Basic Research in Endocrine Dermatology: 3rd Teupitzer Colloquium, Berlin, September 17-20, 2000 (Special Issue: Hormone Research 2000, 5-6)

Basic Research in Endocrine Dermatology 3rd Teupitzer Colloquium 2000 Berlin, September 17–20, 2000

Editor

Christos C. Zouboulis, Berlin

57 figures, 11 in color, 5 tables, 2000

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Vol. 54, No. 5–6, 2000

Contents

217 Preface 218 Intracrinology and The Skin Labrie, F.; Luu-The, V.; Labrie, C.; Pelletier, G.; El-Alfy, M. (Québec City) 230 Human Skin: An Independent Peripheral Endocrine Organ Zouboulis, C.C. (Berlin) 243 The Hair Follicle: A Paradoxical Androgen Target Organ Randall, V.A.; Hibberts, N.A.; Thornton, M.J. (Bradford); Hamada, K. (Odawara City); Merrick, A.E. (Bradford); Kato, S. (Tokushima); Jenner, T.J.; De Oliveira, I. (Bradford); Messenger, A.G. (Sheffield) 251 The SAHA Syndrome Orfanos, C.E.; Adler, Y.D.; Zouboulis, C.C. (Berlin) 259 Oestrogen Receptor Beta Is Not Present in the Pilosebaceous Unit of Red

Deer Skin during the Non-Breeding Season Thornton, M.J. (Bradford); Taylor, A.H.; Mulligan, K.; Al-Azzawi, F. (Leicester) 263 Nuclear Hormone Receptors and Mouse Skin Homeostasis: Implication of

␤ PPAR␤ Michalik, L.; Desvergne, B. (Lausanne); Basu-Modak, S. (Lausanne/Bath); Tan, N.S.; Wahli, W. (Lausanne) 269 Peroxisome Proliferator-Activated Receptors and Skin Development Rosenfield, R.L.; Deplewski, D.; Greene, M.E. (Chicago, Ill.) 275 Regulation of Macrophage Gene Expression by the Peroxisome

␥ Proliferator-Activated Receptor-␥ Ricote, M.; Welch, J.S.; Glass, C.K. (La Jolla, Calif.) 281 Neuroimmunoregulation of Androgens in the Adrenal Gland and the Skin Alesci, S.; Bornstein, S.R. (Bethesda, Md.) 287 The Role of Melanocortins in Skin Homeostasis Böhm, M.; Luger, T.A. (Münster) 294 Prevalence of Endocrine Dysfunction in HIV-Infected Men Brockmeyer, N.H.; Kreuter, A.; Bader, A.; Seemann, U.; Reimann, G. (Bochum) 296 Targeted Somatic Mutagenesis in Mouse Epidermis Indra, A.K.; Li, M.; Brocard, J.; Warot, X.; Bornert, J.-M.; Gérard, C.; Messaddeq, N.; Chambon, P.; Metzger, D. (Illkirch)

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301 Interaction of Vitamin D and Retinoid Receptors on Regulation of Gene

Expression Jimenez-Lara, A.M.; Aranda, A. (Madrid) 306 Development of Efficient Transient Transfection Systems for Introducing

Antisense Oligonucleotides into Human Epithelial Skin Cells Fimmel, S.; Saborowski, A.; Orfanos, C.E.; Zouboulis, C.C. (Berlin) 312 A Novel Pathway for Hormonally Active Calcitriol Lehmann, B.; Knuschke, P.; Meurer, M. (Dresden) 316 Successful Treatment of Non-Segmental Vitiligo: Systemic Therapy with

Sex Hormone-Thyroid Powder Mixture Nagai, K.; Ichimiya, M.; Yokoyama, K.; Hamamoto, Y.; Muto, M. (Ube) 318 Liposomal Ursolic Acid (Merotaine) Increases Ceramides and Collagen in

Human Skin Yarosh, D.B.; Both, D.; Brown, D. (Freeport, N.Y.) 322 Association of Insulin Resistance with Hyperandrogenia in Women Pugeat, M.; Ducluzeau, P.H.; Mallion-Donadieu, M. (Lyon) 327 The Molecular Basis of Androgen Insensitivity Nitsche, E.M.; Hiort, O. (Lübeck) 334 Genetics of Peutz-Jeghers Syndrome, Carney Complex and Other Familial

Lentiginoses Stratakis, C.A. (Bethesda, Md.)

344 Author Index Vol. 54, No. 5–6, 2000 345 Subject Index Vol. 54, No. 5–6, 2000 346 Author Index Vol. 54, 2000 347 Subject Index Vol. 54, 2000

216

Contents

Preface

A Conference on Basic Research in Endocrine Dermatology

It is a great pleasure to present the Proceedings of the 3rd Teupitzer Colloquium 2000 which was dedicated to ‘Basic Research in Endocrine Dermatology’. This nonprofit conference with limited participation, held in the marvellous lakeside Teupitz Castle Hotel, near Berlin on September 17–20, 2000, served as a forum to bring together 61 basic researchers and clinicians inside and outside Dermatology from 12 countries to discuss the newest and most relevant developments in this specific area of skin research. The scientific program included 30 communications, 21 lectures and 9 poster presentations, which were focused on (a) adrenal and gonadal hormones, (b) peroxisome proliferator-activated receptors, (c) immune-endocrine interactions, (d) natural retinoids and vitamin D, and (e) genetics. The dermatological research has experienced a rapid development in the last two decades, however, it has been rarely focused on the endocrine functions of the skin. It is only since a few years that clinicians and researchers in Dermatology started to initially explore skin as target tissue of the most hormones and later as the largest, endocrine, paracrine and autocrine organ of the body. The skin is able to metabolize steroid hormones and to produce derivatives with potentially systemic activity. Disorders of hormone metabolism can either induce direct effects on the skin or indirectly disturb skin homeostasis. Endocrine Dermatology is a new and exciting area of skin research whose scope includes skin diseases due to or associated with endocrine disorders, skin disorders which can be treated with hormones or with compounds with hormone-like activity, and skin disorders which occur as adverse events of hormone treatment or of treatment with compounds exhibiting a hormone-like activity. This thematic issue includes 20 selected manuscripts presented by well-known experts that are representative of

