

Figure 9.2 Endocytic pathway. Schematic representation of organelles of the endosomal system, emphasizing the bestcharacterized compartments found in all cell types. Arrows represent proposed directions of protein and lipid transport between organelles. The clathrin-containing coat of the plasma membrane invagination and the

coat at the cytosolic side of the early sorting endosome (coated endosome) is represented by gray shading. Intralumenal vesicles present in early sorting endosomes and late endosomes are represented by small circles; irregularly shaped membranes and vesicles are generally present in lysosomes.

9.3 Endocytic System and Formation of Melanosomes

to internalized receptors such as those for transferrin (Tf), low-density lipoprotein (LDL), epidermal growth factor (EGF), and asialoglycoprotein (ASGP) [72]. Sorting endosomes consist of tubules that are continuous with closely apposed electronlucent vacuoles with few internal membranes, and are accessed by endocytic cargo within 5–15 min [68]. These membranes are generally associated with the small GTPase Rab5, the Rab5 effector early endosomal antigen 1 (EEA1), and the phosphorylated phosphoinositide phosphatidylinositol-3-phosphate [73]. The tubules transport cargoes into and out of the vacuoles, including receptors that rapidly recycle to the plasma membrane (e.g., Tf receptor). A proportion of such recycling receptors accumulate in and recycle from perinuclear, tubulovesicular elements that are apposed to the Golgi apparatus and are called recycling endosomes [74]. The small GTPases Rab4 and Rab11 are generally involved in the maintenance of these recycling domains, as do many other Rab GTPases (such as Rab14 and Rab22A) and their effectors (for review, see [75]), and they can accumulate recycling cargoes for up to 30 min after internalization [68]. The tubules that emanate from the vacuolar domains of early endosomes are thought to remove membranebound cargoes destined for recycling to the plasma membrane or to other secretory and endosomal compartments, while the vacuolar domains – which mature into late endosomes as these materials are removed – become enriched in soluble cargoes that are destined for degradation in lysosomes. Another sorting event that occurs from the vacuolar domains of early endosomes is the segregation of integral membrane cargoes destined for the lysosomal pathway. This sorting event is mediated by the inclusion of these cargoes on internal membrane vesicles that form by inward budding of the endosomal limiting membrane [76]. Sorting occurs from regions of the vacuolar domain limiting membrane that are adjacent to coated areas on the cytosolic face of the endosome [77]. These bilayered coats are particularly abundant in melanocytic cells [5]. This coat contains clathrin, but is distinct from clathrin coats on buds at the plasma membrane, TGN, and tubular endosomes. The coat also contains components of the so-called ESCRT (endosomal sorting complex required for transport) machinery [78]. Four ESCRT multisubunit complexes are sequentially recruited to the endosomal membrane for the sequestration of membrane proteins that are covalently modified by ubiquitin and for the formation and severing of the internal vesicles from membranes enriched in these cargoes [79]. As the vacuolar endosomes mature, they continue to accumulate internal vesicles; such maturing endosomes are called multivesicular bodies (MVBs) [76]. Fully “matured” MVBs, which typically lack associated membrane tubules, are equivalent to late endosomes. They contain LAMP1 and 2, newly synthesized lysosomal enzymes, and are accessed by fluid-phase endocytic cargoes and ligand–receptor complexes within 30 min [80]. The biogenesis of MVBs and mechanisms involved in the formation of the intraluminal vesicles and sorting of cargo have been particularly well studied in yeast [76] and are largely conserved in mammalian cells during the attenuation of ligand-stimulated receptors such as the EGF receptor (EGFR) [78]. Not all MVBs are similar [81], and the mechanisms involved in the formation of distinct classes of MVBs are likely to be more complex and diverse then

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originally thought. Moreover, not all MVBs are destined for fusion with lysosomes (for review, see [82]). In particular, specialized cells like melanocytes harbor an heterogeneous set of compartments with morphological hallmarks of MVBs that harbor melanosomal proteins (e.g., Pmel17) and that serve as intermediates in melanosome formation. The internal vesicles of these structures appear to form in an ESCRT-independent manner (see Section 9.3.5.3). The effectors involved in cargo sorting and the formation of these intraluminal vesicles are not yet known. While this description of the endocytic system incorporates studies from several laboratories in many cell types, one should keep in mind that endosomal membranes are highly dynamic and constantly remodeling. Distinct endosomal domains vary in prevalence or content based on cell type, differentiation state, or response to extracellular signals. As detailed below, specialized cells – including melanocytes – subcompartmentalize the ubiquitous endosomal system to facilitate regulated cargo sorting to generate cell-type-specific organelles – including melanosomes. 9.3.2 Melanosomes Are LROs but Are Distinct from Lysosomes

The unique morphology and composition of melanosomes pose a challenge for organellogenesis within melanocytes and other pigment cells. To form melanosomes, these cells must either modify a ubiquitous organelle or develop a novel mechanism for sorting specific resident proteins from ubiquitous organelles. Several observations from early immunocytochemical and subcellular fractionation studies had suggested common features for melanosomes and late endosomes/ lysosomes. These include the presence of lysosomal hydrolases and integral membrane proteins, apparent fusion with phagocytosed particles, an acidic pH (for review, see [4]) and the presence of internal vesicles like those in MVBs [83, 84]. Moreover, like lysosomal proteins in other cell types, tyrosinase activity was detected in clathrin-coated vesicles near the TGN in melanocytes [20, 85], and tyrosinase and Tyrp1 localize to late endosomes and lysosomes when expressed ectopically in transfected nonpigment cells [38, 39, 41, 86]. These features of melanosomes led to their classification as LROs, a family of organelles that also include cytolytic granules of cytotoxic T cells, platelet α and dense granules of megakaryocytes, basophilic cell granules of mast cells, major histocompatibility complex (MHC) class II compartments in antigen-presenting cells, lamellar bodies in lung type II epithelial cells, Weibel–Palade Bodies in endothelial cells, and others [87]. Melanosomes, like other LROs, were once considered as transformed lysosomes that act as dual-functional organelles, carrying out both specialized and universal functions [4]. However, it is now clear that all LROs do not necessarily share common biogenetic pathways. While some LROs (such as perhaps cytolytic granules [88]) appear to be modified lysosomes, others (including melanosomes, platelet dense granules, and lamellar bodies) coexist with bona fide late endosomes/

9.3 Endocytic System and Formation of Melanosomes

lysosomes and thus must derive separately from other endocytic and biosynthetic organelles. This distinction explains why LROs are not uniformly disrupted in genetic disorders such as HPS (see Section 9.4) [89]. Evidence that melanosomes coexist with conventional lysosomes – and thus likely represent a separate organelle lineage – was supported by early histochemical observations. Seiji et al. reported that while lysosomal acid phosphatase activity was detected histochemically in melanosomes of melanoma cells, nonmelanosomal structures similar to lysosomes contained higher activity [21]. Moreover, studies from Boissy et al. showed that acid phosphatase activity was absent from melanosomes and premelanosomes of untransformed skin melanocytes [90]. These early observations were extended by studies from our group in skin melanocytic cell models using quantitative immunoelectron microscopy [5]. These analyzes showed that only about 10–20% of cellular LAMP1 and cathepsin D was present in melanosomes, whereas the vast majority of these proteins was detected in separate structures with morphological and temporal characteristics of lysosomes. These compartments were more acidic than mature melanosomes based on the accumulation of the weak base (3-(2,4-dinitroanilino)-3′-amino-Nmethyldipropylamine) (DAMP) generally used as a probe to determine the acidity of intracellular compartments [91]. Moreover, using endocytic tracers such as goldconjugated bovine serum albumin, we showed that stage I melanosomes were accessible to the endocytic system shortly after endocytosis, but that stage II–IV melanosomes were not. These observations indicated that melanosomes diverge and segregate from the endosomal system at the level of stage I melanosomes. These findings have been verified by others [92] and supported by numerous studies in melanocytes from disease models such as HPS (see Section 9.4.2). By contrast, one study in cultured choroidal melanocytes suggested that melanosomes are accessible to endocytic tracers [93], but this might reflect extended incubations of cells with tracers that induce autophagic processes. In other studies, tyrosinase or Tyrp1 were detected largely in subcellular fractions with lysosomal enzyme activity [23, 94, 95], but the “melanosomal” fractions were not assessed for purity and may have contained late endosomes and/or lysosomes. All together the evidence supports the conclusion that eumelanosomes are not merely modified lysosomes, and that sorting mechanisms within pigment cells distinguish between lysosomal and melanosomal cargoes (Figure 9.3). In addition to the differences in protein contents, melanosomes and lysosomes differ in pH. Using DAMP accumulation as an indicator for low pH, premelanosomes are highly acidic, accumulating nearly as much DAMP as lysosomes, but progression from premelanosomes to mature melanosomes is accompanied by a decrease in acidity [5]. This alkalization may be driven by the progressive removal of a vacuolar ATPase during melanosome maturation or by inactivation of a premelanosome-specific proton pump, such as the OCA2 transporter [51]. Tyrosinase is inactive below pH 5 [96, 97]. Thus, a progressive increase in pH from early to late melanosomes might prevent premature tyrosinase activity within earlystage melanosomes but favor higher activity, and consequently facilitate melanin biosynthesis, as melanosomes mature.

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Tyrosinase Tyrp1 Tyrp2/DCT

Early recycling endosome

Pmel17

Stage II

Stage III

Stage IV

Stage I

Early sorting endosome TGN Golgi Apparatus

Late endosome/MVB

Figure 9.3 Melanosomes originate within the endosomal system, but are distinct from lysosomes. Schematic representation of the endocytic and melanosomal organelles, and a working model for the sorting pathways for proteins distributed among them. Pmel17 is delivered to early sorting endosomes either following endocytosis from the plasma membrane or directly from the TGN. Domains of these endosomes then mature into coated endosomes/stage I melanosomes with characteristic internal vesicles and short fibrils and then into stage II melanosomes in which the fibrils organize

Lysosome

into sheets. By contrast, Tyrp1 is delivered to maturing melanosomes from an endosomal intermediate to which it is targeted directly from the TGN. Tyrosinase is also targeted to melanosomes from an endosomal intermediate. Delivery of the melanosomal enzymes results in maturation of the stage II premelanosome to the stage III and IV melanosome. The deposition of black melanin in these compartments is schematized by bold lines on the top of the striations (stage III) or by a black content throughout the organelle (stage IV).

It is likely that the segregation of melanosomal and lysosomal proteins is regulated by melanocyte differentiation and/or by differences in cell type, tissue of origin, or degree or type of pigmentation. In support of this notion, melanosomal proteins like tyrosinase, Tyrp1, OCA2, OA1, and Pmel17 localize to late endosomes/ lysosomes when expressed in nonpigmented cells [38, 39, 41, 44, 86, 98, 99]. Furthermore, different cohorts of melanosomal proteins are expressed in melanocytes during synthesis of pheomelanins and eumelanins [100, 101], and melanosomal proteins are less efficiently segregated from lysosomal proteins in hypopigmented melanocytic cells or brown melanocytes (G. Raposo and M.S. Marks unpublished data). RPE cells have morphologically distinct melanosomes (for review, see [102]) that may fuse more readily with lysosomes. A developmental difference in lysosome/melanosome segregation could explain why phagocytosed latex particles could be observed in melanosomes of RPE cells and choroidal melanocytes [103],

9.3 Endocytic System and Formation of Melanosomes

whereas endocytic tracers failed to access premelanosomes or melanosomes in skin-derived melanocytic cells. 9.3.3 Pmel17 and Generation of Early-Stage Melanosomes

Stage I and II melanosomes lack pigment, and thus are enriched in a distinct set of proteins from those that function in melanogenesis and that are enriched in stage III and IV melanosomes. The most prominent characteristics of early-stage melanosomes are the intralumenal fibrils that mature into parallel sheet-like arrays. These fibrils appear to consist primarily of fragments derived from a single protein, Pmel17. The maturation of Pmel17 from an integral membrane protein to a fibrillar form has been singularly instructive in understanding how early-stage melanosomes form. As discussed below, it has also provided surprising and novel insights into functional amyloid formation. A brief discussion of Pmel17 maturation is therefore warranted here. 9.3.3.1 Pmel17 Structure Like the melanin biosynthetic enzymes tyrosinase, Tyrp1, and Dct, Pmel17 is synthesized as a type I transmembrane protein with a short N-terminal signal sequence, a large lumenal domain, a single membrane spanning domain, and a short cytosolic domain [25]. In humans, two independent alternative mRNA splicing events generate four different Pmel17 protein products that differ within the lumenal domain [104–107], functional differences for these isoforms have not yet been defined. The lumenal domain can be subdivided into four distinct subdomains based on sequence homologies: an N-terminal region (NTR) with no known structural homolog, a PKD domain with homology to a repeated element within the polycystic kidney disease-1 protein, a RPT (proline/serine/threoninerich repeat) domain consisting of 10 imperfect direct repeats of a 13-residue sequence, and a membrane-proximal cysteine-rich Kringle-like domain (KLD) (reviewed in [25]). Hearing et al. further subdivide the lumenal domain to include gap regions (GAP1, GAP2, and GAP3) between the NTR and PKD, between the RPT and KLD, and between the KLD and transmembrane domains [108, 109]; here we will use the more simplified terminology. Pmel17 expression is normally limited to pigment cells. In humans Pmel17 expression is maintained in most melanoma cells [110, 111], but can also be induced in unusual “clear cell” tumors of mixed lineage [112, 113]. Hence, human Pmel17 has been well studied as a tumor-associated antigen, and numerous reagents – including antibodies and T cell clones – have been developed to detect it. These antibodies have proven to be extraordinarily useful in detailing the intracellular itinerary of Pmel17 as it matures within the melanocyte and in detecting Pmel17 by immunoelectron microscopy. 9.3.3.2 Pmel17 Forms the Fibrillar Matrix upon Which Melanins Deposit Unlike other melanosomal proteins discussed earlier, Pmel17 is most easily detected – using monoclonal antibodies to the lumenal domain – in stage I and II

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melanosomes and becomes less easily detected in stages III–IV [5, 114]. Orlow’s group first suggested that Pmel17 might be part of the “melanosome matrix” (i.e., the intralumenal fibrils) based on its insolubility in aqueous nonionic detergents or in the detergent phase of Triton X-114 [115, 116]. Consistently, immunolabeling for Pmel17 within stage II melanosomes was associated with the fibrils [5] and purified fibril-enriched subcellular fractions from melanocytic cells contain Pmel17 fragments [117]. Pmel17 immunoreactivity by microscopy decreases as melanosomes mature to stage III and IV [5], and biochemical detection of Pmel17 is inhibited by melaninization [118], consistent with masking of the Pmel17 fibrils by polymerized melanins during melanosome maturation. Pmel17 is the only pigment cell-specific protein required to generate the fibrils in vertebrate cells, as shown by the arrays of melanosome-like fibrils detected by electron microscopy in endosomes of transfected nonmelanocytic HeLa cells ectopically expressing Pmel17 [98]. Pmel17 is also necessary for fibril formation in melanocytes, as shown by analyses of melanocytic cell lines derived from silver mice in which the Pmel17 cytoplasmic domain is truncated [119]. Finally, recombinant fragments generated in bacterial expression systems and derived from the Pmel17 lumenal domain form premelanosome-like fibrils in vitro [120, 121]. Together, these studies suggest that Pmel17 is the main – if not sole – component of the premelanosome fibrils. Importantly, the fibrils bear biophysical hallmarks of amyloid [120, 121] – a repeating β-sheet-rich protein fold normally associated with misfolded proteins in neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease [122]. Pmel17 thus represents the first of a class of mammalian “functional amyloids” and a model for the formation of pathological amyloids [123]. 9.3.3.3 Pmel17 Biosynthesis and Amyloid Formation How does a transmembrane protein like Pmel17 form amyloid-like fibrils solely within the lumen of stage I–II melanosomes? A key to understanding this remarkable protein transformation is in the intracellular journey of Pmel17 from its point of synthesis in the ER to stage I melanosomes. The results of a number of studies (reviewed in [25] and [124]) paint the following picture of Pmel17 maturation. Pmel17 is synthesized in the ER as a precursor (P1) form with four core N-linked glycans. Three of the N-linked glycans become processed to the mature form upon passage through the Golgi complex. In addition, Pmel17 is extensively modified by O-linked glycosylation within the RPT domain and these oligosaccharides are further modified in the Golgi, including terminal sialic acid (at least in cell culture). Sialylated O-linked oligosaccharides are critical for Pmel17 recognition by the monoclonal antibody HMB45 [125–129], which is often used as a reagent to detect melanoma [126]. The full-length Golgi-matured form of Pmel17 has been referred to as the precursor 2 (P2) form [98]. From the Golgi, at least a fraction of Pmel17 traverses the plasma membrane, where it is internalized into the endosomal system by clathrin/AP-2-dependent endocytosis interacting with a di-leucine-based endocytosis signal within the Pmel17 cytoplasmic domain [119, 130, 131]; a natural truncation mutant of Pmel17 in the silver mouse removes this signal, resulting in

9.3 Endocytic System and Formation of Melanosomes Stage I

Amyloid fibrils Pmel17

Stage II

Mβ Mα

Model for the formation of premelanosome fibrils. The melanocytespecific protein, Pmel17, is present on the limiting membrane and the internal vesicles of early coated endosomes (stage I melanosomes). Within these structures, Pmel17 is cleaved to Mα (purple/orange lumenal domain) and Mβ fragments (blue cytoplasmic domain) by a proprotein convertase (represented by a scissor). Mα fragments that dissociate from membranes are subsequently cleaved by still unknown proteases and begin to assemble in small, irregular fibrils that become fully organized

Figure 9.4

within stage II melanosomes. Only Mα and derived fragments (top right) are reproducibly detected in stage II melanosomes. On the bottom right, a three-dimensional model of a stage II melanosomes obtained by tomographic reconstruction of a region of the cytoplasm of an MNT-1 melanoma cell preserved by high-pressure freezing before freeze substitution and embedding in Epon. Stage II premelanosomes (membrane in pink) bear amyloid fibrillar sheets (yellow/ gold) and intraluminal vesicles (green). Scale bars: 200 nm.

accumulation at the plasma membrane and depletion from melanocytes [119]. Within endosomes or perhaps the TGN, Pmel17 is cleaved at two specific sites within the lumenal domain by two proteases (Figure 9.4). First, a proprotein convertase cleaves Pmel17 at residues 468–469 (human Pmel17), separating Pmel17 into two disulfide-linked fragments: a large lumenal fragment referred to as Mα (mature polypeptide α) – containing the NTR, PKD, and RPT domains – and a smaller, membrane bound fragment called Mβ (mature polypeptide β) – containing the KLD, transmembrane, and cytoplasmic domains [98, 117]. Second, a “site 2 protease” cleaves Mβ within the juxtamembrane region of the lumenal domain to liberate Mα and its associated lumenal part of Mβ from the membrane [132]; the identity of this protease is not yet clear, but depletion of either of two proteases of the ADAM (“a disintegrin and metalloprotease domain”) family of α-secretases

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severely impair this cleavage and downstream events [132]. Indirect evidence suggests that the cleavages occur within specific domains of early endosomes [119], and both seem to require the acidic environment within the late secretory and endocytic pathways [98] (and B. Watt and M.S. Marks, unpublished results). A small fraction of the cleaved forms of Pmel17 is secreted from cells in culture [133, 134], but the functional significance of this cohort is not yet known. The membrane-bound fragment of Mβ remaining after site 2 protease cleavage is a substrate for intramembrane proteolysis by the γ-secretase complex [132], well known for its activity on the Alzheimer precursor protein, Notch, and other clinically important substrates [135]. Once cleaved and released from the membrane within endosomes, Pmel17-Mα has the unusual property of aggregating in an ordered fashion into polymerized fibrils. Indeed, it is Mα (or fragments derived therefrom) that polymerizes into amyloid in vitro [120, 121, 136] and Mα released that is released from Mβ is only detected within fractions of melanocytic cells that are insoluble in nonionic detergents [117]. Fibrillogenesis requires all three subdomains of Mα [108, 119]. However, Mα is only a minor species detected within these fractions. Rather, Mα is further digested to smaller fragments corresponding at least to the RPT and part of the PKD domains [27, 108, 121, 129]. These are the fragments that are detected by immunocytochemistry on stage II melanosomes, and that are likely buried by melanins in stages III and IV; epitopes corresponding to the N- and C-termini of Pmel17 are not detected in melanosomes, likely due to proteolysis (indeed, antibodies to the C-terminus follow Mβ and its derived C-terminal fragment and are excluded from stage II–IV melanosomes) [5, 98, 129]. Thus, the progression of Pmel17 through the secretory pathway and its regulated cleavage within acidic endosomal compartments accounts for the ability of this endogenously synthesized protein to assemble into fibrils only at a given stage of the endosomal pathway. 9.3.3.4 Functional Importance of Fibrillar Melanosomes What is the function of the Pmel17 amyloid fibrils? The answer is not entirely clear. Pmel17 fibrils – and indeed, any amyloid fibril – accelerates melanin polymerization in vitro [106, 114, 120], suggesting that Pmel17 might play a kinetic role in melanization or in absorbing otherwise toxic oxidative melanin intermediates. Pmel17 is responsible for the elliptical shape of melanosomes, as silver mice have round, enlarged melanosomes [119]; this shape might be important for melanosome transfer from melanocytes to keratinocytes or for melanosome motility into the apical processes of RPE [137]. Lastly, the polymerization of melanins into a solid mass composed of fibrillar sheets rather than smaller, separate packages might facilitate melanin transfer to keratinocytes if melanins are released from melanocytes by secretion. However, it should be noted that melanocytes from silver mice are highly pigmented and that the coatcolor defect in these mice is mild [13, 67, 138]. Elucidation of the true function of these fibrils will need to await a complete Pmel17 gene deletion in a tractable experimental system.

9.3 Endocytic System and Formation of Melanosomes

9.3.4 OA Type 1 and Melanosome Biogenesis

The OA1 protein is a pigment cell-specific member of a subfamily of GPCRs [55, 139, 140]. Mutations in the OA1 gene underlie OA type 1, an X-linked disorder that does not impair melanin production but decreases the number of melanosomes in RPE and the choroid [141]. Although skin is pigmented in OA1 patients and mouse models, aberrant giant melanosomes, called “macromelanosomes,” accumulate in both RPE and skin melanocytes, suggesting a defect in melanosome biogenesis [142]. Like other canonical GPCRs, OA1 interacts with arrestins and activates heterotrimeric G-proteins [45, 55, 140]. Null mice for the inhibitory G-protein Gαi3 show similar phenotypes to those observed on OA type I albinism, suggesting that OA1 might control melanosome maturation by activating the G-protein Gαi3 [143]. Unlike other GPCRs that localize mainly to the cell surface, OA1 localizes intracellularly to lysosomes and melanosomes [57, 99] by virtue of specific sorting signals in its cytosolic domain [99]. Thus, whereas other GPCRs bind extracellular ligands, the OA1 ligand is likely exposed in the lumen of the melanosome or other compartments. One report has suggested that l-dopa – the melanin precursor – is the OA1 ligand [56]. Ligand binding triggers a signaling cascade from the organelle lumen to the cytosol. While this cascade remains poorly characterized, the phenotype of OA1 mutant cells predicts that it signals organelle-intrinsic fusion/fission events during melanosome biogenesis, similar to the phagosome intrinsic regulation of antigen processing by Toll-like receptor signaling in dendritic cells [144]. The membrane trafficking steps regulated by OA1 have remained enigmatic. Recent studies by our group suggest that the giant melanosomes observed in OA1deficient melanocytic cells result from aberrant fusion/fission events at early steps of melanosome biogenesis [57]. Transient OA1 downregulation in a eumelanogenic melanoma cell line results in the generation of aberrant premelanosomes that accumulate cargo proteins of immature melanosomes (Pmel17), late-stage melanosomes (Tyrp1), and lysosomes (LAMP1), suggesting that protein sorting and/or segregation of organelles downstream of coated endosomes is somehow regulated by OA1 [57]. Another recent study suggested that OA1 might function not only in the regulation of melanosome biogenesis, but also in melanosome motility. This study showed that melanosomes in skin melanocytes and RPE from Oa1-null mice are abnormally distributed toward the cell periphery [145]. It is possible that both phenotypes are linked, such that the altered motility is a consequence of improper melanosome maturation (for review, see [146]). 9.3.5 Origin of the Melanosome 9.3.5.1 Early-Stage Melanosomes Originate within the Endocytic Pathway Having defined Pmel17 as a major biogenetic constituent of early-stage melanosomes provided a means with which to probe the origins of these unusual organelles within the secretory/endosomal system. Overwhelming evidence now

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supports an early endosomal origin for stage I and II melanosomes. While the details of how these organelles diverge from endosomes is not yet complete, our observations thus far have provided a new paradigm for LRO biogenesis, and highlighted the endocytic system as a unique and highly adaptable platform from which multiple sorting pathways can converge to generate the melanosome. Compelling evidence for an early endosomal origin of the premelanosome came first from immunoelectron microscopy analyzes of Pmel17 distribution in eumelanogenic cells. A cohort of mature Pmel17 was detected in EEA1-positive early endosomal tubules and vacuoles [5]. Some of the vacuoles displayed bilayered clathrin coats on the cytosolic side. These compartments clearly correspond to vacuolar sorting endosome domains based on the presence of EEA1, the flat bilayered coats, a few intralumenal membranes, and accessibility to internalized Tf and gold-conjugated bovine serum albumin after short incubations [5]. These compartments are also structurally equivalent to the stage I premelanosome [5, 10] and are early endosome precursors to stage II premelanosomes based on several lines of evidence, including: (i) the progressive loss of EEA1 correlates with a progressive increase of Pmel17 density during progression from tubular early endosomes to coated endosomes to stage II premelanosomes; (ii) endocytic tracers progressively access early endosomes and then coated endosomes, and are excluded from stage II premelanosomes; and (iii) early and coated endosomes, but not the stage II premelanosome, are labeled by an antibody to the C-terminus of Pmel17, which becomes segregated from the fibrillinogenic lumenal domain after cleavage [5, 117]. Together, these observations suggest that Pmel17 passes through the early endosome and the coated endosome intact before its deposition as fibrils in the stage II premelanosome. Within coated endosomes, Pmel17 accumulates largely on intraluminal membranes, similar to integral membrane cargoes destined for lysosomal degradation [5]. In fact, when expressed in nonpigment cells, Pmel17 accumulates primarily on intraluminal membranes of MVBs [98]. Several lines of evidence support the notion that Pmel17 begins to form fibrils in association with these intraluminal vesicles. First, in transfected nonpigment cells that express Pmel17, fibrils form within MVBs intermingled with the intraluminal membranes [98, 117]. Second, inhibition of sorting onto the intraluminal fibrils by mutagenesis of the Pmel17 NTR or PKD domains ablates fibril formation – as well as the proprotein convertase cleavage required for liberating the fibrillogenic Pmel17-Mα fragment [147]. Third, and most directly, fibrils are seen to emanate from intralumenal vesicles in threedimensional reconstructions from electron tomographic analyzes of coated endosomes/stage I melanosomes in a eumelanogenic melanoma line [11]. Using cryopreservation rather than chemical fixation, intermediates could be visualized ranging between coated vacuolar endosomes with small protofibrils and elongated stage II melanosomes with fibrillar sheets [11]. These observations confirm a direct role for the intraluminal membranes as “seeds” for the formation of Pmel17 amyloid during the biogenesis of early-stage melanosomes. Intraluminal membranes of multivesicular endosomes have also been implicated in the formation of pathogenic amyloid by the Alzheimer’s Aβ peptide and the prion protein [148],

9.3 Endocytic System and Formation of Melanosomes

suggesting that Pmel17 and pathogenic amyloidogenic proteins use a common mechanism in their transformation into fibrils. 9.3.5.2 Melanosomes Do Not Originate from the ER The notion that early-stage melanosomes develop from endosomes has been challenged by an alternative view that stage II melanosomes emerge from the ER. However, the precedents on which this view is based are flawed. The idea originated from an elegant early study by Maul, who used electron microscopy analyses of serial sections of a human melanoma line to show that structures with the characteristic morphologic features of stage II melanosomes could be observed in continuity with smooth tubular membranes near the Golgi apparatus [149]. Maul used the term “smooth ER” to describe these membranes, because at the time this was the only “smooth” tubular membrane system known; the TGN and the endocytic system had not yet been characterized morphologically. The membranes were not further identified; in fact, it is likely that they corresponded to endosomal tubules, which we now understand can fuse with immature melanosomes during maturation (see Section 9.3.5.4). Nevertheless, Hearing et al. have proposed an origin for stage I/II melanosomes from the rough ER based largely on subcellular fractionation of melanocytic cells and immunoblotting analyses. A subcellular fraction of a human eumelanogenic melanoma line that was enriched in stage II melanosomes was shown to contain the full-length, unprocessed P1 form of Pmel17 by immunoblotting using antibodies to the cytoplasmic domain. Maturation from stage II to stage III melanosomes was accompanied by a loss of this form and an accumulation of RPT domaincontaining proteolytic fragments detected by monoclonal antibody HMB45 [27]. Moreover, proteomic analysis of this fraction revealed the presence of traditional ER resident proteins in addition to known constituents of early and late-stage melanosomes, late endosomes/lysosomes, lipid rafts, and secretory granules [42]. While the authors inferred that stage II melanosomes contain ER proteins and stem from the ER, these observations are more likely explained by contaminating ER membranes within these fractions (for a discussion, see [25]). The inability to detect Golgi-processed forms of Pmel17 by immunoblotting with the antibody to the C-terminus in these and other studies [27, 150, 151] was taken as further evidence for a direct formation of melanosomes from the ER. However, these analyses failed to take into account the rapid processing of Pmel17 compared with the slow folding and release from the ER [98], the loss of the C-terminus during maturation [98, 117], and the detergent insolubility of fibrils [108, 117, 129, 152, 153], which together make the P1 form the most predominant by far at steady state within detergent-soluble cell fractions. Moreover, as discussed earlier, HMB45 reactivity with Pmel17-derived bands detected by these authors in stage III melanosome-enriched fractions requires processing by Golgi enzymes. Finally, signal overlap by immunofluorescence microscopy has been used as an argument that the Pmel17 C-terminus and HMB45-reactive fibrils are detected in the same compartment [152, 153], but these analyses failed to account for the wide distribution of the ER throughout the cell or to provide higher resolution views – as in

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other studies [5, 129] – in which the ER and stage II melanosomes could be resolved. Thus, the evidence overwhelmingly supports an origin within the endocytic system. Nevertheless, a role for the ER during the melanosome maturation process should not be disregarded. The generation of melanosomes requires functions that could potentially be effected through specific interactions between maturing melanosomes and the ER, including lipid metabolism, calcium homeostasis, and redox reactions. Tight interactions between the ER and melanosomes could even potentially explain the presence of ER components in isolated fractions of melanosomes [42, 43], as has been shown for phagosomes [154]. 9.3.5.3 Melanosomes Segregate from the Endocytic Pathway beyond Stage I Melanosomes Whereas endocytic tracers such as bovine serum albumin access coated endosomes/ stage I melanosomes, they do not appear to access mature melanosomes (stages II–IV), but rather pass through late endosomes en route to lysosomes with kinetics similar to those in other cells [5]. The presence within the coated endosome of both endocytic tracers and Pmel17 at early timepoints and their segregation at later timepoints indicates that this compartment is a critical sorting point between the endocytic and premelanosomal pathways. This sorting may be required for the segregation of proteins required for the morphogenesis and function of specialized endosomal organelles like the melanosome. The segregation from endosomes, rather than from the TGN, may reflect requirements for (i) the formation of MVBs in premelanosome biogenesis (see Section 9.3.1) and (ii) acidification through a vacuolar ATPase in organelles such as premelanosomes. The latter is supported by the efficient accumulation of the weak base DAMP in coated endosomes and stage II premelanosomes [5] and the disruption of melanosome biogenesis by inhibitors of the vacuolar ATPase ([98] and our unpublished observations). Although coated endosomes appear morphologically similar to vacuolar endosomes in which ubiquitylated cargoes are sorted to intraluminal vesicles, the sorting mechanism appears to be different. In melanocytic cells in which the ESCRT pathway was blocked by a variety of means, a traditional ubiquitylated substrate – the melanocyte-specific protein, MART-1 [155] – was protected from degradation and excluded from internal vesicles, but Pmel17 was still sorted to internal vesicles [147, 156]. Moreover, whereas mutagenesis of all ubiquitylation acceptor sites on the Pmel17 cytoplasmic domain had no effect on its sorting to internal membranes, sorting was completely inhibited by deletion of either of two lumenal subdomains [147]. The mechanism by which Pmel17 becomes incorporated onto the internal membranes remains to be determined, but might be related to its property to aggregate or to potential interactions with lipids such as ceramide [157]. The segregation of Pmel17 and MART-1 into different intralumenal vesicles suggests separate sorting for cargoes fated to degradation or to the melanosomal lineage at the level of the coated endosome. It is tempting to propose that the clathrin-containing coats act as platforms to facilitate the clustering of proteins bound for late endosomes and lysosomes, permitting concentration of Pmel17 to

9.3 Endocytic System and Formation of Melanosomes

levels necessary for ordered aggregation. A similar mechanism has been proposed for the concentration of insulin and other cargoes in nascent secretory granules [158] and in maturing transcytotic vesicles [159]. 9.3.5.4 Components of Mature Melanosomes Are Sorted from Distinct Endosomal Intermediates Melanosomal enzymes and transporters such as tyrosinase, Tyrp1, OCA2, and ATP7A are not detected in stage I and stage II premelanosomes, but are enriched in stage III and stage IV melanosomes [5, 44, 65]. This suggested that cargo sorting to mature melanosomes is a distinct process from cargo sorting to early-stage melanosomes and likely occurs from different sites in pigment cells. The cytochemical detection of tyrosinase activity in clathrin-coated vesicles near the TGN [85], the detection of tyrosinase, Tyrp1 and Dct in clathrin-coated vesicle-enriched subcellular fractions [33], and the colocalization of Tyrp1 and the lysosomal protein LAMP1 in vesicular structures near the TGN [5] led to the proposal that melanosomal proteins might be directly targeted to melanosomes from the TGN [5]. However, more recent results provide strong evidence that these cargoes are trafficked to maturing melanosomes via post-Golgi endosomal intermediates. These studies have exploited either cultured melanocytes derived from mouse models of trafficking disorders such as HPS or genetic manipulation of cultured melanocytes or melanoma cells to interfere with endosomal transport. As discussed further in Section 9.4.2, the products of the genes that are defective in HPS localize primarily to endosomal compartments and mature melanosomal cargoes in these models become trapped in endosomal intermediates, whereas stage II melanosomes are largely unimpaired. Evidence from analyses of wild-type and genetically altered melanocytic cells (see Section 9.4) suggests that cargoes are delivered to melanosomes from endosomes via two distinct classes of intermediates – vesicles that bud from tubular endosomal domains, and tubules that make direct contacts between endosomal vacuoles and melanosomes (Figure 9.5). Whereas vesicles have been thought to be the main mediators of anterograde traffic within the endosomal system [160], direct tubular contacts between endosomal organelles have been observed in some cases [161] (Figure 9.5). In melanocytes, direct tubular contacts between vacuolar endosomes and maturing stage III/IV melanosomes have been observed using both three-dimensional reconstructions from electron tomography analyses and live cell imaging [162]. These tubules contain cargoes of recycling endosomes and thus likely correspond to specialized recycling endosomal domains that derive from the vacuoles. Membrane continuities between the tubules and both organelles suggest that they transiently fuse with melanosomes, and thus likely allow for the exchange of contents between endosomes and melanosomes [162]. Consistently, accumulating evidence suggests that at least one major cargo transport pathway from endosomes to maturing melanosomes requires components known to regulate recycling endosomal sorting and positioning (see Section 9.4.2.1). Therefore, the data suggest that these tubules are specialized endosomal domains that serve as conduits for the delivery of cargoes to maturing stage III/IV melanosomes.