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the scope and level of the 3rd Teupitzer Colloquium. They are addressed to clinicians and researchers, especially in the fields of Dermatology, Endocrinology, Gynaecology, Paediatrics, Genetics, Biology, Biochemistry, Pharmacology and Molecular Biology. I am grateful to all contributors for the quality of their papers and for making possible the presentation of this volume. I want to express my sincere thanks to Prof. Michael B. Ranke, Thübingen, Germany, Editor-in-Chief of Hormone Research, and S. Karger AG and its staff who agreed to publish the Proceedings of the 3rd Teupitzer Colloquium 2000 as peer-reviewed papers under most favourable conditions. I am pleased to acknowledge here the support of Galderma Laboratorium GmbH, Freiburg and Schering AG, Berlin, as well as of Merck & Co., Inc., N.J., USA; Pfizer GmbH, Karlsruhe; Pacific Corporation R & D Center, Kyounggi-do, Korea; Galderma R + D, Valbonne, France, and Yamanouchi Pharma GmbH, Heidelberg to the 3rd Teupitzer Colloquium 2000. Last but not least, I want to cordially thank Prof. Constantin E. Orfanos, Berlin for his continuous support, advice and encouragement and the colleagues of our Department for their contribution towards the success of the Colloquium. Hoping that you will find the following articles interesting and stimulating, I wish you a pleasant lecture. Christos C. Zouboulis, Berlin

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Intracrinology and The Skin Fernand Labrie Van Luu-The Claude Labrie Georges Pelletier Mohamed El-Alfy Oncology and Molecular Endocrinology Research Center, Laval University Medical Center (CHUL) and Laval University, Québec City, Canada

Key Words Skin W Sebaceous glands W Androgens W Estrogens W Intracrinology W Dehydroepiandrosterone (DHEA)

Abstract The skin, the largest organ in the human body, is composed of a series of androgen-sensitive components that all express the steroidogenic enzymes required to transform dehydroepiandrosterone (DHEA) into dihydrotestosterone (DHT). In fact, in post-menopausal women, all sex steroids made in the skin are from adrenal steroid precursors, especially DHEA. Secretion of this precursor steroid by the adrenals decreases progressively from the age of 30 years to less than 50% of its maximal value at the age of 60 years. DHEA applied topically or by the oral route stimulates sebaceous gland activity, the changes observed being completely blocked in the rat by a pure antiandrogen while a pure antiestrogen has no significant effect, thus indicating a predominant or almost exclusive androgenic effect. In human skin, the enzyme that transforms DHEA into androstenedione is type 1 3ßhydroxysteroid dehydrogenase (type 1 3ß-HSD) as revealed by RNase protection and immunocytochemistry. The conversion of androstenedione into testosterone is then catalyzed in the human skin by type 5 17ß-HSD. All the epidermal cells and cells of the sebaceous glands are

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labelled by type 5 17ß-HSD. This enzyme is also present at a high level in the hair follicles. Type 1 is the 5·-reductase isoform responsible in human skin for the conversion of testosterone into DHT. In the vagina, on the other hand, DHEA exerts mainly an estrogenic effect, this effect having been demonstrated in the rat as well as in post-menopausal women. On the other hand, in experimental animals as well as in post-menopausal women, DHEA, at physiological doses, does not affect the endometrial epithelium, thus indicating the absence of DHEAconverting enzymes in this tissue, and avoiding the need for progestins when DHEA is used as hormone replacement therapy. Copyright © 2001 S. Karger AG, Basel

Introduction

An important finding in the field of sex steroids is that a large proportion of androgens and estrogens in men and women are synthesized locally in peripheral target tissues from the inactive adrenal precursors dehydroepiandrosterone (DHEA), DHEA-sulfate (DHEA-S), and androstenedione (4-dione) (fig. 1). In fact, in post-menopausal women, almost 100% of sex steroids are synthesized in peripheral tissues from DHEA and DHEA-S of adrenal origin except for a small contribution from ovarian and/or adre-

Prof. Fernand Labrie Oncology and Molecular Endocrinology Research Center Laval University Medical Center (CHUL) 2705 Laurier Boulevard, Quebec City, Quebec, G1V 4G2, Canada Tel. +1 418 654 2704, Fax +1 418 654 2735, E-Mail [email protected]

Fig. 1. Schematic representation of the role of gonadal (testicular and ovarian) and adrenal sources of sex steroids in men and women. After menopause, the secretion of estradiol by the ovaries ceases and almost 100% of sex steroids are made locally in peripheral target intracrine tissues. LH, luteinizing hormone; ACTH, adrenocorticotropin.

nal testosterone and androstenedione. Thus, in postmenopausal women, almost all active sex steroids are made in target tissues by an intracrine mechanism. The secretion of DHEA and DHEA-S by the adrenals increases during the adrenarche in children at the age of 6–8 years and elevated values of circulating DHEA and DHEA-S are maintained throughout adulthood, thus providing the high level of substrates required for conversion into potent androgens and estrogens in peripheral tissues. In fact, plasma DHEA-S levels in adult men and women are 100–500 times higher than those of testosterone and 1,000 to 10,000 times higher than those of estradiol, thus providing a large reservoir of substrate for conversion into androgens and/or estrogens in peripheral intracrine tissues. This local formation of sex steroids provides autonomous control to target tissues which are thus able to adjust the formation and metabolism of sex steroids according to local needs [1]. The situation of a high secretion rate of adrenal precursor sex steroids in men and women is completely different from the animal models used in the laboratory, namely the rat, mouse, guinea pig and all others

(except monkeys) where the secretion of sex steroids takes place exclusively in the gonads [2–6, and ref. therein]. In these lower animal species, no significant amounts of androgens or estrogens are made outside the testes or ovaries and no sex steroid is left after castration. The term intracrinology was coined in 1988 [7] to focus our attention on the synthesis of active steroids in peripheral tissues where the active steroids exert their action in the same cells where synthesis takes place without release in the extracellular space and in the general circulation [1] (fig. 2). The rate of formation of each sex steroid thus depends upon the level of expression of each of the specific androgen- and estrogen-synthesizing enzymes in each cell of each tissue [1, 2, 8–10]. The skin, the largest organ in the human body, contains a series of androgen-sensitive components, namely the hair follicles [11, 12], sebaceous glands [13], sweat glands [14, 15], epidermis [15], and dermis [16]. All the above-mentioned skin appendages and components contain 17ß- and 3·-hydroxysteroid dehydrogenase (HSD) activities, as well as 3ß-hydroxysteroid dehydrogenase-¢4¢5isomerase (3ß-HSD) and 5·-reductase activities [17–

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Fig. 2. Schematic representation of endocrine, para-

crine, autocrine and intracrine secretion. Classically, endocrine activity includes the hormones secreted in specialized glands, called the endocrine glands. These hormones are released in the general circulation and are transported to distant target cells. On the other hand, hormones released from one cell can influence neighboring cells (paracrine activity) or can exert a positive or negative action on the cell of origin (autocrine activity). Intracrine activity describes the formation of active hormones which exert their action in the same cells where synthesis took place without release into the pericellular compartment.

23]. The presence in skin of the main steroidogenic enzymes [17, 18, 24–29] makes it likely that this tissue, in the human, synthesizes a significant proportion of total sex steroids including the potent androgen dihydrotestosterone (DHT) from DHEA [1, 29–32]. Moreover, receptors for androgens and estrogens have been described in squamous cells, sweat and sebaceous gland cells as well as in the hair follicles [33–38]. There is already convincing evidence that DHEA of adrenal origin may have an important androgenic influence on sebaceous gland activity. In fact, transformation of DHEA into testosterone and other metabolites has been observed in the skin [21, 22, 26, 39–42]. Although the steroids synthesized in various skin compartments could possibly have some systemic effects, it is more likely that the steroids synthesized locally in each cell type from adrenal precursors exert their effects locally in the same cells where they are produced, without being released outside their cells of origin. Abnormalities of androgen action in the skin are frequent and are cosmetically important. These include acne, seborrhea, hirsutism and androgenic alopecia. On the other hand, an even more frequent anomaly is skin atrophy which accompanies ageing.

metabolizing enzymes in each of these tissues. Knowledge in this area has recently made rapid progress with the elucidation of the structure of most of the tissue-specific genes that encode the steroidogenic enzymes responsible for the transformation of DHEA-S and DHEA into androgens and/or estrogens in peripheral tissues [5, 10, 43–45] (fig. 3). The particular importance of DHEA and DHEAS is illustrated by the finding that approximately 50% of total androgens in the prostate of adult men derive from these adrenal precursor steroids [2, 46, 47]. As mentioned above, our best estimate of the intracrine formation of estrogens in peripheral tissues in women is in the order of 75% before menopause and close to 100% after menopause [1]. Because the molecular structure of most of the key non-P-450-dependent enzymes required for sex steroid formation had not been elucidated and knowing that local formation of sex steroids is most likely to play a major role in both normal and tumoral hormone-sensitive tissues, an important proportion of our research program and that of other groups has been devoted to this exciting and therapeutically promising area [5, 10, 29–31, 48].

Androgenic Action of DHEA in the Skin Structure of the Human Steroidogenic Enzymes

As mentioned above, transformation of the adrenal precursor steroids DHEA-S and DHEA into androgens and/or estrogens in peripheral target tissues depends upon the level of expression of the various steroidogenic and

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In the present study, we used the ovariectomized female Sprague-Dawely rat as a model to evaluate the effect of DHEA given percutaneously for 3, 6 or 12 months, on the histomorphology of the skin and its appendages and especially the sebaceous glands. In addition, we have administered the combination of DHEA

Labrie/Luu-The/Labrie/Pelletier/El-Alfy

Fig. 3. Human steroidogenic enzymes in peripheral intracrine tissues.

with the pure antiandrogen Flutamide [49, 50] or the pure antiestrogen EM-800 [51–56] for 12 months in order to analyze the androgenic and/or estrogenic component(s) in the action of DHEA in rat skin [57]. In control ovariectomized untreated animals, 3, 6 or 12 months after ovariectomy, the sebaceous glands of both the dorsal and ventral skin are composed of small cells with poor staining of the nucleus and cytoplasm, with a small number of acidophilic granules and an absence of lipid droplets. The acini have a small lumen in the immediate vicinity of the ducts of the acini [57]. The appearance is not different from that of intact female rats of the same age. After 3, 6 or 12 months of DHEA administration to ovariectomized animals, a mild to moderate increase in the number and size of the sebaceous glands is seen at a similar degree in both the dorsal and ventral skin. This change is due to the enlargement and budding of the acini as well as to an increase in the size of the individual sebaceous cells. Interestingly, no differences were observed in the degree of the above-indicated histological changes of the sebaceous glands after either 3, 6 or 12 months of DHEA administration. The quantitative analysis performed after 6 months of treatment demonstrates that DHEA caused increases of 170 and 175% in the number of glands in the dorsal and ventral skin, respectively [57]. The total surface area of

the sebaceous glands was similarly stimulated by DHEA at both sites, the increase being of 225 and 260% in the dorsal and ventral skin, respectively (fig. 4A and B) [57]. In order to differentiate between androgenic and/or estrogenic effects of DHEA, the pure antiandrogen Flutamide and the pure antiestrogen EM-800 were administered concomitantly with DHEA. Concomitant administration of Flutamide with DHEA abolished the effects of DHEA on the number and surface of sebaceous glands in both dorsal and ventral skin. In the group of animals who received DHEA and Flutamide, the histological pattern was similar to that seen in ovariectomized control animals (fig. 5D). It is noteworthy that the effect of Flutamide was somewhat more striking on ventral skin glands. On the contrary, the addition of EM-800 to DHEA had no influence on the effects of DHEA on skin histomorphology (fig. 5E), the values being not significantly different from those obtained in animals which received only DHEA. The presence of androgen receptors [35] in several structures of the skin, including the sebaceous glands, is in agreement with the findings that androgens regulate the activity of the sebaceous glands. The intracrine steroidogenic activity provides an explanation for the observation that the sebaceous glands develop fully in both boys and girls in utero and at puberty [58, 59]. At puberty, the increase in the secretion of DHEA, and especially of

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Fig. 4. Effects of DHEA administered alone or in combination with Flutamide or EM-800 on the surface area of sebaceous glands in dorsal (A) and ventral (B) skin of ovariectomized (OVX) rats. The results are expressed as the means B SEM. *** p ! 0.001, ovariectomized controls vs. the other experimental groups (8 animals per group) [57].