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9 Biogenesis of Melanosomes Melanosome

Melanosome

Recycling endosome

Figure 9.5 Endosomal tubules and buds are close to and continuous to melanosomes. (A) Electron micrograph of an ultrathin cryosection of a MNT-1 cell showing tubular membranes harboring coated buds (small arrows) closely apposed to a melanin containing melanosomes. (B) Schematic representation of the endosomal– melanosomal network, with clathrin coats represented by gray shading. (C) Threedimensional model obtained after tomographic reconstruction of a region of the

cytoplasm of a MNT-1 cell fixed by highpressure freezing before freeze substitution and embedding in Epon. Left panel: tubular membranes (green) that are continuous with the melanosome limiting membrane (red) are indicated by arrows. In white, the ER. On the right panel are shown two different orientations of the three-dimensional model highlighting continuities between the endosome and the melanosome (arrows). Scale bars: 200 nm.

As melanosomes mature from stage II to stage IV, melanosome size is maintained relatively constant. Thus, the delivery of membrane to melanosomes by anterograde fusion events that facilitate cargo delivery must be balanced by retrograde transport out of melanosomes. It is possible that the endosomal tubules observed between endosomal vacuoles and melanosomes provide for both anterograde and retrograde events. Indeed, these carriers harbor endosomal cargoes that do not accumulate in melanosomes [162]; thus, either the entry into melanosomes is “gated” such that only certain cargoes are permitted to flow from the tubule membrane into melanosomes or the bulk flow of membrane through these tubules is from the melanosome to the endosome, with only specialized cargoes transferring in the other direction. Future live cell imaging analyses will be required to differentiate between these views.

9.4 Melanosome Maturation: Cargo Sorting to Mature Melanosomes

9.4 Melanosome Maturation: Cargo Sorting to Mature Melanosomes

Most known components of mature melanosomes are integral membrane proteins that bear cytoplasmic targeting signals required for their delivery to melanosomes [37–39, 41, 44, 99]. What cellular components recognize these signals and coordinate the intracellular transport events required to accumulate these proteins specifically within melanosomes? Key clues have come from analyses of cells derived from patients and mouse models in which OCA is one component of a syndrome of disorders characterized by generalized defects in LROs. Other clues derive from inhibitory RNA approaches in model cultured melanocytic cells (Table 9.2). 9.4.1 Griscelli Syndrome and CHS

Among diverse pathological conditions that disrupt lysosomes and related organelles there are three genetic disorders that affect melanosomes: Griscelli syndrome (GS), CHS, and HPS. GS and corresponding mouse models have been invaluable to decipher the molecular mechanisms that underlie the motility of melanosomes and other LROs [163]. Genetic, cellular, and biochemical analyses of GS-derived melanocytes revealed that an actin-based motor protein, myosin Va, is recruited to mature melanosomes by the concerted action of a member of the Rab family of small GTPases, Rab27a, and the Rab27a effector, melanophilin. These three proteins coordinate melanosome translocation from microtubules to actin filaments in the cell periphery for their ultimate transfer to keratinocytes [163, 164]. A similar complex with a different motor (myosin VIIa) and Rab27a effector (Myrip) regulate diurnal light cycle-dependent melanosome motility in mouse retinal pigment epithelia [137] (for further details, see Chapter 10). CHS is a rare genetic disorder of multiple organ systems characterized minimally by severe immunodeficiency, neuropathy, and OCA. The gene targeted in CHS (and the corresponding mouse beige mutation in mouse) encodes an approximately 400-kDa protein cytosolic protein called LYST (lysosomal trafficking regulator) or CHS1p [165, 166]. Many cell types from CHS patients or beige mice harbor giant lysosomes and LROs (including melanosomes in melanocytes), indicating a defect in organelle biogenesis, but the exact function of LYST within pathways of organelle formation is not yet clear. Owing to its unusually large size, LYST is difficult to express in physiologically relevant models. Dominant-negative approaches suggest that LYST might play a role in phosphoinositide metabolism [167]. Consistently, our studies in B lymphocyte cell lines derived from CHS patients suggest that LYST is required for sorting endosomal resident proteins into multivesicular endosomes [168]. Since multivesicular endosomes are also intermediates in melanosome formation, defects in sorting to MVBs are likely to impact melanosome biogenesis. More work needs to be done to define how LYST might regulate this process in pigment cells and whether CHS reflects a defect in the formation of early-stage melanosomes, late stage melanosomes, or both.

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Table 9.2 Molecular complexes and proteins playing key roles in melanocyte trafficking.

Intracellular trafficking protein

Organelle localization

Function

Human disease/mouse model

BLOC-1

early endosome (tubules)

trafficking from endosomes to melanosomes

HPS7 (h)–HPS8 (h)/muted (m)–pallid (m)–reduced pigmentation (m)–capuccino (m)–sandy (m)

Tyrp1–ATP7A BLOC-2

BLOC-3

AP-3A

RABGGTaseII

early endosome (tubules)

trafficking from endosomes to melanosomes

melanosome

vesicular trafficking

Golgi vesicles

Tyrp1–tyrosinase

early endosome (buds)

trafficking from endosomes to melanosomes

cytosol

prenylation of Rab

Tyrp1–tyrosinase?

HPS3 (h)–HPS5 (h)–HPS6 (h)/cocoa (m)–ruby eye2 (m)–ruby eye (m) HPS1 (h)–HPS4 (h)/pale ear (m)–light ear (m) HPS2 (h)/pearl (m)–mocha (m)

interacts with cytoplasmic tail of tyrosinase HPS-like phenotype gunmetal (m)

HOPS (VPS33A)

cytosol

fusion events

HPS-like phenotype

tethering factor for SNAREs

Rab38

Rab32

Tyrp1

buff (m)

melanosome

trafficking from TGN/endosomes to melanosomes

HPS-like phenotype in rats/chocolate (m)

post-Golgi tubular membranes

Tyrp1–Tyrosinase

unknown

trafficking from TGN/endosomes to melanosomes

unknown

Tyrp1–tyrosinase AP-1A

KIF13A

RAB7

early endosome (buds)

trafficking from endosomes to melanosomes

early endosome (tubules)

trafficking from endosomes to melanosomes

unknown/embryonic lethal in mice

interacts with cytoplasmic tail of Tyrp1–tyrosinase unknown

interacts with AP-1 and Tyrp1

melanosome

vesicular trafficking

late endosome

Tyrp1

unknown

9.4 Melanosome Maturation: Cargo Sorting to Mature Melanosomes

271

Table 9.2 (Continued)

Intracellular trafficking protein

Organelle localization

Function

Human disease/mouse model

TSG101 (ESCRT-I)

endosomes

component of the ESCRT machinery

unknown

trafficking from endosomes to melanosomes Tyrp1 Myosin Ib

endosomes

actin-associated motor

unknown

facilitates endosomal sorting of Pmel17 BLOC: biogenesis of lysosome-related organelle complex; HPS: Hermansky – Pudlak syndrome; RABGGTaseII: Rab geranyl geranyl transferase (prenylation of small GTPases of the Rab superfamily); ESCRT: endosomal sorting complex responsible for transport.

9.4.2 HPS

Whereas CHS remains largely mysterious, studies on HPS genes and their corresponding mouse models have greatly accelerated our understanding of melanosome formation and the role of novel trafficking regulators in endosomal dynamics [169]. HPS is a multisystem disorder that is characterized by partial albinism, excessive bleeding, and often lung fibrosis, sometimes accompanied by immunodeficiency and/or granulomatous colitis [89]. These systemic symptoms result from the malformation or malfunction of LROs, including melanosomes, platelet dense granules, and, in some cases, lung lamellar bodies, cytotoxic T cell and natural killer cell granules, and perhaps other organelles. HPS results from mutations in any of at least eight different genes; mutations in at least 15 different genes, including the orthologs of the genes that are mutated in human HPS, result in a similar disorder in mice, first identified based on coat-color dilution [89, 170, 171]. Although HPS-associated genes are ubiquitously expressed, they appear to be functionally essential for the generation of a restricted number of LROs such as platelet-dense granules and melanosomes [87]. This cell-type specificity in penetrance suggests that an ubiquitous set of proteins, perhaps by interacting with additional tissue-specific machineries, is exploited in specialized cell types to generate a novel lineage of organelles. Understanding how these proteins function in LRO-generating cells such as the melanocyte has already provided insight into novel membrane trafficking pathways that underlie endosomal dynamics and melanosomal enzyme trafficking. 9.4.2.1 Adaptor Protein (AP) Complexes The transfer of macromolecules between organelles of the endosomal system is mediated by tubulovesicular carriers that bud from one membrane and fuse with

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another. One critical step in this process is the concentration of proteins into emerging transport carriers through the recognition of sorting signals present in the cytoplasmic domain of transmembrane cargo proteins. Heterotetrameric APs are a family of macromolecular protein complexes that mediate this sorting event. Four distinct APs (AP-1 to -4) are ubiquitously expressed in mammals and each consists of four subunits: one small (σ1–4), one medium (μ1–4), and two large (α, γ, δ, and ε, and β1–4) [172]. The tissue-specific expression of some “replacement” subunits, such as μ1B and μ3B, leads to the formation of additional unique APs in certain cell types, such as AP-1B and AP-3B in epithelia and neurons, respectively. AP-2 is thought to function exclusively in endocytosis from the plasma membrane by coupling cargo sorting to clathrin-coated vesicle formation. By contrast, AP-1, AP-3, and AP-4 function intracellularly in protein sorting among endosomal organelles and the TGN. AP-3 was the first adaptor shown to be involved in melanogenesis. The gene encoding the AP-3 β3A subunit is mutated in HPS2 patients [173] and its mouse model, pearl [174], and the AP-3 δ subunit is mutated in the mouse HPS model, mocha [175]. The mocha and pearl mice are mildly hypopigmented compared to wild-type mice, reflecting a diminution in mature melanosomes within melanocytes [176, 177]. AP-3 interacts with di-leucine-based melanosomal sorting signals in the cytoplasmic domains of tyrosinase [28, 40] and OCA2 [44]. Melanocytes derived from HPS2 patients or from pearl mice accumulate tyrosinase in endosomes [28, 178], suggesting a function for AP-3 in sorting tyrosinase from endosomes toward melanosomes. Accordingly, in melanocytes (as in other cell types [179, 180]), the bulk of AP-3 in wild-type melanocytes localizes to clathrin-coated vesicles and buds that are associated with tubular domains of endosomes, and that are often enriched in tyrosinase [28]. While its function in tyrosinase sorting is indisputable, several observations indicate that AP-3 is not required for the transport of other melanosomal enzymes or even for the entire cohort of tyrosinase. (i) Hypopigmentation of pearl and mocha mice is mild compared with other HPS models [169]. (ii) AP-3-deficient melanocytes are still able to generate morphologically intact melanosomes [28, 176, 178] that harbor a reduced but significant cohort of tyrosinase [28]. (iii) Other melanosomal proteins, including Tyrp1 and the copper transporter ATP7A, do not interact with AP-3, and their transport to melanosomes is not substantially impaired in AP-3-deficient melanocytes [28, 65, 178] (although misrouting of Tyrp1 through the cell surface seems to be enhanced [181]). Thus, additional sorting molecules must function in melanosome biogenesis. Another critical member of the AP family in melanocytes is AP-1. Genes encoding AP-1 subunits have not been shown to be targets of HPS, but this is likely due to embryonic lethality as observed in mice with targeted deletion in the genes encoding the γ-adaptin [182] or μ1A [183] subunits. The cytoplasmic sorting signals of tyrosinase, Tyrp1, and OCA2 interact with AP-1 [28, 44], and both Tyrp1 and tyrosinase can be detected in vesicles coated with AP-1 in melanocytes [5, 28]. Using in vitro assays, AP-1 and AP-3 have been proposed to mediate the budding of distinct cargo-containing carriers destined for melanosomes [184]. In

9.4 Melanosome Maturation: Cargo Sorting to Mature Melanosomes

melanocytes, AP-1 has been shown to mainly localize to tubulovesicular endosomes that are closely apposed to melanosomes and that derive from the endosomal recycling system; these endosomes likely correspond at least in part to the tubules that contact melanosomes as observed by electron tomography [162]. Whereas in most cells, recycling endosomes accumulate near the microtubule organizing center in the perinuclear region [68], similar domains in melanocytes are positioned in the periphery, apposed to melanosomes. This peripheral polarization of recycling endosomal domains requires AP-1, and facilitates a “dialog” between endosomes and melanosomes that ultimately results in the transfer of protein cargoes, such as Tyrp1, from endosomes to melanosomes[162]. This conclusion is supported by the entrapment of Tyrp1 in endosomes upon small interfering RNA-mediated depletion of AP-1 or an interacting motor protein, KIF13A, in a melanocytic cell line [162, 185]. Together, the data suggest that AP-1 functions both as a cargo sorting molecule and as an effector of endosome positioning to facilitate cargo transfer from recycling endosomal intermediates toward melanosomes. 9.4.2.2 BLOC Complexes In most cell types, AP-1-dependent cargoes are delivered to early endosomes or the TGN and AP-3-dependent cargoes are delivered to late endosomes or lysosomes. By contrast, even though the same compartments are present in melanocytes, a specific cohort of AP-1- and AP-3-dependent cargoes are efficiently delivered to a distinct destination – maturing melanosomes. This suggests that additional effectors must participate in diverting melanosomal cargoes toward this destination. Studies in melanocytes from mouse HPS models have established the biogenesis of lysosome-related organelle complexes (BLOCs) as key effectors in these pathways. The BLOCs are peripheral membrane multisubunit protein complexes that are ubiquitously expressed but, like AP-3, play specialized roles in cells harboring LROs. Genes encoding BLOC subunits constitute the majority of targets of mutation in human and mouse HPS models. There are three different BLOCs: BLOC-1, -2, and -3. Proteins that comprise these multisubunit complexes lack common structural motifs or homology to proteins of known functions [186] and so their molecular functions are not understood. 9.4.2.2.1 BLOC-1 BLOC-1 is an approximately 250-kDa protein complex that consists of eight subunits. Two of the subunits (dysbindin and BLOS3) are encoded by genes that are mutated in human HPS (HPS type 7 and type 8, respectively), and these subunits and three others (Muted, Pallidin, and Cappuccino) are encoded by genes that bear inactivating mutations in mouse HPS models (sandy, reduced pigmentation, muted, pallid, and cappuccino) [170]. Inactivating mutations in any subunit (except BLOS3 in reduced pigmentation mice) destabilize the entire complex, resulting in a complete loss of BLOC-1. BLOC-1 mutant mice are the most severely hypopigmented of all of the mouse HPS models [89, 170] and correspondingly

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melanocytes derived from them are nearly completely depleted of pigmented melanosomes [176, 177, 187]. By contrast to the substantial missorting of tyrosinase in AP-3-deficient cells, tyrosinase in BLOC-1-deficient melanocytes is only mildly depleted from fibrous stage II-like melanosomes and only partially missorted to endosomes. However, other proteins, including Tyrp1, are nearly completely excluded from melanosomes in BLOC-1-deficient cells and trapped in a continual recycling loop through enlarged vacuolar endosomes [187]; under some conditions, a cohort of Tyrp1 is also misrouted to lysosomes ([181] and our unpublished results). This suggests that BLOC-1 regulates a cargo delivery pathway distinct from that of AP-3. Strikingly, although a substantial cohort of tyrosinase is present in melanosomes in BLOC-1-deficient melanocytes, it is inactive due to an absence of its cofactor, copper [65]. Copper must be pumped into melanosomes by the copper transporter, ATP7A, which itself is a cargo of the BLOC-1dependent transport pathway as shown by its exclusion from melanosomes in BLOC-1-deficient melanocytes; incubation of fixed BLOC-1-deficient cells with excess copper restores tyrosinase activity [65]. Thus, the severe hypopigmentation observed in BLOC-1 mutant mice results from impaired trafficking of Tyrp1, ATP7A, and perhaps other cargoes from endosomes toward melanosomes in an AP-3-independent manner [65, 187]. Like APs, BLOC-1 localizes to early endosomes [181], but consistent with their distinct functions, AP-3 and BLOC-1 are spatially segregated on endosomal membranes: AP-3 is enriched in buds, whereas BLOC-1 localizes to tubular endosomal domains [181], perhaps similar to those that connect vacuolar endosomes with melanosomes [162]. What molecular function BLOC-1 serves on endosomal membrane is still not clear. Analyses of endosomal recycling and localization of Tyrp1 in BLOC-1- and AP-3-deficient melanocytes suggest that BLOC-1 is required for cargo exit from endosomal intermediates, but it remains to be established whether BLOC-1, like AP-1 and AP-3, interacts directly with cargo. BLOC-1 and some of its component subunits have been shown to interact directly with the recycling endosomal tSNARE component syntaxin-13 [188, 189] and the neuronal tSNARE component SNAP-25 [189, 190], suggesting a potential role in regulating fusion steps during melanosomal cargo delivery. BLOC-1 also shows genetic interactions with the recycling endosomal GTPase, Rab11 [189], perhaps suggesting a role in regulating recycling endosomal membrane dynamics. This would be consistent with the role of AP-1 in both recycling endosome dynamics and cargo transport; indeed, the morphological and protein-sorting defects in AP-1-depleted melanocytes are similar to those of BLOC-1-deficient cells [162]. Nevertheless, BLOC-1 has not yet been shown to interact physically with AP-1. Moreover, despite the distinct functions and localization in melanocytes, a cohort of BLOC-1 can be coimmunoprecipitated with AP-3, but not AP-1, from membrane fractions of various cell types [181] and from a subpopulation of synaptic-like microvesicles in neuronal cells [191]. The details of this interaction are still not clear, but one plausible hypothesis is that BLOC-1 and AP-3 might reciprocally regulate their association with membranes [181], and consequently regulate trafficking steps sequentially from the same organelle [181, 191].