DHEA-S [60], is associated with an increase in sebaceous gland size and sebum production [61, 62] which frequently leads to problems of acne [63]. Although the presence of estrogen receptors has been described in both human and mouse skin [33] and estrogen has been reported to increase vascularization of skin in rodents [64], the effects of DHEA on rat skin dermis appear to result exclusively from an androgenic action, as demonstrated by the reversal of the above-described effects by the addition of the specific antiandrogen Flutamide. On the contrary, the coadministration of the specific antiestrogen EM-800 did not alter the histological changes induced by DHEA, thus excluding a significant estrogenic action of DHEA on skin, at least on the parameters measured. The present study provides morphological evidence that DHEA secreted by the adrenal cortex has an exclusive androgenic influence on the histomorphology and function of the sebaceous glands, most likely via its intracrine conversion into testosterone and DHT. The development of inhibitors acting locally in the skin to block the intracrine formation of androgens offers the potential for an effective treatment of common skin diseases, such as acne and hirsutism, while avoiding systemic effects.

Cell-Specific Expression of the Steroidogenic Enzymes in the Skin

3ß-Hydroxysteroid Dehydrogenase (3ß-HSD) The first step in the conversion of DHEA and androst5-ene-3ß,17ß-diol (5-diol) into 4-dione and testosterone, respectively, is catalyzed by 3ß-HSD. In order to determine with precision the level of expression of the type I and type II 3ß-HSD genes in human skin, a ribonuclease assay was performed. As could be seen, type I 3ß-HSD mRNA is the exclusive mRNA species detectable in the skin and placenta while in the adrenal, type II 3ß-HSD mRNA was the only RNA species detected [18]. As illustrated in fig. 6, a high level of 3ß-HSD immunoreactivity is seen in the sebaceous glands as well as sebaceous gland ducts while the surrounding tissue shows no significant immunoreactivity. The present data show that the exclusive 3ß-HSD gene expressed in human skin is type I, the same gene exclusively expressed in the placenta [65]. When compared to the cDNA previously isolated from human placenta, the cDNA obtained from a human skin library contains, however, an additional 131 nucleotides in the 5)-noncoding region, thus corresponding almost entirely to the 5)-noncoding region which contains 138 nucleotides [18]. 17ß-Hydroxysteroid Dehydrogenase (17ß-HSD) As illustrated in fig. 3, the enzymes of the 17ß-HSD gene family are responsible for the inter-conversion of

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Labrie/Luu-The/Labrie/Pelletier/El-Alfy

Fig. 5. Antagonism of the effect of DHEA on ventral skin by the antiandrogen Flutamide. The histology of ventral skin in intact (A), ovariectomized (B) and ovariectomized rats treated with DHEA alone (C) or with the combination of DHEA and Flutamide (D) or DHEA and EM-800 (E) for 12 months. The stimulatory effects of DHEA on the

growth and size as well as the secretory activity of the sebaceous glands of ventral skin, seen after 12 months of treatment of ovariectomized animals (C), were completely abolished by the concomitant administration of Flutamide (D). On the other hand, the addition of EM-800 had no effect on the DHEA-induced histological changes in the skin (E). Compare with intact (A) and ovariectomized controls (B). Bar = 50 ÌM [57].

DHEA and 5-diol, 4-dione and testosterone, as well as estrone (E1) and E2. The interconversion of androstanedione and DHT as well as androsterone (ADT) and androstane-3·,17ß-diol is catalyzed by the same enzymes. The various 17ß-HSDs are therefore required for the synthesis of all active androgens and all active estrogens as

well as for their inactivation. That 17ß-HSD activity is widely distributed is illustrated by the finding that in a study of 25 tissues in the monkey, both estrogenic and androgenic 17ß-HSD activities were found in all the tissues examined, thus indicating the generalized role of 17ß-HSDs in the formation of androgens and estrogens in

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Fig. 6. Immunohistochemical localization of 3ß-HSD in human skin. Immunostaining for 3ß-HSD can be seen in the cytoplasm of sebaceous gland (SG) and sebaceous duct (SD) cells. !200 [18].

Fig. 7. Autoradiographs of 3H-labeled type 5 17ß-HSD antisense and sense riboprobes, hybridized in situ to normal human skin. Antisense (a and c): all the epidermal cells are well labeled except the cells of the stratum corneum. In the dermis (c), the endothelial cells of blood vessels are also labeled. Sense (b and d): similar areas of the same skin area with few scattered silver grains.

peripheral target intracrine tissues [22]. On the other hand, androgenic 17ß-HSD activity has been measured in the fifteen human tissues examined [21]. The highest level of androgenic 17ß-HSD activity was found in the placenta and liver followed by the testis, endometrium, prostate, adipose tissue, adrenal, prostate and skin.

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The structure and characteristics of six human 17ßHSDs are presently known (see Labrie et al., 2000 for review) [10]. Since androgens play the major role in skin physiology [57], we will briefly examine the properties of type 5 17ß-HSD, the enzyme that catalyses the transformation of 4-dione into testosterone in peripheral tissues.

Labrie/Luu-The/Labrie/Pelletier/El-Alfy

Fig. 8. Autoradiographs of 3H-labeled type 5 17ß-HSD anti-

sense and sense riboprobes, hybridized in situ to normal human skin. Antisense (left picture): in the hair follicle, the cells of the cortex are strongly labeled. It can be seen that most of the silver grains are located over the cytoplasm and not the nuclei. Sense (right picture): in a similar area of a hair follicle, background labeling with a sense probe.

While type 3 17ß-HSD synthesizes testosterone from 4dione in the Leydig cells of the testicles, thus providing approximately 50% of the androgens acting in the prostate and other androgen-sensitive tissues in men, the same enzymatic reaction is catalyzed in peripheral tissues of both men and women by another enzyme, namely type 5 17ß-HSD. This enzyme belongs to the aldo-keto reductase family. In fact, type 5 17ßHSD is highly homologous with types 1 and 3 3·-HSDs as well as 20·-HSD [66]. As illustrated in fig. 7, all the epidermal cells of human skin are well labeled for type 5 17ß-HSD, except the cells of the stratum corneum. Type 5 17ß-HSD is also present at a high level in the cortex of the hair follicles (fig. 8). 5·-Reductase In addition to its function in normal skin physiology, 5·-reductase has been suggested as being involved in androgen-dependent disorders such as acne vulgaris, seborrhea, male pattern baldness and idiopathic hirsutism [67–69]. Precise knowledge of the type(s) of 5·-reductase expressed in the skin thus becomes important for a better understanding, not only of human skin physiology but also for a more rational therapy of androgen-sensitive skin disorders. cDNA clones encoding two types of 5·-reductase have been isolated from human prostate cDNA libraries [70, 71] and have been chronologically identified as type I and type II 5·-reductases. We have thus cloned 5·-reductase cDNAs from adult human keratinocyte and skin fibroblast cDNA libraries in order to identify and gain better knowledge of the 5·-

reductase(s) expressed in normal human skin. Nucleotide sequence analysis shows that the clones obtained correspond to the type I 5·-reductase [20]. When the type I antisense cRNA probe was used, the protected fragment of 155 bp was observed almost exclusively with keratinocyte mRNA, while only a barely detectable protected band could be detected with prostate mRNA. On the contrary, when the type II antisense cRNA probe was used, the expected protected band was observed at 319 bp with prostatic mRNA while no signal could be detected with keratinocyte mRNA [20]. Using antibodies raised against the synthetic peptide covering amino acids 227–240 of type I 5·-reductase to perform immunohistochemical localization of 5·-reductase in human skin, a high level of immunoreactivity was found in the epidermal cell layers (fig. 9A), sebaceous glands (fig. 9B), and sweat glands (fig. 9C) while no reaction could be detected in an immunoabsorbed control (fig. 9D). Similarly, type 1 5·-reductase activity has been detected at a high level in the cytoplasm of cultured human sebocytes, keratinocytes, fibroblasts, dermal microvascular endothelial cells, hair dermal papilla cells and melanonocytes [72]. Using in situ hybridization, type 1 5·reductase was found to be highly expressed in all the layers of the epidermis with the exception of the stratum corneum while a lower labelling was observed in some fibroblasts as well as in the secretory cells of sebaceous glands and excretory ducts of sweat glands in preputial skin [73].

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Predominant Estrogenic Effect of DHEA in the Vagina and No Effect in the Endometrium

In order to obtain additional knowledge on the tissuespecific activity of DHEA, we first used the rat as a model to study the effects of DHEA administration on the histomorphology and anatomical integrity of sex-steroid target tissues such as the uterus and vagina. In the past, analysis of the effect of DHEA on the uterus and vagina had been limited to the effect on total organ weight [74, 75] while cell-specific effects have not been investigated. DHEA applied on the dorsal skin of ovariectomized animals, at the twice daily dose of 30 mg, resulted in a complete reversal of the vaginal atrophy seen 1, 3 and 6 months after ovariectomy, and induced proliferation and mucification of the vaginal epithelium. At histopathological examination, following DHEA administration to intact animals, atrophy, involving especially the endometrium, became apparent after 3 and 6 months of treatment. In ovariectomized animals, the endometrium remained atrophic at all time intervals during DHEA treatment. Comparison of the effect of DHEA on the vaginal epithelium indicates that local application of DHEA on the vaginal mucosa is approximately 10-fold more efficient than application at a distant site on the skin. The present data suggest that DHEA possesses a tissue-specific action, through its local transformation into active estrogens in the vaginal epithelium while the uterine epithelium remains atrophic, due, most likely, to the absence of the steroidogenic enzymes required to transform DHEA. In addition, the site of administration of DHEA appears to be a significant factor, at least for its stimulatory effect on the vaginal mucosa.

It is most interesting that similar observations have been made in postmenopausal women [76]. In fact, in a 12-month study performed in postmenopausal women, vaginal cytology was examined as specific parameter of the estrogenic action of DHEA [76]. Before treatment, ten women had a completely atrophic vaginal smear exclusively composed of parabasal cells (fig. 10A and B, as example). In eight of these 10 women, under DHEA treatment, the vaginal cytology was converted into a pattern typical of normal cycling women showing mainly the presence of superficial pyknotic cells (fig. 11A and B, as example). In two of the ten women having a zero maturation index value at start of treatment, no significant change in the cytological maturation value was observed up to 12 months of treatment (data not shown). In the three women who had a maturation value between 1 and 40 at start of treatment, stimulation was also observed and the cytology became typical of the normal reproductive range in all of all them at 3 months, the first time interval measured after the start of DHEA administration (data not shown). In the last two women, the maturation value was already in the normal range of reproductive age at pretreatment and it remained unchanged during treatment (data not shown). Considering the major concern related to the stimulatory effect of estrogens on endometrial proliferation with the related risk of endometrial carcinoma [77, 78], an endometrial biopsy was performed before starting treatment and after 12 months of DHEA administration. As can be seen in fig. 12, the endometrial atrophy seen in all women at start of treatment remained unaffected by 12 months of DHEA administration.

Fig. 9. Immunohistochemical localization of 5·-reductase in human skin. Immunostaining for 5·-reductase can be seen in the cytoplasm and the nucleus of the epidermal cell layers (A), sebaceous glands (arrow) (B) and sweat glands (arrow) (C). An immunoabsorbed control is shown in (D). Bar represents 10 Ìm (A, D) and 20 Ìm (B, C), respectively. Fig. 10. A Atrophic vaginal smear with numerous parabasal cells in a 65-year-old woman before starting treatment with DHEA (!100). B Atrophic vaginal smear with numerous parabasal cells in a 65-yearold woman before starting treatment with DHEA at higher magnification (!300) [76]. Fig. 11. A Vaginal smear from the same patient as fig. 10A after 12 months of DHEA treatment showing superficial pyknotic cells (!100). B Vaginal smear from the same patient as fig. 10B after 12 months of DHEA administration showing superficial pyknotic cells at higher magnification (!300) [76]. Fig. 12. Atrophic endometrium after 12 months of DHEA treatment in a representative 65-year-old woman (!50) [76].