9.4 Melanosome Maturation: Cargo Sorting to Mature Melanosomes

9.4.2.2.2 BLOC-2 AP-3, AP-1, and BLOC-1 regulate the sorting of melanosomal cargoes into transport carriers, but what effectors regulate the delivery of these carriers to melanosomes? BLOC-2 might participate in this process. BLOC-2 is a 340-kDa complex that consists of at least three subunits: HPS3, HPS5, and HPS6 [192, 193]. Each subunit is encoded by a gene that is mutated in HPS patients (HPS types 3, 5, and 6) and corresponding mouse models (cocoa, ruby-eye2, and ruby-eye, respectively) [89, 170]. The only recognizable motif on any BLOC-2 subunit is a clathrinbinding domain on HPS3; HPS3 can coimmunoprecipitate with clathrin from melanocyte cell lysates [194], but whether this interaction is physiologically relevant is not clear. BLOC-2-deficient mice are more mildly hypopigmented than BLOC-1 mutants, and defects in melanosome architecture vary from dramatic in the RPE and choroid to less severe in hair-bulb melanocytes [176, 177]. The subcellular distribution of several melanosomal proteins is altered in melanocytes from BLOC-2-deficient patients and mouse models [187, 195–198], but as in BLOC-1deficient cells, Tyrp1 is particularly depleted from melanosomes. Both Tyrp1 and tyrosinase accumulate in vesicles [195, 197] and in large tubulovesicular endosomal structures that are distinct from those that accumulate in BLOC-1 mutants [187], perhaps corresponding to transport intermediates between endosomes and melanosomes [187]. Moreover, Tyrp1 is more rapidly degraded in BLOC-2-deficient melanocytes than in controls [181, 197], suggesting increased missorting to lysosomes. Together, these phenotypes suggest that BLOC-2 might function in the same pathway as BLOC-1 but downstream, perhaps by regulating the directed targeting or fusion of BLOC-1-dependent transport carriers with maturing melanosomes. Consistently, BLOC-2 can be detected on endosomal tubules and can physically interact with a cohort of BLOC-1 [181]. Further analysis is needed to determine whether BLOC-2 indeed functions at this or a distinct step in cargo transport to melanosomes. 9.4.2.2.3 BLOC-3 Although most HPS-associated protein complexes clearly function in vesicular trafficking of melanogenic enzymes, the function of BLOC-3 is less clear. BLOC-3 is an approximately 146-kDa complex consisting of two subunits, HPS1 and HPS4 [199–202], and is largely present in the cytosol with a small fraction associated with membranes [201, 202]. The genes encoding these subunits are mutated in HPS types 1 and 4 – the most prevalent forms of HPS – and corresponding mouse models pale ear and light ear [89, 170]. Melanosomes in RPE and melanocytes of BLOC-3-deficient patients and mice are often misshapen – either grossly enlarged or excessively small and deformed, depending on the tissue [196, 203–206] – but cargo localization defects have not yet been observed [196]. Melanocytes from patients with HPS type 1 display increased autophagy and accumulate melanosomes in compartments with features of autophagic vacuoles [207]. This suggests that the morphological disruption of melanosomes may be a consequence of a defect in a process other than trafficking of melanogenic enzymes. Indeed, in nonpigmented cells, BLOC-3 has been reported to regulate late endosome/

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lysosome motility and distribution [208], which may have secondary consequences on endosomal membrane dynamics. HPS1 has been localized to the melanosomal membrane and to tubulovesicular membranes close to the Golgi [209]. Interestingly, the only identified interacting partner of BLOC-3 is the small GTPase Rab9 [200], which functions in recycling from late endosomes/lysosomes. However, despite the GTP dependence of this interaction, suggesting that BLOC-3 is a Rab9 effector [200], it is not clear whether Rab9 functions in melanogenesis. 9.4.3 Molecular Motors and the Cytoskeleton

In melanocytes, fully pigmented stage IV melanosomes accumulate towards the cellular periphery after capture and immobilization on peripheral actin filaments, ensuring their efficient transfer to keratinocytes (see Chapter 10). However, during their formation and maturation, they undergo rapid bidirectional movements along microtubules, both toward the cell center and toward the cell periphery, as well as shorter localized movements along actin filaments [210]. These movements are coordinated by molecular motors – myosins on actin, and kinesins and dynein on microtubules. Recent studies suggest that one microtubule motor, the kinesin KIF13A, plays an important role in melanosome maturation. KIF13A is an ubiquitously-expressed plus end-directed kinesin belonging to the kinesin-3 superfamily [211]. KIF13A has been shown to facilitate the transport of mannose 6-phosphate receptors from the TGN to the plasma membrane by virtue of an interaction with the ear domain of the β1-adaptin subunit of AP-1 [211]. KIF13A was identified in a proteomic analysis of melanosomes [43]. We have shown that in melanocytic cells, KIF13A localizes largely to recycling endosomes and coordinates endosomal positioning in the cell periphery near melanosomes [162]. Consistent with the physical interaction between KIF13A and AP-1, KIF13A depletion from melanocytic cells by small interfering RNA mimics AP-1 depletion by inducing hypopigmentation, perinuclear clustering of recycling endosomes, and entrapment of Tyrp1 on enlarged endosomal vacuoles. This interaction between an AP complex and a molecular motor provides a previously unappreciated link between cargo sorting and endosome positioning to create specialized subdomains of the endosomal recycling system. These specialized domains can be used as gated conduits through which cell-type specific cargoes are delivered to maturing melanosomes. Our observations suggest that AP-1 and KIF13A might cooperate with AP-3, BLOC-1, and BLOC-2 to control the sorting of melanosomal cargoes from early endosomes and their transport to maturing melanosomes. Defining how these complexes are coordinated during cargo transfer is a challenge for the future. Actin-based motility plays important roles in organelle maturation and fusion events as well. The actin-based motor, myosin Ib, has been proposed to facilitate maturation of stage I melanosomes [212]. Myosin Ib is a monomeric and nonprocessive motor, and has been detected in association with endosomes and the plasma membrane [212]. In melanocytic cells, myosin Ib was shown to coimmu-

9.4 Melanosome Maturation: Cargo Sorting to Mature Melanosomes

noprecipitate with Pmel17, and overexpression of a dominant-negative form of myosin Ib inhibited Pmel17 proteolytic maturation and incorporation in intraluminal vesicles [212]. It is possible that myosin Ib functions to tether Pmel17 to endosomal subdomains destined for invagination to form intralumenal vesicles. Alternatively, it might anchor endosomal subdomains to the actin cytoskeleton to allow for force generation sufficient to drive membrane deformations that are required for intralumenal vesicle formation. In either case, myosin Ib is a candidate to control the formation of stage I melanosomes. 9.4.4 SNAREs, Rabs, and Other Regulators

The directed targeting of AP-3-, AP-1-, and/or BLOC-1-dependent cargo transport carriers toward maturing melanosomes and their fusion with these compartments is likely to require a large cohort of additional molecular components. Membrane fusion processes throughout the secretory and endocytic pathways are mediated by SNARE family proteins [213]. Distinct sets of SNAREs localize to each membrane compartment and transport intermediate, and can be classified as either vesicle or vSNAREs or components of target or tSNAREs. Fusion occurs when the α-helical regions of cognate vSNAREs on the vesicle/source membrane and tSNARE complexes on the target membrane assemble into a four-helix bundle, resulting in close apposition and destabilization of the two membranes that ultimately results in fusion. To date, the SNAREs that mediate fusion of transport intermediates with maturing melanosomes have not yet been defined. However, multiple endosomal SNAREs are upregulated upon differentiation of a melanocytic cell line [214]. One of them, syntaxin-13, is a strong candidate for facilitating the fusion of recycling endosomal transport intermediates with maturing melanosomes. Although syntaxin-13 has been proposed to function in homotypic endosome–endosome fusion [215], it is predominantly localized to tubular recycling endosomes in many cell types [216] and likely functions in fusion events in the endocytic recycling pathway. Syntaxin-13 has been shown to directly interact in vitro with BLOC-1 via the pallidin subunit [188, 189, 217] and is mislocalized in AP-3-deficient melanocytes [187]. Another excellent candidate is the vesicle-associated membrane protein, VAMP7 (also called TIVAMP for tetanus-insensitive vesicle-associated membrane protein). VAMP7 physically and functionally interacts with AP-3 [218], and is mislocalized and selectively degraded in neuronal cells that lack AP-3 or BLOC-1 [191], suggesting that it might be a key target of these HPS-associated protein complexes. It is tempting to speculate that VAMP7 and syntaxin-13 are components of the SNARE complex that mediates fusion of recycling endosomal tubules with maturing melanosomes. Another likely SNARE regulator is the homotypic fusion and vacuole protein sorting (HOPS) complex. HOPS is an evolutionary conserved complex that appears to function both in membrane tethering and in regulating SNARE-dependent fusion in yeast during homotypic vacuole fusion [219] and likely plays a similar

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role to regulate endosomal dynamics in mammals [220]. HOPS is comprised of six different subunits. One of them, Vps33, belongs to a family of proteins that interact directly with SNAREs [221], and facilitates SNARE pairing and fusogenic activity [222]. At least two Vps33 isoforms are present in higher eukaryotes and an amino acid substitution in one of them – Vps33a – underlies the HPS-like phenotype of buff mice [223]. Vps33a binds to a number of endosomal SNAREs in mammals, including syntaxin-13 [220], and is therefore poised to regulate fusion between endosomal transport carriers and maturing melanosomes either by recruiting and/or activating SNAREs or by participating directly in the fusion process itself [224]. Melanosomes in RPE of buff mice are small and sparse, and melanocytes derived from buff mice are severely hypopigmented [223], but hair-bulb melanocytes in buff mice show minor defects in melanosome architecture [177]. The Rab family of small GTPases are also key regulators for many membrane trafficking events, particularly in modulating the function and homeostasis of membrane domains [75, 225]. Over 60 different Rabs in mammals function on distinct membranes as molecular switches: when bound to GTP they recruit effectors to their resident membranes, but they release these effectors upon GTP hydrolysis. A number of Rab proteins are selectively expressed at high levels in melanocytes as compared to other tissues [226]. Among them, Rab32 and Rab38 have been shown to be required for the efficient delivery of tyrosinase and Tyrp1 to melanosomes. Rab32 and Rab38 are highly homologous proteins that are expressed in a limited set of cell types, including melanocytes, and that localize in melanocytes to mature melanosomes and tubulovesicular structures that likely correspond to endosomal domains [227]. The Rab38 gene is mutated in ruby rats [228], which suffer from a HPS-like phenotype, and in chocolate mice, which are hypopigmented [229]. Moreover, a mutation in lightoid, the Drosophila melanogaster ortholog of both Rab32 and Rab38, causes defects in eye pigmentation [230]. Interestingly, whereas melanocytes derived from mutant chocolate mice are only mildly hypopigmented, additional depletion of Rab32 severely impairs the delivery of Tyrp1 and tyrosinase to melanosomes, melanosome morphology and pigmentation [227]. These Rab proteins likely function in a redundant way in melanocytes, although perhaps less redundant in RPE [14], to regulate the stability and/or targeting of melanogenic cargo transport intermediates [14]. Additional evidence for the importance of Rabs during melanogenesis comes from analysis of the gunmetal mouse, another of the mouse HPS models. The phenotype results from a mutation in the gene encoding the α subunit of Rab geranylgeranyl transferase II [231], an enzyme that catalyzes prenylation of Rab proteins, which is required for their membrane anchoring and stability. The membrane recruitment of a number of Rabs was greatly reduced in platelets and melanocytes from gunmetal mice relative to controls [226, 231], suggesting that some of them might participate in melanosome biogenesis or function. One was Rab27a, which functions in melanosome motility (see Chapter 10). Another was Rab11, which regulates membrane dynamics within the endocytic recycling

9.5 Conclusions

system [75]. Rab11 was recently predicted to be a BLOC-1-interacting partner [232], and a genetic interaction between Rab11 and BLOC-1 has been observed in D. melanogaster [189]. It is tempting to speculate that Rab11 might regulate BLOC-1dependent cargo delivery between recycling endosomes and melanosomes [162]. The ubiquitously expressed Rab7, which functions in late endosome membrane dynamics and fusion with lysosomes [75], has been implicated in regulating Tyrp1 trafficking [233], but it is not clear whether this reflects a direct role for Rab7 in regulating the dynamics or formation of transport intermediates or a more direct role in premelanosome motility [234] or early endosome maturation [235]. Proteomics analyzes show that a number of other Rab proteins associate with melanosomes [42, 43], but whether any of them play functional roles in melanosome formation or motility is not yet clear. 9.4.5 Lipids

One other contributing factor to melanosomal enzyme sorting is the lipid environment of the late secretory and endocytic pathway. Glycosphingolipids are essential components of these membranes, and accumulate in “lipid rafts” at the plasma membrane and in endosomal compartments. Two studies have shown that melanocytes that lack a key enzyme in glycosphingolipid synthesis, ceramide glucosyltransferase, are severely hypopigmented due to missorting of tyrosinase and other melanogenic proteins such as Tyrp1 [236, 237]. Whereas Tyrp1 and tyrosinase in wild-type melanocytes are directly transported from the TGN to endosomal transport intermediates for delivery to melanosomes [156, 237], both proteins are diverted through the plasma membrane in glycosphingolipid-deficient melanocytes [236, 237]. These effects appear to be mediated via the lumenal domains of the melanosomal proteins [236], suggesting a potential interaction between glycosphingolipid head groups and melanosomal enzymes. How these effects are mediated is not yet clear, but represent exciting implications for the role of the lipid bilayer in controlling protein trafficking fates.

9.5 Conclusions

The findings from the past recent years have started to unravel how melanocytes integrate unique and ubiquitous molecular mechanisms in exploiting the endosomal system to generate melanosomes. The subversion of the endocytic pathway to form melanosomes in melanocytes parallels similar processes for the generation of other LROs, but also additional specialized organelles such as synaptic vesicles in neurons that also originate from specialized endosomal trafficking. In melanocytes, as in other cells hosting LROs, such as platelets, B lymphocytes, and dendritic cells, the multivesicular endosome is a common intermediate playing a key role in the concentration of cargo involved in organelle morphogenesis and

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function. In melanocytes, these MVBs offer an optimal environment for the formation of Pmel17-driven amyloid fibrils with features of pathological amyloid. The mechanisms, membrane domains, and molecular components involved in the endosomal sorting and the subsequent cleavage of the protein are still far from being fully elucidated. Further studies will help to unravel how within the endosomal membrane this amyloidogenic protein segregates together with proteases, what are the fates of the two domains (amyloidogenic lumenal domain and the C-terminal fragment), and what are the major effectors of these processes. A better comprehension of these mechanisms operating for a physiological amyloid is also certainly an asset to understand the molecular events leading to the formation of pathological amyloid fibrils and their intermediates accumulating in neurodegenerative diseases (Alzheimer’s, prion diseases). In late stages of melanogenesis pigment synthesis is under the control of HPS gene products. Some of these molecular components have been highlighted as novel trafficking regulators. It is unclear how these molecular complexes (BLOC) operate to associate with membranes and regulate cargo sorting. Some of their major interactors have started to be pinpointed mostly from studies in Drosophila and plants (SNAREs, Rab GTPases, retromer components). Additional studies are certainly required to decipher how these ubiquitous components operate in concert with the endosomal trafficking machinery for the generation of tubulovesicular carriers and their transfer to target membranes (i.e., the melanosome). We also need to know more about how melanosome membrane transporters modulate membrane dynamics via the lumenal environment of source and target compartments. Most of the current models for melanosome biogenesis arise from studies on melanocytic cell models of eumelanogenesis. Suitable cell models will be invaluable to enable the elucidation of the basic mechanisms involved in the biogenesis of pheomelanosomes, which will allow conceptual working models to be drawn in parallel for both types of melanogenesis. Finally, given the importance of the keratinocyte in driving melanogenesis, it will be of interest to understand how establishment of the pigmentation synapse influences melanosome biogenesis and melanosome maturation before final transfer.