Sex Steroid Formation in the Skin

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39 Chrousos GP, Peck GL, Gross EG, Cutler GB Jr, Loriaux DL: Adrenal function in women with idiopathic acne. J Invest Dermatol 1982; 78:468–471. 40 Faredin I, Fazekas AG, Toth I, Kokai K, Julesz M: Transformation in vitro of [4-14C]-dehydroepiandrosterone into 7-oxygenated derivatives by normal human male and female skin tissue. J Invest Dermatol 1969;52:357–361. 41 Voigt W, Sawaya M, Hsia SL: Mechanism of action of dehydroepiandrosterone in the androgenic stimulation of sebaceous glands. International Symposium on Regulation on Androgen Action, Montréal, 1984. 42 Gallegos AJ, Berliner DL: Transformation and conjugation of dehydroepiandrosterone by human skin. J Clin Endocrinol Metab 1967;27: 1214–1218. 43 Labrie F, Sugimoto Y, Luu-The V, Simard J, Lachance Y, Bachvarov D, Leblanc G, Durocher F, Paquet N: Structure of human type II 5·-reductase. Endocrinology 1992;131:1571– 1573. 44 Luu-The V, Zhang Y, Poirier D, Labrie F: Characteristics of human types 1, 2 and 3 17ßhydroxysteroid dehydrogenase activities: Oxidation-reduction and inhibition. J Steroid Biochem Molec Biol 1995;55:581–587. 45 Labrie Y, Durocher F, Lachance Y, Turgeon C, Simard J, Labrie C, Labrie F: The human type II 17ß-hydroxysteroid dehydrogenase gene encodes two alternatively-spliced messenger RNA species. DNA Cell Biol 1995;14:849– 861. 46 Bélanger A, Brochu M, Cliche J: Levels of plasma steroid glucuronides in intact and castrated men with prostatic cancer. J Clin Endocrinol Metab 1986;62:812–815. 47 Labrie F, Bélanger A, Dupont A, Luu-The V, Simard J, Labrie C: Science behind total androgen blockade: From gene to combination therapy. Clin Invest Med 1993;16:475–492. 48 Pelletier G, Dupont E, Simard J, Luu-The V, Bélanger A, Labrie F: Ontogeny and subcellular localization of 3ß-hydroxysteroid dehydrogenase (3ß-HSD) in the human and rat adrenal, ovary and testis. J Steroid Biochem Mol Biol 1992;43:451–467. 49 Neri R, Monahan MD, Meyer JG, Afonso BA, Tabachnick IA: Biological studies of an antiandrogen (SH-714). Eur J Pharmacol 1967;1: 438–444. 50 Simard J, Luthy I, Guay J, Bélanger A, Labrie F: Characteristics of interaction of the antiandrogen Flutamide with the androgen receptor in various target tissues. Mol Cell Endocrinol 1986;44:261–270. 51 Simard J, Labrie C, Bélanger A, Gauthier S, Singh SM, Mérand Y, Labrie F: Characterization of the effects of the novel non-steroidal antiestrogen EM-800 on basal and estrogeninduced proliferation of T-47D, ZR-75-1 and MCF-7 human breast cancer cells in vitro. Int J Cancer 1997;73:104–112.

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52 Luo S, Stojanovic M, Labrie C, Labrie F: Inhibitory effect of the novel antiestrogen EM-800 and medroxyprogesterone acetate (MPA) on estrone-stimulated growth of dimethylbenz (a)anthracene (DMBA)-induced mammary carcinoma in the rat. Int J Cancer 1998;73: 580–586. 53 Luo S, Martel C, Gauthier S, Mérand Y, Bélanger A, Labrie C, Labrie F: Long term inhibitory effects of a novel antiestrogen on the growth of ZR-75-1 and MCF-7 human breast cancer tumors in nude mice. Int J Cancer 1997;73:735– 739. 54 Luo S, Sourla A, Labrie C, Bélanger A, Labrie F: Combined effects of dehydroepiandrosterone and EM-800 on bone mass, serum lipids, and the development of dimethylbenz(a)anthracene (DMBA)-induced mammary carcinoma in the rat. Endocrinology 1997;138:4435– 4444. 55 Gauthier S, Caron B, Cloutier J, Dory YL, Favre A, Larouche D, Mailhot J, Ouellet C, Schwerdtfeger A, Leblanc G, Martel C, Simard J, Mérand Y, Bélanger A, Labrie C, Labrie F: (S)-(+)-4-[7-(2,2-dimethyl-1-oxopropoxy)-4methyl-2-[4-[2-(1-piperidinyl)-ethoxy]phenyl]2H-1-benzopyran-3-yl]-phenyl 2,2-dimethylpropanoate (EM-800): A highly potent, specific, and orally active nonsteroidal antiestrogen. J Med Chem 1997;40:2117–2122. 56 Labrie F, Labrie C, Bélanger A, Simard J, Gauthier S, Luu-The V, Mérand Y, Giguère V, Candas B, Luo S, Martel C, Singh SM, Fournier M, Coquet A, Richard V, Charbonneau R, Charpenet G, Tremblay A, Tremblay G, Cusan L, Veilleux R: EM-652 (SCH 57068), a third generation serm acting as pure antiestrogen in the mammary gland and endometrium. J Steroid Biochem Mol Biol 1999;69:51–84. 57 Sourla A, Richard V, Labrie C, Labrie F: Exclusive androgenic effect of dehydroepiandrosterone in sebaceous glands of rat skin. J Endocrinol 2000;166:455–462. 58 Serri F, Huber WM: The development of sebaceous glands in man; in Montagna W, Ellis RA, Silver AF (eds): Advances in Biology of Skin: The Sebaceous Glands. Oxford, Pergamon Press, 1963, pp 1–18. 59 Sharp F, Hay JB, Hudgins MB: Metabolism of androgens in vitro by human foetal skin. J Endocrinol 1976;70:491–499. 60 de Peretti E, Forest MG: Pattern of plasma dehydroepiandrosterone sulfate levels in human from birth to adulthood: Evidence for testicular production. J Clin Endocrinol Metab 1978;47:572–577. 61 Ramasastry P, Downing DT, Pochi PE, Strauss JS: Chemical composition of human skin surface lipids from birth to puberty. J Invest Dermatol 1970;54:139–144. 62 Pochi PE, Strauss JS, Downing DT: Skin surface lipid composition, acne, pubertal development and urinary excretion of testosterone and 17-ketosteroids in children. J Invest Dermatol 1977;69:485–489. 63 Milne JA: The metabolism of androgens by sebaceous glands. Br J Dermatol 1969;81 (suppl 2): 22–28.

64 Reynolds SRM, Foster FI: Peripheral vascular action of estrogen observed in the ear of the rabbit. J Pharmacol Exp Ther 1940;68:173– 184. 65 Lachance Y, Luu-The V, Labrie C, Simard J, Dumont M, de Launoit Y, Guérin S, Leblanc G, Labrie F: Characterization of human 3ß-hydroxysteroid dehydrogenase/¢5-¢4 isomerase gene and its expression in mammalian cells. J Biol Chem 1990;265:20469–20475. 66 Dufort I, Rheault P, Huang XF, Soucy P, LuuThe V: Characteristics of a highly labile human type 5 17beta-hydroxysteroid dehydrogenase. Endocrinology 1999;140:568–574. 67 Sansone G, Reisner RM: Differential rates of conversion of testosterone to dihydrotestosterone in acne and in normal skin: A possible pathogenic factor in acne. J Invest Dermatol 1971;56:366–372. 68 Bingham KD, Shaw DA: The metabolism of testosterone by human male scalp skin. J Endocrinol 1973;57:111–121. 69 Kuttenn F, Mowszowicz I, Schaison G, Mauvais-Jarvis P: Androgen production and skin metabolism in hirsutism. J Endocrinol 1977; 75:83–91. 70 Andersson S, Russel DW: Structural and biochemical properties of cloned and expressed human and rat steroid 5a-reductases. Proc Natl Acad Sci USA 1990;87:3640–3644. 71 Andersson S, Bergman DM, Jenkins EP, Russel DW: Deletion of steroid 5·-reductase 2 gene in male pseudophermaphroditism. Nature 1991;354:159–161. 72 Chen W, Zouboulis CC, Fritsch M, Blume-Peytavi U, Kodelja V, Goerdt S, Luu-The V, Orfanos CE: Evidence of heterogeneity and quantitative differences of the type 1 5alpha-reductase expression in cultured human skin cells– evidence of its presence in melanocytes. J Invest Dermatol 1998;110:84–89. 73 Pelletier G, Luu-The V, Huang XF, Lapointe H, Labrie F: Localization by in situ hybridization of steroid 5·-reductase isozyme gene expression in the human prostate and preputial skin. J Urol 1998;160:577–582. 74 Roy S, Mahesh VB, Greenblatt KB: Effect of dehydroepiandrosterone and D4-androstenedione on the reproductive organs of female rats: Production of cystic changes in the ovary. Nature 1962;196:42–43. 75 Ward RC, Costoff A, Mahesh VB: The induction of polycystic ovaries in mature cycling rats by the administration of dehydroepiandrosterone (DHEA). Biol Reprod 1978;18:614–623. 76 Labrie F, Diamond P, Cusan L, Gomez JL, Bélanger A: Effect of 12-month DHEA replacement therapy on bone, vaginum, and endometrium in postmenopausal women. J Clin Endocrinol Metab 1997;82:3498–3505. 77 Franceschi S: The epidemiology of endometrial cancer. Gynecol Oncol 1991;41:1–16. 78 Friedl A, Jordan VC: What do we know and what don’t we know about tamoxifen in the human uterus. Breast Cancer Res Treat 1994; 31:27–39.

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Human Skin: An Independent Peripheral Endocrine Organ Christos C. Zouboulis Department of Dermatology, University Medical Center Benjamin Franklin, The Free University of Berlin, Berlin, Germany

Key Words Endocrinology W Hormone synthesis W Hormone receptors W Hormone metabolism W Hormone activity

Abstract The historical picture of the endocrine system as a set of discrete hormone-producing organs has been substituted by organs regarded as organized communities in which the cells emit, receive and coordinate molecular signals from established endocrine organs, other distant sources, their neighbors, and themselves. In this wide sense, the human skin and its tissues are targets as well as producers of hormones. Although the role of hormones in the development of human skin and its capacity to produce and release hormones are well established, little attention has been drawn to the ability of human skin to fulfil the requirements of a classic endocrine organ. Indeed, human skin cells produce insulinlike growth factors and -binding proteins, propiomelanocortin derivatives, catecholamines, steroid hormones and vitamin D from cholesterol, retinoids from diet carotenoids, and eicosanoids from fatty acids. Hormones exert their biological effects on the skin through interaction with high-affinity receptors, such as receptors for peptide hormones, neurotransmitters, steroid hormones and thyroid hormones. In addition, the human skin is

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able to metabolize hormones and to activate and inactivate them. These steps are overtaken in most cases by different skin cell populations in a coordinated way indicating the endocrine autonomy of the skin. Characteristic examples are the metabolic pathways of the corticotropin-releasing hormone/propiomelanocortin axis, steroidogenesis, vitamin D, and retinoids. Hormones exhibit a wide range of biological activities on the skin, with major effects caused by growth hormone/insulin-like growth factor-1, neuropeptides, sex steroids, glucocorticoids, retinoids, vitamin D, peroxisome proliferator-activated receptor ligands, and eicosanoids. At last, human skin produces hormones which are released in the circulation and are important for functions of the entire organism, such as sex hormones, especially in aged individuals, and insulin-like growth factor-binding proteins. Therefore, the human skin fulfils all requirements for being the largest, independent peripheral endocrine organ. Copyright © 2001 S. Karger AG, Basel

Introduction

The human skin is the target for a wide range of chemical messengers. These include several hormones, which in the classical sense are defined as substances secreted into

Prof. Dr. Christos C. Zouboulis Department of Dermatology, University Medical Center Benjamin Franklin The Free University of Berlin, Fabeckstrasse 60–62 D–14195 Berlin (Germany) Tel. +49 30 8445 6910, Fax +49 30 8445 6908, E-Mail [email protected]