Acknowledgments

We thank all members of our laboratories for their contributions to many of the studies described here, including Figures 9.1, 9.4, and 9.5 (I. Hurbain), Figure 9.4 (G. van Niel), and Figure 9.5 (D. Tenza). We are grateful for all the daily stimulating discussions. We apologize to those authors whom we did not cite. This work was funded by grants R01 EY015625 and R01 AR048155 from the National Institutes of Health (to M.S.M. and G.R.), and Institut Curie, CNRS, Fondation pour la Recherche Médicale, Institut National du cancer (INCA), Association pour la Recherche sur le Cancer (to G.R.).

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E.K., Panthier, J.J., Suprabha, P.G., Vida, T., Swank, R.T., and Spritz, R.A. (2003) The mouse organellar biogenesis mutant buff results from a mutation in Vps33a, a homologue of yeast vps33 and Drosophila carnation. Proc. Natl. Acad. Sci. USA, 21, 21. Nickerson, D.P., Brett, C.L., and Merz, A.J. (2009) Vps-C complexes: gatekeepers of endolysosomal traffic. Curr. Opin. Cell Biol., 21, 543–551. Zerial, M. and McBride, H. (2001) Rab proteins as membrane organizers. Nat. Rev. Mol. Cell Biol., 2, 107–119. Zhang, Q., Zhen, L., Li, W., Novak, E.K., Collinson, L.M., Jang, E.K., Haslam, R.J., Elliott, R.W., and Swank, R.T. (2002) Cell-specific abnormal prenylation of Rab proteins in platelets and melanocytes of the gunmetal mouse. Br. J. Haematol., 117, 414–423. Wasmeier, C., Romao, M., Plowright, L., Bennett, D.C., Raposo, G., and Seabra, M.C. (2006) Rab38 and Rab32 control post-Golgi trafficking of melanogenic enzymes. J. Cell Biol., 175, 271–281. Oiso, N., Riddle, S.R., Serikawa, T., Kuramoto, T., and Spritz, R.A. (2004) The rat Ruby (R) locus is Rab38: identical mutations in Fawn-hooded and Tester-Moriyama rats derived from an ancestral Long Evans rat sub-strain. Mamm. Genome, 15, 307–314. Loftus, S.K., Larson, D.M., Baxter, L.L., Antonellis, A., Chen, Y., Wu, X., Jiang, Y., Bittner, M., Hammer, J.A., 3rd, and Pavan, W.J. (2002) Mutation of melanosome protein RAB38 in chocolate mice. Proc. Natl. Acad. Sci. USA, 99, 4471–4476. Ma, J., Plesken, H., Treisman, J.E., Edelman-Novemsky, I., and Ren, M. (2004) Lightoid and Claret: a rab GTPase and its putative guanine nucleotide exchange factor in biogenesis of Drosophila eye pigment granules. Proc. Natl. Acad. Sci. USA, 101, 11652–11657. Detter, J.C., Zhang, Q., Mules, E.H., Novak, E.K., Mishra, V.S., Li, W., McMurtrie, E.B., Tchernev, V.T., Wallace, M.R., Seabra, M.C., et al. (2000) Rab geranylgeranyl transferase alpha mutation in the gunmetal mouse

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9 Biogenesis of Melanosomes reduces Rab prenylation and platelet synthesis. Proc. Natl. Acad. Sci. USA, 97, 4144–4149. 232 Rodriguez-Fernandez, I.A. and Dell’Angelica, E.C. (2009) A data-mining approach to rank candidate proteinbinding partners – the case of biogenesis of lysosome-related organelles complex-1 (BLOC-1). J. Inherit. Metab. Dis., 32, 190–203. 233 Hirosaki, K., Yamashita, T., Wada, I., Jin, H.Y., and Jimbow, K. (2002) Tyrosinase and tyrosinase-related protein 1 require Rab7 for their intracellular transport. J. Invest. Dermatol., 119, 475–480. 234 Jordens, I., Westbroek, W., Marsman, M., Rocha, N., Mommaas, M., Huizing, M., Lambert, J., Naeyaert, J.M., and Neefjes, J. (2006) Rab7 and Rab27a control two motor protein activities involved in melanosomal transport. Pigment Cell Res., 19, 412–423.

235 Rink, J., Ghigo, E., Kalaidzidis, Y., and

Zerial, M. (2005) Rab conversion as a mechanism of progression from early to late endosomes. Cell, 122, 735–749. 236 Groux-Degroote, S., van Dijk, S.M., Wolthoorn, J., Neumann, S., Theos, A.C., De Mazière, A.M., Klumperman, J., van Meer, G., and Sprong, H. (2008) Glycolipid-dependent sorting of melanosomal from lysosomal membrane proteins by lumenal determinants. Traffic, 9, 951–963. 237 Sprong, H., Degroote, S., Claessens, T., van Drunen, J., Oorschot, V., Westerink, B.H., Hirabayashi, Y., Klumperman, J., van der Sluijs, P., and van Meer, G. (2001) Glycosphingolipids are required for sorting melanosomal proteins in the Golgi complex. J. Cell Biol., 155, 369–380.

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10 Transport and Distribution of Melanosomes Mireille Van Gele and Jo Lambert

10.1 Introduction

The light-absorbing pigment melanin is produced and stored within specialized lysosome-related organelles termed melanosomes, present in the melanocytes [1]. Two major types of melanin are synthesized inside melanosomes: eumelanin (black/brown) and pheomelanin (yellow/red). The formation of both pigments starts with the enzymatic oxidation of tyrosine, but then follows different metabolic pathways (see Chapter 4). Each epidermal melanocyte provides melanin to approximately 36 keratinocytes using its dendrites. After transfer, melanin is transported to the apical face of the keratinocyte nucleus, where it protects the genetic material against UV damage. This symbiotic system is called the “epidermal melanin unit” and is primarily responsible for skin pigmentation. Extensive studies on pigment cells of lower vertebrates and mammals revealed that the transport of melanosomes from their site of synthesis (i.e. cell center) to the cell periphery occurs via fast, long-range transport along microtubules, followed by a short-range movement along actin filaments. Three different classes of motor proteins were shown to be involved in this intracellular transport: kinesins, dyneins, and myosin Va (Myo5a). In melanophores of fish and frogs they are responsible for fast aggregation and dispersion of melanosomes in response to variations in the environment [2]. In retinal pigment epithelium (RPE) cells of the eye these motor proteins guarantee transport of melanosomes into the apical processes in response to light [3]. Studies of naturally occurring mouse color mutants revealed that the transport of melanosomes along actin filaments and their capture in the cell periphery is accomplished by the action of a Rab27a–Mlph (melanophilin)– Myo5a tripartite complex. The importance of this complex was also confirmed in human melanocytes and mutations in MYO5A, RAB27A, or MLPH result in Griscelli syndrome (GS) types I, II, and III, respectively. These patients are characterized by pigment dilution of the skin and hair, due to defects involving melanosome transport in melanocytes. A similar tripartite complex, consisting of Rab27a–Myrip–Myo7a (myosin VIIa), was discovered in mouse RPE cells.

Melanins and Melanosomes: Biosynthesis, Biogenesis, Physiological, and Pathological Functions, First Edition. Edited by Jan Borovanský and Patrick A. Riley. © 2011 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2011 by Wiley-VCH Verlag GmbH & Co. KGaA.

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Likewise, this complex is responsible for the transport of melanosomes along actin filaments and their tethering in actin-rich apical processes. Epidermal melanocytes transfer mature melanosomes along the dendritic processes to neighboring keratinocytes. The mechanisms behind this transfer process have proved to be difficult to unravel. Different hypotheses, such as exocytosis, cytophagocytosis, and tunnel formation, were proposed. With the development of high-resolution assays (in space as well as in time) the evidence is growing that filopodia are used as conduits for intercellular transfer of melanosomes. Whether filopodia eventually connect cytoplasms forming a tunnel or they are phagocytosed by the receiving cell (the keratinocyte), as argued by Singh et al., remains to be further substantiated [4]. Inside keratinocytes the melanosomes continue their journey and travel along microtubules in the direction of the cell center. They tend to aggregate above the keratinocyte nucleus in lightly pigmented skin, while in darker phototypes they are dispersed throughout the cytoplasm. Little is known about the signals that regulate this distribution pattern. Communication and melanin transfer between individual keratinocytes may play an important role in this process.

10.2 Model Systems to Study Pigment Transport 10.2.1 Melanophores from Fish and Amphibians

In fish and amphibians the melanosomes are found in melanophores – large black pigmented cells present in the dermis and epidermis. The major function of these cells involves aggregation of pigment granules in the center of the cell (resulting in skin lightening) or dispersal of them throughout the cytoplasm (causing a dark appearance of the skin). This bidirectional and coordinated transport of pigment granules (i.e., physiological color change) allows the animal to effect color changes important for camouflage and social interactions. Similar to mammals, the melanosomes of amphibians can also be dispersed and transferred to surrounding cells, providing long-term morphological changes. Much of our current understanding of melanosome motility and its regulation is derived from studies of fish and frog melanophores (Figure 10.1a). They are regarded as excellent model systems for the analysis of organelle transport for several reasons: (i) intracellular movements of their large pigment granules can easily be monitored by conventional bright-field microscopy, (ii) bidirectional transport can be experimentally manipulated by treatment of the cells with neurotransmitters (fish) or hormonal stimuli (fish and frogs), and (iii) the availability of an immortalized cell line of Xenopus melanophores [7]. In fish, melanosome movements are fast and tightly regulated due to the longer and unidirectional movements that they undergo. In frog melanophores, melanosome transport is bidirectional, including frequent pauses and reversals that

10.2 Model Systems to Study Pigment Transport a)

b)

c)

Figure 10.1 (a) Bright field images of frog melanophores (Xenopus laevis) illustrating

dispersed pigment (left) and aggregated pigment (right). Bars = 10 μm. (Reproduced from [5].) (b) Schematic view of a melanophore showing the dispersal and aggregation of pigment granules that occur along microtubules under the influence of cAMP. (c) Bright field images of wild-type (left) and ashen mouse melanocytes (right). Bars = 12 μm. (Reproduced from [6].)

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increase the amount of time necessary to aggregate or disperse. In this context, transport in frog melanophores resembles more the organelle transport occurring in other cell types. In both cases, however, melanosome dispersion is induced by elevation of intracellular cAMP levels, while aggregation is triggered by depression of cAMP (Figure 10.1b). The downstream steps of these second messengers are poorly understood but seem to involve protein kinases and phosphatases [5]. Several morphological and functional transport studies in melanophores revealed that two elements of the cytoskeleton – microtubules and actin filaments – are required for the movement of pigment in these cells. Microtubuledependence of melanosome transport could be demonstrated by treating fish melanophores with low temperature, high hydrostatic pressure, or microtubuledisrupting drugs. These treatments resulted in the inhibition of both aggregation and dispersion [8, 9]. Additional studies showed that dynein, a minus-end-directed microtubule motor, colocalized with fish [10] and amphibian melanosomes [11], and that it was involved in the aggregation of these melanosomes [12]. By using cultured Xenopus melanophores, Rogers et al. were also able to show that kinesin-2 was present on melanosomes [11]. A year later, Tuma et al. showed, by using a dominant-negative mutant of kinesin-2, that it was also the motor protein responsible for dispersion of melanosomes in Xenopus melanophores [13]. The same authors suggested that kinesin-2, and not conventional kinesin, is responsible for dispersion in fish melanophores as well [14]. Parallel to the studies described above, other research groups performed studies on fish melanophores that could, however, demonstrate that removal of microtubules did not completely abolish pigment movement, suggesting also a role for the actin cytoskeleton in pigment transport [15]. In addition, studies on frog melanophores have shown that removal of actin filaments inhibited dispersion of melanosomes [16, 17] and that actin filaments were indeed present on the cell periphery of melanophores [18]. Later, it was demonstrated that the motor protein Myo5a was responsible for actin-based transport of melanosomes in Xenopus melanophores [19]. Moreover, the actin filaments were mainly required for proper dispersion and for keeping the melanophores dispersed [17]. Next, Sköld et al. demonstrated that Myo5a was present on fish melanosomes as well, but the actinbased transport seemed to play a less important role for the overall bidirectional translocations in fish [20]. To discover in more detail how the motor proteins dynein, kinesin, and Myo5a are regulated in melanophores and cooperate with each other to move along their cytoskeleton tracks we refer the reader to two excellent reviews on this topic [2, 21]. 10.2.2 Mammalian Melanocytes

Mammalian melanocytes have been widely used to study the transport of melanosomes. Extensive studies of mouse and human melanocytes showed that, similar to what is observed in melanophores of lower vertebrates, both microtubules and actin filaments are required for melanosome transport. A major advantage of

10.3 Intracellular Melanosome Transport

mammalian melanocytes lies in the fact that a number of naturally occurring mouse coat color mutants with defects in melanosome transport exist: dilute, ashen, and leaden [22–24]. Normal levels of melanin are synthesized in these mouse mutants, but melanosomes are not efficiently distributed to neighboring keratinocytes. Instead they clump around the melanocyte cell nucleus (Figure 10.1c). Molecular genetic studies led to the discovery of the genes involved and uncovered the role of the Rab27a–Mlph–Myo5a complex in melanosome transport. Myo5a is the actin-based motor protein that transports melanosomes along the sublemmal and filopodial network. Two molecules – Mlph and Rab27a – are necessary for the attachment of Myo5a to the melanosome surface [25]. The important role of this tripartite complex in melanosome transport was also confirmed in human melanocytes and the mouse coat color mutants find their human counterparts in GS type 1 (MYO5A), 2 (RAB27A), and 3 (MLPH) [26]. 10.2.3 RPE Cells

RPE cells lie at the back of the vertebrate eye, and they synthesize melanin pigments and store them within numerous melanosomes. RPE cells closely interact with photoreceptors – a critical action for the maintenance of visual function. In eyes of lower vertebrates, which do not have dilatable pupils, the melanosomes in the RPE cells undergo massive, light-dependent migrations, whereas the movements in mammals are more subtle. Some of the properties of pigment transport in RPE cells are similar to those observed in melanophores. For instance, the bidirectional transport of melanosomes and the induction of pigment aggregation or dispersion by treatment with cAMP or dopamine, respectively. Fish RPE cells have mainly been used to study the dual role of microtubules and actin filaments in melanosome transport [27]. Studies on mouse cells led to the discovery that the actin-based motor Myo7a is involved in melanosome transport in RPE cells, whereas Myo5a accomplishes this function in epidermal melanocytes [28]. This finding provided evidence that Rab27a can link several classes of myosin motors to the organelle surface [3, 29] (see Section 10.4).

10.3 Intracellular Melanosome Transport

Inside mammalian melanocytes, mature melanosomes are transported from the perinuclear area to the cell periphery and dendritic tips along the microtubular network. This fast, long-distance transport is mediated by two classes of cytoskeletal motor proteins: dyneins and kinesins. Upon arrival at the cell periphery, melanosomes undergo “short-range” movements along the sublemmal actin network through the association with the processive motor protein Myo5a, which attaches to melanosomes through interaction with Mlph and Rab27a (Figure 10.2).

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Intracellular transport of melanosomes. The tip of a melanocyte dendrite is depicted. Two motor proteins transport melanosomes along microtubules: kinesin centrifugally and dynein centripetally. Dynein mainly localizes to early melanosomes, and probably binds them via interaction with Rilp and Rab7. Mature melanosomes travel towards the dendrite

Figure 10.2

tips where they hop onto the sublemmal actin network. Transport along the actin filaments is accomplished by the action of the Rab27a–Mlph–Myo5a complex. When Rab27a binds another effector, Slp2-a, melanosomes attach to the plasma membrane. Melanosomes are now ready for release and transfer to keratinocytes.

10.3.1 Microtubule-Based Transport 10.3.1.1 Kinesin and Dynein Two motor proteins orchestrate the transport of melanosomes along microtubules: kinesin for anterograde movement towards the microtubule plus-ends located at the cell periphery and dynein for retrograde movement towards the microtubule minus-ends located at the cell center [30, 31]. This area is actually less extensively studied in melanocytes and knowledge from other cell systems, such as neurons, opens areas for future research around these motors in melanocytes [32]. Melanocytic dendrites consist of a central core of microtubules and a subcortical actin network. In previous reports we showed the presence of microtubuleassociated motor proteins kinesin and cytoplasmic dynein on the melanosomal surface, forming a link with microtubules [33, 34]. We could also demonstrate the association of kinectin, the kinesin receptor, with melanosomes. The interaction of cytoplasmic dynein with its cargoes is thought to be indirectly mediated by dynactin, a complex that binds to the dynein intermediate chain. The dynactin subunits P150Glued and P50 colocalize with the melanosomal membrane [35]. Spe-

10.3 Intracellular Melanosome Transport

cifically, Gross et al. propose that dynactin enables dynein to participate efficiently in bidirectional transport, increasing its ability to stay “on” during minus-end motion and keeping it “off” during plus-end motion [36]. Dynein is the common motor protein found in early, unpigmented, melanosomes. Probably, Rab7 recruits dynein via its interaction with Rab7-interacting lysosomal protein (Rilp). Rab7 mainly localizes to early stage melanosomes and these melanosomes locate to the cell center upon overexpression of Rilp [37]. Instead, kinesin is enriched in mature melanosomes [38]. This is consistent with the ongoing maturation of early melanosomes in the perinuclear area and the transport of mature melanosomes, ready for delivery to keratinocytes, to the dendrite tips (Figure 10.2). Next to their role in melanosomal organelle transport, these motors also play a role in positioning and correctly localizing endosomal proteins and domains during the biogenesis of melanosomes [38, 39] 10.3.2 Actin-Based Transport 10.3.2.1 MYO5A MYO5A functions as a dimeric, actin-based molecular motor protein, and is divided into three separate domains termed “head”, “neck, ” and “tail” (Figure 10.3). First,

Figure 10.3 Schematic view of active MYO5A. At the N-terminus MYO5A is composed of two motor domains (MD) that contain ATP- and actin-binding sites followed by a neck domain consisting of six IQ motifs. The tail region comprises a proximal and

medial domain, and contains three major α-helical coiled-coil segments interrupted by two small uncoiled regions. The distal or globular tail domain at the C-terminus is responsible for cargo binding via cargospecific receptors.

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MYO5A is composed out of two separate N-terminal (or “head”) motor domains, containing ATP-binding sites as well as having the capability to bind to actin fibers. These ATP-binding sites can convert energy released by multiple catalytic cycles of ATP hydrolysis, generating a mechanical movement along the actin filaments. Following these two separate head domains are two identical heavy chains that dimerize into each other. These two α-helical coils termed the “neck” domains are comprised of six subsequent amino acid residues termed IQ motifs with the consensus sequence IQXXXRGXXXR. These IQ motifs act as binding sites for light chains, which are either calmodulin or calmodulin-like light chains and have a regulatory function by controlling the ATPase activity of the globular heads [40]. The “tail” (proximal/medial) domain commences at the site where both α-helical coils dimerize to form a homodimer made up of a series of coiled-coil segments interrupted by small flexible globular regions (around 500 amino acids). The length and composition of the medial tail region varies depending on alternative splicing. In humans, six different isoforms have been identified that are expressed in certain cell types, and each isoform specifies which cargo is bound and transported. Transcripts containing the exon F were found to be the isoforms intervening in melanosome transport [41]. Finally, the last approximately 400 amino acids distinguish the distal/globular tail region, also known as the cargo-binding site, where the interaction with different cargos is mediated by organelle specific receptors. Mercer et al. were the first to report that the murine Myo5a (dilute) locus encodes the heavy chain of Myo5a, a processive molecular motor [23]. More then 20 years ago, the GS was mapped by Pastural et al. to 15q21, the region where MYO5A is located [42]. The presence of mutations in MYO5A [42] and in its murine counterpart [23] led to the identification of MYO5A to be the first gene involved in GS. This disease was therefore termed GS1 (OMIM #214450). In general, these patients show hypomelanosis with a primary neurological deficit and without immunological impairment or manifestations of hemophagocytic syndrome. MYO5A mutations were identified in only two GS patients [42, 43]. However, the low detection frequency of MYO5A mutations in other patients [44] suggested the existence of a second GS gene also located at 15q21 [31, 43]. 10.3.2.2 RAB27A The Ras super family of small GTPase monomeric G-proteins (20–25 kDa) is essential in regulating a wide variety of cell processes, including vesicular transport pathways, cellular differentiation, and motility [45]. RAB27A is a member of this family and has the ability to act as a molecular switch by cycling between two conformations through the binding of GTP, rendering it in an “active” state or hydrolyzing GTP to GDP, translating it to its “inactive” form. Two regions have been shown to change conformation upon GDP or GTP binding and have been termed switch I and switch II regions. A guanine nucleotide exchange factor (GEF), called Rab3GEF, is responsible for the activation of Rab27a in melanocytes [46]. Rab27a is a substrate for prenylation by an enzyme called Rab geranylgeranyl transferase. These prenyl groups are anchored to mature melanosomes and bind

10.3 Intracellular Melanosome Transport

to two conserved cysteine residues located at the C-terminal end, also referred to as geranylgeranylation motifs of Rab27a. This interaction can be considered similar to the labeling or tagging of membranes of vesicles, thus defining the identity and future routing of these vesicles. This implies that Rab27a is involved in targeting, docking and fusion of transport vesicles with their appropriate acceptor membranes [47]. RAB27A, like MYO5A, localizes to melanosomes and is mostly concentrated in melanosome-rich dendritic tips of wild-type melanocytes. Rab27a-deficient ashen melanocytes exhibit normal dendritic morphology and melanosome biogenesis but an abnormal accumulation of end-stage melanosomes in the cell center. Ménasché et al. were the first to demonstrate that RAB27A mutations found in GS patients were linked to GS2 [48], which accounts for most cases of GS to date [26]. Re-expression of RAB27A in GS melanocytes (or ashen melanocytes) restores the proper distribution of melanosomes to the dendritic tips, stressing its importance in melanosome trafficking. Coimmunoprecipitation studies with antibodies against Rab27a proved an association between Rab27a and Myo5a. This association was found to be indirect, and Mlph was later identified as the linker protein connecting both Rab27a and Myo5a (see Section 10.3.2.3). As mentioned above, the second type of GS (GS2, OMIM #607624) is caused by mutations in RAB27A, which is also located on chromosome 15q21. GS2 patients suffer from pigmentary dilution of skin and hair, and also develop immunodeficiency due to impaired lytic granule exocytosis in cytotoxic T lymphocytes. This can lead to episodes of a life-threatening uncontrolled T lymphocyte and macrophage activation syndrome also known as hemophagocytic syndrome or hemophagocytic lymphohistiocytosis [26]. Apart from its role in melanosome transport, the RAB27A protein is thus also involved in docking and release of lytic granules in cytotoxic T lymphocytes. 10.3.2.3 MLPH Matesic et al. identified melanophilin (Mlph) as the mutated gene in leaden (ln) mice [22]. Since the phenotype of the leaden mouse is similar to the ashen and dilute mouse, Mlph was considered as a potential new candidate gene that was mutated in GS. As perinuclear clustering of melanosomes was also observed in leaden melanocytes it was suggested that Mlph might function as part of a transport complex with Myo5a and Rab27a [22]. The newly identified Mlph or the synaptotagmin-like protein (Slp) lacking C2-a domains (Slac2-a) represented a novel class of the Slp family. All members of the Slp family contain an N-terminal Slp homology domain (SHD) consisting of two conserved potential α-helical regions (SHD1 and SHD2) often separated by two zinc finger motifs. In contrast to other Slp proteins, the Slac2 family (Slac2-a/ Mlph, Slac2-b, and Slac2-c/Myrip) lacks C-terminal tandem C2 domains (termed C2A and C2B). Instead, MLPH contains two unique coiled-coil domains (Coil 1 and Coil 2) at its C-terminal end [49]. Biochemical and cell biological analyses have now revealed that MLPH is the specific linker protein between MYO5A and RAB27A (for more details, see below).

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In 2003, the sole mutation in the human MLPH gene (located on 2q37), giving rise to GS type 3 (GS3, OMIM #609227), was documented [50]. Interestingly, phenotypic expression of GS3 is restricted to the characteristic hypopigmentation of this syndrome. Loss of function of MLPH does not cause neurological nor immunological defects. GS3-associated hypomelanosis is indistinguishable from that described in GS1 and GS2. A second Rab27a effector was also identified in melanocytes: Slp2-a. Slp2-a contains two C2 domains and silencing experiments demonstrated that the C2A domain binds phosphatidylserine, thereby attaching melanosomes to the plasma membrane [51]. Both Rab27a effectors thus act successively in melanosome transfer: Mlph in controlling transfer from microtubules to actin filaments as well as actin-based melanosome transport and Slp2-a in anchoring of melanosomes to the plasma membrane (see also Figure 10.2). This sequential action is accompanied by differences in Rab27a binding affinities, with low-affinity binding of Mlph and high-affinity binding of Slp2-a [52]. 10.3.2.4 RAB27A–MLPH–MYO5A Tripartite Protein Complex In the past, we have reported that human MYO5A undergoes tissue-specific alternative splicing located at its medial tail region leading to an alternate usage of three exons designated as B, D, and F [41]. Of the six known human isoforms (ABCDEF, ABCEF, ACDEF, ABCDE, ABCE, and ACE), three contain exon F. These exon F-containing isoforms are most abundantly expressed in melanocytes and were shown to colocalize with melanosomes [41, 53, 54]. Molecular studies (use of dominant-negative constructs, rescue experiments, yeast two-hybrid screenings) revealed that both the C-terminal globular tail domain and the exon F sequence are essentially required for Myo5a to colocalize with and influence the position of melanosomes in melanocytes [53, 54]. It became clear that Rab27a and Myo5a exon F-containing isoforms function as a receptor complex involving a specific linker protein. Yeast two-hybrid screenings containing exon F and globular tail constructs revealed specific interactions with a Rab effector protein termed Mlph [54, 55]. Other groups confirmed these observations by performing colocalization studies in leaden or ashen melanocytes [25, 56] or in vitro binding assays [57]. The region of Mlph responsible for binding of both Rab27a and Myo5a was mapped to its N- and C-terminus, respectively [49, 55, 57]. Based on the results summarized above, the recruitment of Myo5a to melanosomes occurs as follows: activated Rab27a first binds to the surface of the melanosomes and then recruits Mlph, which subsequently recruits the Myo5a exon F-containing isoforms (Figure 10.4a). It is now generally accepted that the Rab27a–Mlph– Myo5a tripartite complex is essential for the transportation of melanosomes from the microtubules to the actin filaments and for the retention of the melanosomes on the sublemmal actin cytoskeleton [54, 55, 57, 58]. Moreover, in vitro reconstitution of the Myo5a receptor complex by use of dominant active Rab27a and Mlph demonstrated that these proteins are not only necessary, but also sufficient, to form a transport complex that moves processively on actin [59]. Loss of one of the components of the tripartite complex results in GS, biologically

10.3 Intracellular Melanosome Transport a)

b)

Figure 10.4 Functional role of the Rab27a– Mlph–Myo5a tripartite complex in intracellular melanosome transport. (a) In melanocytes, activated Rab27a is present on mature melanosomes that move to the cell periphery on microtubules via the motor protein kinesin. Once arrived at the cell periphery, Mlph is directly assembled via its SHD1 domain (part of the Rab27a-binding domain, R27BD) to the switch II region of Rab27a. Finally, Myo5a is recruited to the Rab27a–Mlph complex through a direct interaction of its globular tail and exon F sequence with two distinct regions located in middle domain of Mlph (MBD). The microtubule end-binding protein (EB-1) physically interacts with the ABD of Mlph, but its exact function in melanosome transport still needs to be elucidated.

Eventually, a stable Rab27a–Mlph–Myo5a tripartite protein complex is formed that captures the melanosomes in the actin-rich dendritic tips, a necessary step before transfer of melanosomes to the surrounding keratinocytes. Loss of function of the tripartite complex due to mutations in MYO5A, RAB27A, or MLPH/SLAC2-A leads respectively to GS types 1 (GS1), 2 (GS2), or 3 (GS3). (b) Phase-contrast microscopy images of a normal melanocyte (a) compared to a GS-derived melanocyte (b) where the abnormal perinuclear accumulation of melanosomes is depicted. Bars: 25 μm. ZnF, zinc finger; GTBD, globular tail-binding domain; EFBD, exon F-binding domain; C, coil; 1–590, the amino acid sequence of mouse MLPH. (Reproduced from [26].)

characterized by a perinuclear accumulation of melanosomes in the melanocytes (Figure 10.4b). Several binding domains (and critical residues) of Mlph have been identified as being essential for the correct binding of Rab27a and proper recruitment of Myo5a. The N-terminal part of Mlph, containing SHD1, SHD2, and an intervening zinc

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finger domain, is responsible for interaction with Rab27a and is termed R27BD (RAB27A-binding domain). Within this region, SHD1 binds directly with the switch II region of the GTP-bound active form of Rab27a present on melanosomes [60, 61]. The globular tail and exon F sequence of Myo5a bind two distinct domains of Mlph; Myo5a-GT binds to a newly characterized region adjacent to SHD2 (mouse amino acids 147–240, termed GTBD). In comparison, Myo5a-exon F has been proven to bind to the middle region of Mlph (mouse amino acids 320–406, termed EFBD) (see also Figure 10.4a). The interaction between Mlph and Myo5aGT seems to be less stable and much weaker compared to the interaction between Mlph and Myo5a-exon F. Interestingly, studies with dilute missense mutations in Myo5a-GT impaired only the former interaction and not the latter, indicating that the Mlph–Myo5a-GT is physiologically relevant. As a consequence, both domains are essential and probably function in a synergistic manner during melanosome transport in melanocytes [62]. Hume et al. extended the results shown above by demonstrating that melanosomal Rab27a-GTP recruits Mlph to the melanosomes via interaction with both SHD1 and SHD2 [63]. In addition, the authors identified a coiled-coil structure in the C-terminal end (mouse amino acids 440–483), termed the actin-binding domain (ABD) of Mlph which is essential for the recruitment of Myo5a to the melanosomes by increasing the interaction of the Myo5a-binding domain (MBD) of Mlph with Myo5a. Additional binding partners, apart from the ones of the tripartite complex, were also identified for Mlph. The ABD of Mlph interacts with actin and with EB-1, the microtubule plus-end tracking protein (Figure 10.4a). It is suggested that interaction with actin might enhance the Myo5a motor activity [64]. The binding of EB-1 is proposed to allow transport of Mlph to the peripheral dendrite tips on the plusend tips of growing microtubules, where it would reside in complex with actinassociated Myo5a until a suitable Rab27a-associated melanosome could be captured [65]. The role of actin and EB-1 binding by Mlph in melanosome transport is disputed by recent research. Expression of Mlph mutants that lack the ABD in leaden melanocytes are able to rescue peripheral melanosome transport just like wild-type Mlph, and depletion of EB-1 using small interfering RNA does not affect melanosome transport [63, 66]. Further research will be necessary to determine if these interactions play functionally redundant roles or if they are involved in other processes (e.g., melanosome transfer). 10.3.2.5 RAB27A as a New MITF Target Gene In vitro studies in B16 mouse melanoma cells demonstrated that the α-melanocyte stimulating hormone, by means of activating the cAMP pathway, rapidly induces an increase in the interaction of Mlph with actin, resulting in a fast accumulation of melanosomes around the actin-rich region of the dendrite extremities. Additionally, cAMP stimulates the expression of Rab27a that could facilitate the interaction of melanosomes with cortical actin [67]. In melanocytes the effects of cAMP on melanin synthesis are mediated by microphtalmia-associated transcription factor (MITF) that plays an essential role in survival, migration, proliferation and differentiation of melanocytes during development. MITF controls the expression of

10.4 Melanosome Motility in RPE: The Rab27a–Myrip–Myo7a Tripartite Complex

genes essential for melanin synthesis (such as TYR (tyrosinase), TYRP1 (tyrosinaserelated protein 1), and DCT (dopachrome tautomerase; also known as TYRP2)) and for the maturation of melanosomes (MART-1, PMEL17 and GPR143 (OA1)) [68]. Interestingly, one study also demonstrated that Mitf contributes to melanosome distribution and dendrite formation of frog melanophores [69]. Based on the knowledge described above, Ballotti et al. decided to examine the possible role of MITF in melanosome transport. Silencing of MITF in human and mouse melanoma cells induced perinuclear aggregation of melanosomes, including a relocalization of RAB27A, MLPH, and MYO5A to the cell body caused by a dramatic decrease in RAB27A expression. Functional analysis of the RAB27A promoter revealed that MITF directly binds to two E-boxes in the proximal region of the RAB27A promoter, thereby stimulating the expression of RAB27A and facilitating its interaction with MLPH [70]. In this manner, loss of MITF inhibits the expression of RAB27A (and blocks the effect of cAMP on RAB27A), which consequently results in the impairment of the RAB27A–MLPH–MYO5A tripartite complex, thus explaining the abnormal melanosome distribution in MITF-deficient melanoma cells. The identification of RAB27A as a new MITF target gene links MITF to actin-dependent melanosome transport, an important step in melanocyte differentiation.

10.4 Melanosome Motility in RPE: The Rab27a–Myrip–Myo7a Tripartite Complex

In mammals, melanosomes are not solely found in epidermal melanocytes, but are also present in choroidal melanocytes of the eye and RPE cells. In the latter cell type, melanosome transport into apical processes, which surround the photoreceptor outer segments, is regulated by incident light and/or circardian rhythms [3, 29]. The molecular mechanisms involved in melanosome distribution have been examined in detail in mammalian RPE cells. Liu et al. demonstrated that RPE cells from shaker-1 mice, which carry a mutation in the gene encoding Myo7a, exhibit a perinuclear distribution of their melanosomes [71]. They are excluded from the apical processes indicating that Myo7a is required for movement. Further, Myo7a was detected on retinal melanosomes by immunoelectron microscopy [28, 71]. A human counterpart for Shaker-1 mice is found in Usher syndrome 1B and the role of Myo7a in human and mouse RPE cells is accepted as comparable [72]. Rab27a is also present on RPE melanosomes and the phenotype of ashen cells is similar to that of shaker-1 cells, with perinuclear aggregation of melanosomes [73, 74]. A Mlph analog, Myrip (also know as Slac2-c), was detected as a linker protein between Rab27a and Myo7a [28, 75]. In vitro studies revealed that Myrip acts as a multifunctional myosin-activating protein able to interact and activate both Myo7a and Myo5a, through different binding domains [76]. Conclusive evidence for the function of a Rab27a–Myrip–Myo7a tripartite complex in the transport of RPE melanosomes, similar to the function of Rab27a–Mlph–Myo5a in the

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transport of epidermal melanosomes, came from RNA interference studies. Loss of any of the three components resulted in a redistribution of melanosomes from the actin-rich apical region and processes to the microtubule-rich cell body, the same phenotype as observed in ashen and shaker-1 cells [77]. Interestingly, nocodazole treatment (to disrupt microtubules) of RPE cells leads to an almost complete loss of melanosome movement suggesting that, as in epidermal melanocytes, melanosome transport also requires microtubular motors such as kinesins and dyneins [77]. See Figure 10.5.

Melanosome transport in mouse RPE cells. Melanosomes display fast, bidirectional, microtubule-dependent long-range movements in the cell body driven by kinesin/dynein motor proteins. Rab27a, which is targeted to the melano-

Figure 10.5

some membrane, interacts with its effector, Myrip, which in turn binds to Myo7a. Myo7a then enables the retention and/or local movement of the melanosomes along the actin filaments of the RPE apical processes.

10.5 Melanosome Transfer

10.5 Melanosome Transfer

Melanosomes need to be delivered to surrounding skin cells in order to fulfill their function – protecting genetic material of skin cells against UV damage. This process of delivery of melanosomes from melanocytes to surrounding skin cells (i.e., keratinocytes) is called melanosome transfer. In contrast to the extensive knowledge on the transport of melanosomes inside the melanocyte, relatively little is known about the processes and molecules involved in their transfer. Different hypotheses have been postulated. They are (i) (cyto)phagocytosis, (ii) the release of melanosomes by the melanocytes (exocytosis) and the subsequent endocytosis (or phagocytosis) of naked melanin by the keratinocytes, (ii) the active transfer of melanosome-containing vesicles by the melanocyte into the keratinocyte, and (iv) fusion of plasma membranes with tunnel formation [78]. Transfer of melanin in the hair follicle from mature melanocytes residing at the hair bulb to cortical and medullary keratinocytes is presumed to involve the same mechanisms as in the epidermal melanin unit [79]. 10.5.1 Modes of Transfer 10.5.1.1 Cytophagocytosis Phagocytosis is the cellular internalization of particles with a diameter of more than 0.5 μm. The process is receptor-mediated and activation of phagocytic receptors, such as the Fcγ receptors, leads to local reorganization of the actin cytoskeleton. Phagocytosis is mainly associated with neutrophils, macrophages, and monocytes – all “professional” phagocytes functioning to eliminate infectious agents, apoptotic cells, and cellular debris. Phagocytic activity of keratinocytes has been shown previously by use of different approaches [80–82] Cytophagocytosis stands for phagocytosis of a viable cell or an intact part of a viable cell. The cytophagocytosis hypothesis of melanin transfer describes phagocytosis of an intact melanocytic cell part (i.e., the tip of a dendrite) by the keratinocyte. At the first stage, the melanocyte extends its dendrite towards and makes contact with a surrounding keratinocyte. The keratinocyte reacts with extensive membrane ruffling and engulfing of the dendrite tip with villus-like cytoplasmic projections. In the second stage, the dendrite tip is being squeezed and pinched off, resulting in the formation of a cytoplasmic poach filled with melanosomes. In the third stage, a phagolysosome is formed by fusion of lysosomes, degradation of the melanocyte membranes and cytoplasmic constituents takes place and, meanwhile, the phagolysosome is transported to the supranuclear region. In the fourth and last stage, the phagolysosome disintegrates into smaller vesicles containing a single melanin granule or aggregates of melanin granules, which are then dispersed over the cytoplasm (Figure 10.6a) [78]. The hypothesis of cytophagocytosis has basically been supported by electron microscopy and time-lapse video microscopy studies [78].

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10 Transport and Distribution of Melanosomes a)

b)

c)

Different modes of melanosome transfer. (a) Cytophagocytosis: a melanocyte (MC) dendrite is pinched off and phagocytosed, leading to a phagolysosome from which melanosomes disperse throughout the cytoplasm. (b) Exocytosis: melanin is externalized by fusion of the melanosomal membrane with the plasma membrane and is then taken up by endocytosis or phagocy-

Figure 10.6

tosis. (Adapted from [78].) (c) Filopodialphagocytosis model: after formation and elongation, melanocyte filopodia (containing melanosomes) adhere to and are inserted into the keratinocyte (KC) plasma membrane. The melanocyte filopodia with melanosomes are finally taken up by the keratinocyte through phagocytosis. (Adapted from [4].)

10.5.1.2 Exocytosis All eukaryotic cells undergo constitutive exocytosis. However, specialized secretory cells, such as neurons, and cells of the endocrine, exocrine, and immune system show regulated exocytosis – a process in which the membranes of cytoplasmic organelles fuse with the plasma membrane in response to stimulation. The process functions to secrete products that are segregated in the organelle lumen to the extracellular space [83]. Figure 10.6(b) depicts the exocytosis process for melanin transfer: fusion of the melanosomal membrane with the melanocyte plasma membrane results in extracellular melanin, which is subsequently endocytosed or phagocytosed by surrounding keratinocytes. Evidence suggesting that (regulated) exocytosis is involved in the transfer process was derived from electron microscopic analyses of human skin and hair follicles, demonstrating naked melanin (i.e., not surrounded with a membrane) in the intercellular space and the enfolding of these pigment granules by kerati-

10.5 Melanosome Transfer

nocyte pseudopods or clathrin-coated pits [84]. Further, in vitro studies demonstrated that melanocytes discharged melanin in the extracellular space, especially when stimulated with α-melanocyte-stimulating hormone or UV [81]. Moreover, SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins and Rab GTPases, involved in exocytosis in other cell types, were shown to be expressed by melanocytes. SNARE proteins are implicated in almost all intracellular membrane trafficking events [85]. The synaptic proteins syntaxin (STX), SNAP25 (25-kDa synaptosome-associated protein), and VAMP (vesicle-associated membrane protein, also called synaptobrevin) were the first SNAREs to be discovered. These three conserved SNARE families act late in the events of membrane fusion. They associate into core complexes, and usually SNAP25 and syntaxin on the plasma membrane bind VAMP on the vesicle membrane. Different SNAREs were identified in melanosome-enriched fractions: SNAP23, SNAP25, VAMP2, STX4, and STX6 [86, 87]. Immunoprecipitation shows association of VAMP2 and SNAP23, but not STX4. Possibly, VAMP2 on melanosomes interacts with SNAP23 and an as yet to be identified syntaxin on the melanocyte plasma membrane to achieve fusion. Other proteins that play a role in membrane fusion, particularly in tethering and docking of membranes before actual fusion, are Rab GTPases. The Rab3 proteins consisting of Rab3a–d are the central Rabs in regulated exocytosis. Rab3a is expressed in melanocytes and downregulation of its expression is effected by UV irradiation [86]. Interestingly, downregulation of Rab3 in other cell types has proved to stimulate regulated exocytosis [88, 89]. 10.5.1.3 Filopodial-Phagocytosis Model Filopodia are narrow (200- to 300-nm diameter) highly motile tubular membrane extensions that contain long actin filaments organized as bundles, with plus-ends/ barbed ends (fast-growing ends) in the direction of protrusions. Filopodia seem to be used by many cell types as a sensing organelle to explore the extracellular matrix and the surface of other cells, to identify appropriate targets for adhesion, and then generate guidance cues and traction forces to move the cell body. Several studies revealed their involvement in the transport of different organelles such as membrane vesicles [90–93]. Filopodia extent from the dendrite tips and cell body of melanocytes, adhere to the surface of neighboring keratinocytes, and allow transport of melanosomes towards the keratinocyte membrane. In some instances, transfer was seen via these protrusions, suggesting the formation of a tunnel between melanocyte and keratinocyte cytoplasms [94, 95]. Definitive proof of membrane fusion could not be given, though. The role of filopodia in melanosome transfer was further explored in great detail by Singh et al. Scanning electron microscopy studies of epidermal melanocyte–keratinocyte cocultures revealed numerous intraluminal ovoid bodies present in filopodia extending from melanocytes to abut adjacent keratinocytes. These bodies were shown to be gp100-positive and were thereby identified as melanosomes [4]. Time-lapse video micrography confirmed their transfer from melanocytes to keratinocytes. Interestingly, transfer was inhibited

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when silencing a regulator of filopodium formation, MyoX [96, 97], in melanocytes, and when blocking filopodium formation by addition of low-dose cytochalasin B. As MyoX is also an effector of phagocytosis [98], Singh et al. decided to examine its role in the interaction of the melanocyte filopodia with the keratinocyte plasma membrane – an interaction that is crucial for the successful transfer of melanosomes. Knockdown of MyoX in keratinocytes resulted in an almost complete inhibition of melanosome uptake by keratinocytes. Based on these findings the authors suggested a dual role for MyoX in melanosome transfer: formation of filopodia in melanocytes and subsequent phagocytosis of filopodia by keratinocytes. This new model of melanosome transfer between human skin cells was termed the “filopodial-phagocytosis” model (Figure 10.6c) [4]. The same authors reported the unexpected finding that keratinocyte filopodia are also conduits for melanin between adjacent keratinocytes. At the moment one can only speculate about the role(s) of this homotypic melanin transfer. It might, for example, facilitate melanin degradation – a process largely uncharacterized. Further research will be necessary to sustain such or other hypotheses. 10.5.2 Molecular Players 10.5.2.1 PAR-2 and KGF Protease activated receptor-2 (PAR-2) belongs to a family of transmembrane G-protein-coupled receptors (PAR-1 to -4) that are proteolytically activated by serine proteases. These enzymes (including trypsin or mast cell tryptase) cleave the extracellular amino terminal domain of PAR-2. The newly formed N-termini are tethered ligands, they undergo a conformational change and bind the receptors leading to activation. The use of synthetic peptides, corresponding to the sequence of the N-terminus of the cleaved receptor, also enables activation of PAR-2 independent of receptor cleavage. A role for PAR-2 in melanin transfer has been established by the group of Seiberg [99]. They showed that stimulation of this receptor, which is expressed in keratinocytes [100], but not in melanocytes [101], enhances the phagocytosis rate of keratinocytes and leads to increased melanin transfer [101–103]. Melanocyte–keratinocyte contact is a prerequisite for this function [101]. UV irradiation induces PAR-2 and, conversely, blocking of the PAR-2 receptor inhibits UV-induced pigmentation [104]. Further, PAR-2 expression and induction by UV seem to depend on skin type, with a higher expression and more pronounced induction in dark-skinned individuals [105]. It has been shown in vitro that activation of PAR-2 leads to serine protease secretion by keratinocytes, creating a positive feedback loop. UV-B induces a similar effect [104]. However, melanin transfer cannot be completely inhibited by treatment with serine protease inhibitors [102]. This suggests that PAR-2 is probably not the only molecular player involved in phagocytosis by keratinocytes. In fact, the keratinocyte growth factor receptor (KGFR) has been allocated a similar role [106]. Activation of KGFR enhances keratinocyte phagocytosis of latex beads and addition of KGF to cocultures induces transfer of tyrosinase-positive granules.

10.6 Fate of Melanin in the Keratinocyte

Apart from phagocytosis, PAR-2 affects skin pigmentation by stimulation of melanocyte dendricity. Prostaglandins E2 and F2α are released by keratinocyte upon stimulation. They bind the surface of melanocytes, thereby inducing dendrite formation [107]. 10.5.2.2 Adhesion Molecules: Cadherins and Lectins Cell–cell contact between melanocytes and keratinocytes is a prerequisite for melanosome transfer. Different adhesion molecules are involved at the melanocyte– keratinocyte contact site. Cadherins are a family of glycoproteins that function in promoting Ca2+-dependent cell–cell adhesion and serve as the transmembrane components of cell–cell adherent junctions. E-cadherin and P-cadherin are expressed in human melanocytes, and both mediate melanocyte adhesion to keratinocytes. P-cadherin seems to play a minor role as opposed to E-cadherin, which is the major mediator of melanocyte–keratinocyte adhesion [108]. A role for E-cadherin in pigment transfer has been suggested based on findings in Darier’s disease. This acantholytic disorder, caused by a mutation in the (sarco)endoplasmic reticulum pump SERCA2, is sometimes associated with confetti-like hypopigmented macules in dark-skinned individuals. The ultrastructure of these hypopigmented lesions, with basal and suprabasal keratinocytes being “empty” despite being surrounded by melanosome-filled dendrites, suggests a defect in melanosome transfer. Considering the importance of E-cadherin in melanocyte– keratinocyte adhesion and the fact that it is dissociated in acantholytic lesions of Darier’s disease points to a possible involvement of E-cadherin in the transfer process [109]. Lectins are adhesion receptors that bind sugar residues present on the surface of delimiting membranes. When added to keratinocyte–melanocyte cultures, lectins and neoglycoproteins inhibit melanin transfer, as shown by flow cytometry and electron microscopy [110]. The inhibition is reversible and can be enhanced by addition of niacinamide [111]. Lectins that bind galactose residues are more effective than lectins binding mannose residues, while α-l-fucose receptors display a variable effect. Cerdan et al. studied the role of these molecules in binding of melanin-containing vesicles shed by melanoma cells to keratinocytes [112]. Binding is inhibited by neoglycoproteins, showing a role for α-l-fucose-specific lectins of keratinocytes, on the one hand, and for 6-phospho-β-d-galactose-specific lectins of melanocyte-derived vesicles, on the other hand.

10.6 Fate of Melanin in the Keratinocyte

Once transferred, melanin granules distribute differently according to the skin phototype. In light skin, melanin granules cluster in membrane-bound organelles that border on the nucleus, while in dark-skinned individuals melanin granules are distributed throughout the cell body [113]. Light skin predominantly contains smaller melanosomes than dark skin (around 0.5 versus 0.8 μm in diameter) and

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traditionally the distribution pattern was considered to be size-dependent. Small melanosomes cluster and form melanosome complexes, whereas larger ones are packed individually [114]. The size-dependency of the distribution pattern was contradicted by later studies. Analysis of cocultures composed of cells derived from different phototypes showed that the origin of the recipient keratinocyte is the decisive factor [114]. The significant role of keratinocytes in the determination of skin pigmentation became even more evident when studying human skin reconstructs composed of cells derived from different skin phototypes. Reconstructs with light skin-derived melanocytes appeared darker, produced more melanogenic cytokines as well as melanin, and contained more mature melanosomes, which had a higher individual/clustered distribution ratio, when the keratinocytes originated from dark skin versus light skin [114, 115]. Apparently, the keratinocyte influences the quality of melanosome production and it determines the distribution pattern of melanosomes after transfer. Keratinocytes can adopt a pigment recipient phenotype, leading to recruitment of melanocytes and inducing their own pigmentation. At least this was shown for epidermal keratinocytes in mouse skin. When the transcription factor Foxn1 is transgenically expressed in basal keratinocytes of mouse skin, numerous melanocytes are found in the interfollicular epidermis and melanization of this epidermis follows (whereas normally murine interfollicular epidermis is not pigmented) [116]. Keratinocytes are also crucial for the response of melanocytes to UV irradiation, and thus for the determination of facultative skin pigmentation [117]. Increased melanogenesis, for example, requires a 10-fold higher UV-B dose when melanocytes are cultured alone versus together with keratinocytes [117]. Inside keratinocytes, melanin is stored in melano-phagosomes that eventually fuse with lysosomes to form melano-phagolysosomes. Aggregation of melanophagolysosomes above and adjacent to the nucleus of suprabasal keratinocytes results in the formation of the “microparasol” or “supranuclear melanin cap,” which protects keratinocyte DNA from UV damage [118]. Supranuclear caps are also formed in cultured skin equivalents and their formation increases after UV irradiation and 3-isobutyl-1-methylxanthine treatment [119]. Transport to the cell center is accomplished by the microtubule-dependent minus-end motor protein dynein that attaches to the melano-phagolysosome via the dynactin complex [120]. Knockdown of the p150Glued subunit of dynactin, that binds dynein as well as microtubules, impairs the perinuclear targeting of ingested polystyrene microspheres in keratinocytes [121]. This underlines the importance of the dynein–dynactin complex for the formation and maintenance of the microparasol, and indicates that the transport is independent of any melanosomal component or signal, since phagocytosed microspheres behave just like melano-phagolysosomes. Data on melanin and melanosome degradation are limited. Melanosomes mainly populate the basal layers of fair-skinned as well as dark-skinned individuals and they are no longer visible in the differentiated keratinocytes from the upper epidermal layers. Only in dark skin types can some scattered melanosomes be

10.7 Conclusions

distinguished up to the stratum corneum. Hydrolytic reactions are responsible for the breakdown of the lipid and protein components of melanosomes, but melanin is a hardy, resistant structure and degradation via hydrolysis would be impossible [122]. Oxidative reactions could be responsible, for example, via NADPH oxidase, which resides on the phagosomal membrane. There is still doubt, though, on whether melanin as a chemical compound is degraded in vivo.

10.7 Conclusions

Skin pigmentation depends on the amount and type of melanin synthesized by the melanocyte, the subsequent transfer of melanosomes to the keratinocytes, and eventually the distribution of pigment in the skin. Prior to the transfer of melanosomes to keratinocytes, mature melanosomes need to be moved from the perinuclear area to the cell periphery and dendritic tips of the melanocytes. This intracellular melanosome transport is dependent on an intact cytoskeleton, and is functional in pigment cells derived from lower vertebrates and mammals. Detailed studies in both melanophores and melanocytes revealed that melanosomes undergo transport by means of a coupled system of microtubule-dependent transport to the periphery, followed by actin-dependent retention. Three different classes of motor protein were shown to be involved in this movement: kinesins, dyneins, and Myo5a. In mammals, melanosomes are captured below the sublemmal actin network through the formation of a Rab27a–Mlph–Myo5a tripartite protein complex. From there, melanosomes have to pass from the dendrites of the melanocytes to neighboring keratinocytes. This transfer process appears difficult to unravel and the mechanisms involved remain poorly understood. The development of reliable coculture assays to study melanosome transfer, combined with high-resolution microscopy, provides growing evidence that filopodia from melanocytes are involved in melanosome transfer [4]. Moreover, filopodia were also identified as conduits for melanosome transfer between individual keratinocytes. This unexpected result might point at a much greater role for keratinocytes in the control of mammalian skin pigmentation than hitherto assumed. Interestingly, the use of long-term model systems (e.g., grafting of melanocytes and keratinocytes derived from skin of different racial/ethnic origin onto SCID mice) revealed that keratinocytes play an important role in determining the amount and type of melanin produced by melanocytes, the number of melanosomes that are transferred, and their eventually distribution pattern in keratinocytes [115]. Today, there is great interest to develop new tools in order to darken or lighten skin coloration or to treat skin pigmentary disorders in an efficient and safe manner. In this context, our group is focusing on the development of RNA interference-based therapeutics in order to reduce pigmentation by silencing the tyrosinase gene, for example, or genes involved in the intracellular melanosome transport pathway. As novel insights into the mechanisms of melanosome transfer

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become available, it will be interesting to interfere at this particular level of the pigmentary process in order to modulate pigmentation.

Acknowledgments

We would like to thank Dr Karolien Van Den Bossche for critical comments on this text and Raphael van den Eerenbeemt for drawing Figure 10.5. M.V.G. is a postdoctoral researcher of the Fund for Scientific Research-Flanders (Belgium).

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11 Genetics of Melanosome Structure and Function Vincent J. Hearing

11.1 Introduction

In mammals, melanins are synthesized within specialized membrane-bound organelles termed melanosomes that are exclusively produced in melanocytes, as detailed in other chapters throughout this book. The eventual colors and patterns generated, be they in the hair, skin, and/or eye, depend on a complex series of physiologically regulated events. Those begin during the development of melanoblasts, continue throughout the differentiation and function of melanocytes in their destination tissues, and culminate with the synthesis and distribution of melanin pigments in those tissues to produce the wide variety of colors in animals that we are familiar with. Each of those steps is regulated by a number of distinct genes that control those processes in very sensitive and specific manners. Currently, more than 250 genes are known to affect mammalian pigmentation either directly or indirectly (see http://www.espcr.org/micemut/ for the current list) and even minor perturbations in the functions of their encoded proteins can have dramatic consequences on eventual color, and often on other cellular and tissue processes that depend in some context on melanocyte function. There has been a recent surge in interest in studying the genetics of pigmentation, not only because of the important functions of melanins (e.g., camouflage and photoprotection), but also because the highly visible colors that are produced provide an easy visible output assay to characterize the full complement of processes (e.g., intracellular signaling and transcription factors) that regulate various cellular functions. This chapter focuses on genes that regulate melanosome structure and function; however, to put those in context, will begin with a general review of genes important to the development, growth, survival, and differentiation of melanocytes – the cells that produce the melanins and melanosomes. It is important to keep in mind that colors (and their patterns) ultimately depend upon the presence of functional melanocytes in any given area (e.g., consider the black and white stripes on a zebra), and also how much and what types of melanin they are producing (e.g., consider the black versus orange stripes of a tiger and even the decreased pigmentation in ventral areas). It is the sum of the development, migration, proliferation, Melanins and Melanosomes: Biosynthesis, Biogenesis, Physiological, and Pathological Functions, First Edition. Edited by Jan Borovanský and Patrick A. Riley. © 2011 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2011 by Wiley-VCH Verlag GmbH & Co. KGaA.

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Figure 11.1

Processes critical to getting functional melanocytes in place.

survival, and even regulated function of melanocytes in various areas of tissues that gives rise to the wide diversity of pigmentation patterns seen in nature. This is not merely a cosmetic issue, since melanins in lower species play critical roles in survival, such as camouflage for predator and prey, and in regulating body temperature in amphibians and snakes; in man, they play important roles in the absorption of toxic materials, in the scavenging of free radicals, and in photoprotection against UV damage. More complete reference lists and further information can be found in [1–3], and a schematic summarizing these processes is shown in Figure 11.1.

11.2 Genes Involved in Melanoblast Development, Migration, and Specification

Melanocytes that eventually populate the skin and other locations in the body begin as precursor cells termed melanoblasts. In vertebrates, all pigment cells (except for those destined for the retinal pigment epithelium of the eye) are initially derived from the dorsal neural tube during embryonic development. To get to their eventual destinations in the body, cues must be provided to these precursor cells to begin migrating from the neural tube along defined pathways that take them to their distant destinations, they must continue to migrate along those pathways

11.3 Genes Involved in Melanocyte Differentiation, Survival, and Proliferation

until they reach their targeted destination, and then obviously they must stop migrating and begin to transition into functional melanocytes (the latter process being termed “specification”). Each of those steps is under the continual control of many independent factors (including intrinsic and environmental factors) and the overall timing of these processes is critical to achieve the adult pigmentation patterns in a given normal individual of any species. The genes that function at this level encode, among other things, transcription factors (e.g., PAX3, SOX10, and MITF), receptors and their ligands (e.g., EDNRB/EDN3, KIT/KITL, and FZD4/WNT3a), and other factors important to the start/continue/stop signals for migration, specification, etc (e.g., ADAMTS20, MCOLN3, and ITGB1). Mutations in those genes and/or disruptions in their functions frequently perturb normal development and lead to developmental diseases that not only affect pigmentation, but in many cases also affect tissues that depend in some fashion on the functions of cells derived from the neural tube. Regarding effects on pigmentation, those diseases typically lead to white-spotted areas where melanoblasts failed to develop and migrate to (typically on the forehead and abdomen) that are stable (i.e., present at birth and remain through adulthood). Examples of such developmental diseases include Waardenburg syndrome, Hirschsprung’s disease, and piebaldism. It is beyond the scope of this chapter to provide details of the genes and processes involved or the pigmentary diseases that result, and interested readers are referred to recent reviews for pertinent details [4–11] and also to relevant chapters in this book. A summary of genes involved in melanoblast development, migration, and specification, along with the functions of their encoded proteins and associated diseases where known is provided in Table 11.1.

11.3 Genes Involved in Melanocyte Differentiation, Survival, and Proliferation

Once melanoblasts have arrived at their final destinations in the tissues, they must then differentiate to functional melanocytes, they must survive, and then must proliferate to populate the tissue to achieve the correct eventual density of melanocytes required for normal function. Again, a relatively large number of genes are involved in those processes and disruption in any of them leads to decreased melanocyte function in the adult. Again, the genes involved in these processes encode, among other things, transcription factors (e.g., MITF and FOXD3), receptors and their ligands (e.g., EDNRB/EDN3 and KIT/KITL), and other factors important to the signals that regulate proliferation, survival, and so on (e.g., BCL2, SEMA3C, and NLRP1). Note that many of these critical genes function at multiple stages, ranging from developmental processes (as noted in the previous section) through differentiation processes (as discussed below). Genes that are critical to many different levels that affect pigmentation include transcription factors (such as MITF and SOX10) and receptor/ligand pairs (such as MC1R/POMC and EDNRB/EDN3). Regarding effects on pigmentation, mutations in those genes typically lead to hypopigmented areas where melanoblasts failed to differentiate or

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11 Genetics of Melanosome Structure and Function Genes and diseases involved with melanoblast development, migration, and specification.

Table 11.1

Gene

Encoded protein

Function

Associated disease/ condition

OMIM #

PAX3

splotch

transcription factor

Waardenburg syndrome types 1, 3

193500

SOX9

Sry box gene 9

transcription factor

unknown

SOX10

Sry box gene 10

transcription factor

Waardenburg–Shah syndrome

277580

SNAI2

snail homolog 2

transcription factor

Waardenburg syndrome type 2

193510

MITF

microphthalmia

transcription factor

Waardenburg syndrome type 2

193510

PAX6

paired box gene 6

transcription factor

aniridia

106210

LEF1

lymphoid binding factor 1

transcription factor

sebaceous adenoma

153245

TCFAP2A

AP2α

transcription factor

unknown

FOXD3

forkhead box D3

transcription factor

vitiligo

607836

EDNRB

endothelin B receptor

receptor

Hirschsprung’s disease type 2

600155

EDN3

endothelin 3

EDNRB ligand

Waardenburg–Shah syndrome

277580

EDN1

endothelin 1

EDNRB ligand

unknown

FGFR2

fibroblast growth factor receptor 2

receptor

Crouzon syndrome

FGF

fibroblast growth factor

FGFR2 ligand

unknown

KIT

Kit oncogene

receptor

piebaldism

KITL/SCF

stem cell factor

KIT ligand

unknown

FZD4

frizzled homolog 4

receptor

exudative vitreoretinopathy

WNT1

wingless related 1

FZD4 ligand

unknown

WNT3A

wingless related 3a

FZD4 ligand

unknown

123500

172800

133780

11.4 Genes Involved in Regulating Melanocyte Function Table 11.1 (Continued)

Gene

Encoded protein

Function

Associated disease/ condition

MET

Met oncogene

receptor

unknown

HGF

hepatocyte growth factor

MET ligand

unknown

EGFR

epidermal growth factor receptor

receptor

unknown

ITGB1

integrin β1

receptor

unknown

MCOLN3

mucolipin 3

cation channel

unknown

ADAMTS20

disintegrin protease 20

metalloprotease

unknown

OMIM #

survive that are progressive (i.e., pigmentation may be normal at birth, but become more severe with age). Examples of such diseases include vitiligo and retinitis pigmentosum. Again, it is beyond the scope of this chapter to go into further detail about these processes and how the relevant genes are involved, and interested readers are encouraged to look at recent review articles [12–18] and also the relevant chapters in this book. A partial list of genes involved with these processes at this level and diseases associated with their dysfunction is shown in Table 11.2.

11.4 Genes Involved in Regulating Melanocyte Function

Once functional melanocytes have been established and become active in the appropriate locations and densities in various tissues, the next level of control involves the regulation of their functions (i.e., their phenotypic characteristics such as their ability to produce melanin and melanosomes, and what types of melanins are produced and how much). Interestingly, many of the same genes discussed above (and their encoded products) are involved at this level of regulation. Obviously, mutations in those multilevel genes affect pigmentation in developmental stages initially and are associated with developmental diseases as noted above, but the functions of their encoded proteins also regulate melanocyte function, and thus modulation of their expression and function in adult tissues leads to often dramatic changes in pigmentation. This list of genes is far from complete at this point and the bulk of as yet uncloned pigment genes (see http://www.espcr.org/ micemut/ for an updated list) no doubt includes a large number of genes that operate at this level, such as genes that regulated constitutive colors in the skin, hair, and eyes or that regulate environmental responses such as those modulated by UV exposure, as summarized in Table 11.3.

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11 Genetics of Melanosome Structure and Function Genes and diseases involved with melanocyte differentiation, survival and proliferation.

Table 11.2

Gene

Encoded protein

Function

Associated disease/condition

OMIM #

MITF

microphthalmia

transcription factor

Waardenburg syndrome type 2

193510

FOXD3

forkhead box D3

transcription factor

vitiligo

607836

EDNRB

endothelin B receptor

receptor

Hirschsprung’s disease type 2

600155

EDN3

endothelin 3

EDNRB ligand

Waardenburg– Shah syndrome

277580

EDN1

endothelin 1

EDNRB ligand

unknown

KIT

Kit oncogene

receptor

piebaldism

KITL/SCF

stem cell factor

KIT ligand

unknown

BCL2

B cell leukemia 2

inhibit apoptosis

follicular lymphoma

GNAQ

GNA subunit Gaq

G-protein-coupled receptor signaling

unknown

GNA11

GNA subunit Ga11

G-protein-coupled receptor signaling

unknown

RB1

retinoblastoma 1

growth inhibitor

retinoblastoma

SEMA3C

semaphorin 3c

signaling factor

unknown

SEMA4A

semaphorin 4a

signaling factor

retinitis pigmentosum

610282

NLRP1

NLR protein 1

immune regulator

vitiligo

606579

172800

151430

180200

Visible pigmentation is regulated physiologically under normal conditions, such as to provide the wide range of constitutive skin, hair, and eye colors in various races and individuals, and also to respond to various stresses, such as exposure to UV radiation and/or injury. Effects on visible pigmentation can be short-term (e.g., temporary increases (tans) due to UV exposure) and/or more permanent (e.g., age spots and postinflammatory hyperpigmentation). Again, transcription factors and receptor/ligand interactions play major roles in regulating these responses, but a number of other factors, such as membrane-bound transporters and proteolytic components, also have important functions in this respect, as noted in Table 11.3. The following is a brief consideration of genes and processes involved with the regulation of hair, skin, and eye color under “normal” physiological conditions,

11.4 Genes Involved in Regulating Melanocyte Function Table 11.3 Genes and diseases involved with regulating melanocyte function.

Gene

Encoded protein

Function

Associated disease/condition

MITF

microphthalmia

SOX9

Sry box gene 9

SOX2

Sry box gene 2

hair, skin, and eye color skin phenotype, response to UV unknown

SOX10

Sry box gene 10

SOX18

Sry box gene 18

PAX6

paired box gene 6

FOXD3

forkhead box D3

MC1R

KIT

melanocortin 1 receptor proopiomelanocortin agouti signaling protein Kit oncogene

transcription factor transcription factor transcription factor transcription factor transcription factor transcription factor transcription factor receptor MC1R ligand MC1R antagonist receptor

KITL

stem cell factor

KIT ligand

EDNRB

endothelin B receptor

receptor

EDN3 EDN1

endothelin 3 endothelin 1

EDNRB ligand EDNRB ligand

MET

receptor growth factor

UV-A melanosis

DKK1

hepatocyte growth factor receptor granulocyte macrophage colony-stimulating factor dickkopf 1

hair and skin color hair, skin, and eye color UV-B melanosis, lentigo senilis UV-B melanosis, lentigo senilis, hair color UV-B melanosis, lentigo senilis unknown UV-B melanosis, lentigo senilis UV-A melanosis

Wnt inhibitor

NRG1

neuregulin 1

P

P protein

TYR

tyrosinase

TYRP1

tyrosinase-related protein 1

Erb receptor ligand transport of ??? tyrosinase enzyme tyrosinase stability

skin color and thickness skin color

POMC ASP

GM-CSF

OMIM #

unknown unknown aniridia, eye color

607108

vitiligo

607836

hair and skin color

eye and skin color skin color eye and hair color

(Continued)

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11 Genetics of Melanosome Structure and Function Table 11.3

(Continued)

Gene

Encoded protein

Function

Associated disease/condition

SLC45A2

solute carrier 45 A2

eye and skin color

SLC7A11

solute carrier 7 A11

SLC24A5

solute carrier 24 A5

ATP7A

ATPase 7α

ATP7B

ATPase 7β

CTNS

cystinosin

ATOX1

antioxidant protein 1

ATRN MGRN1 DEFB103A

mahagony mahogunin ring finger 1 β-defensin 3

PMEL17

gp100/silver

RAB7 IL1

Ras-associated protein 7 interleukin-1