the blood stream by specific ductless glands. Their effects have long been recognized and in some instances well characterized. For example, hair follicles and sebaceous glands are the targets for androgen steroids secreted by the gonads and the adrenal cortex [1, 2] and melanocytes are directly influenced by polypeptide hormones of the pituitary [3]. However, the historical picture of the endocrine system as a set of discrete hormone-producing organs, most of them under the control of a master gland, the pituitary, has become extended and modified to the point of metamorphosis. The skin and other tissues can no longer be regarded simply as the recipients of signals from distant transmitters. They must rather be viewed as organized communities in which the cells emit, receive and coordinate molecular signals from a seemingly unlimited number of distant sources in addition to the established endocrine organs (modern and classic endocrine functions, respectively), their neighbors (paracrine and juxtacrine function), and themselves (autocrine and intracrine function) (fig. 1). In the widest sense the human skin and its tissues are thus the targets as well as the producers of hormones. In addition to the modified determination of the endocrine skin functions, the results of current research have blurred the distinction between hormones secreted into the blood stream and locally active factors. Epithelial skin cells share common properties with secretory neurons exhibiting a complete hypothalamic-pituitary-like axis [4] and the skin converts the circulating androgen steroids dehydroepiandrosterone (DHEA) and androstenedione to testosterone and further to 5·-dihydrotestosterone (5·DHT) by the intracellular enzyme 5·-reductase but is also responsible for large amounts of the circulating testosterone and 5·-DHT levels [2, 5]. Finally, the identification of a number of pharmacologically active peptides in a range of tissues throughout the body focused attention on the ubiquity of locally acting hormones. Although the role of hormones in the development of human skin tissues and their capacity to produce and release further hormones are well established [6, 7], little attention has been drawn to the ability of human skin to fulfil the requirements of a classic endocrine organ, namely synthesis of hormones from major classes of compounds used by the body for general purposes, binding and regulation of specific receptors by the derived hormones, organized metabolism, activation, inactivation, and elimination of the hormones in specialized cells of the tissue, exertion of biological activity, and release of hormones in the circulation.

Human Skin as Endocrine Organ

Fig. 1. Modes of hormone action. Classical and modern endocrine functions: Hormones produced by established endocrine organs or other distant sources, respectively, reach target tissues through the circulation. Paracrine function: Hormones act locally on cells other than those that produce them. Juxtacrine function: Hormones produced in one cell interact directly with a receptor of an immediate neighboring cell. Autocrine function: Hormones act on the cell in which they are produced. Intracrine function: Hormones get activated in the cell in which they are produced and act on it by binding to nuclear receptors.

Synthesis of Hormones in Human Skin

All types of small molecules can practically represent precursors of skin hormones which can be proteins, including glycoproteins, smaller peptides or peptide derivatives, amino acid analogs or lipids (fig. 2). Polypeptide hormones are direct translation products of specific mRNA, such as growth hormone (GH), and cleavage products of large precursor proteins, such as propiomelanocortin (POMC) derivatives and prolactin. Although there is no evidence that GH or GH-like peptides are produced in the skin, its downstream peptide, insulinlike growth factor-I (IGF-I), is synthesized in the skin, mainly by dermal fibroblasts and melanocytes and also possibly by keratinocytes of the stratum granulosum [8, 9]. Dermal fibroblasts are also source for IGF-II and IGFbinding protein (IGFBP)-3 [10, 11]. POMC derivatives, such as adrenocorticotropic hormone (ACTH), melanocyte stimulating hormone (MSH) isotypes and ß-endorphin are produced in several skin cell types in vivo and in vitro [4, 12–15]. ACTH and ·-MSH are mainly expressed in epidermal keratinocytes, melanocytes, the outer root sheath of the anagen hair follicle,

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Fig. 2. Synthesis of hormones in

human skin. ➀ Keratinocytes; ➁ hair follicles; ➂ cutaneous nerves; ➃ sebaceous glands; ➄ melanocytes; ➅ fibroblasts; ➆ endothelial cells. ACTH = Adrenocorticotropic hormone; ·-MSH = ·-melanocyte stimulating hormone; CRH = corticotropin releasing hormone; Vit. D = vitamin D; atRA = all-trans retinoic acid; IGF-I = insulin-like growth factor I; IGFBP-3 = insulin-like growth factor binding protein-3.

dermal fibroblasts and microvascular endothelial cells. ß-Endorphin is mainly produced by the outer root sheath of the anagen hair follicle and dermal fibroblasts. The few data existing on prolactin synthesis in human skin are controversial. While dermal fibroblasts in vitro have been shown to synthesize prolactin [16], no prolactin mRNA was detected in human skin in another study [17]. Catecholamines – norepinephrine and epinephrine – which are modified amino acids and natural activators of cAMP pathway, are produced in human keratinocytes but not in melanocytes [18]. Another type of modified amino acid, the corticotropin releasing hormone (CRH), has been detected in epidermal and follicular keratinocytes, melanocytes, endothelial cells, and dermal nerves but not in sebocytes or fibroblasts [4, 19]. Steroid hormones and vitamin D are derived from cholesterol. The skin, especially the sebaceous glands, is capable of synthesizing cholesterol – from two-carbon fragments such as acetate [20, 21] – which is utilized in cell membranes, formation of the epidermal barrier, in sebum, and as substrate for steroid hormone synthesis [22]. Skin is also source of corticosteroids [23] and the unique site of vitamin D3 (cholecalciferol) production [24, 25]. Retinoids and eicosanoids, such as prostaglandins, prostacyclins and leukotrienes, are fatty acid derivatives. In humans, vitamin A (retinol) and natural retinoids are derived from carotenoids in the diet that are modified by the body; in the skin, excess retinol is mainly esterified [26]. Human keratinocytes in vitro are able to produce

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low amounts of the intracellularly active metabolite alltrans retinoic acid (atRA) [27–29]. Eicosanoid synthesis can also be induced in human keratinocytes by several proinflammatory signals [30, 31].

Hormone Receptors in Human Skin

Hormones exert their biological effects on the skin through interaction with high-affinity receptors, which are, in turn, linked to one or more effector systems within cells. These effectors involve many different components of the cellular metabolic machinery, ranging from ion transport at the cell surface to stimulation of the nuclear transcriptional apparatus. In general, receptors for the peptide hormones and neurotransmitters are aligned on the cell surface, while those for the steroid and thyroid hormones are found in the cytoplasmic or nuclear compartments. Receptors for the Peptide Hormones and Neurotransmitters The peptide hormone and neurotransmitter receptors fall into four major groups; three of them are represented in human skin. The first includes the so-called serpentine or ‘seven transmembrane domain’ receptors which contain an amino terminal extracellular domain followed by seven hydrophobic amino acid segments, each of which is believed to span the membrane bilayer. The seventh segment is followed by a hydrophilic carboxyl terminal

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domain that resides within the cytoplasmic compartment. To this group belong the parathyroid hormone (PTH)/ parathyroid hormone-related peptide (PTHrP) receptor which is expressed in dermal fibroblasts but not in epidermal keratinocytes [32, 33], the thyroid-stimulating hormone (TSH) receptor which is present in dermal fibroblasts [34], the CRH receptors from which type 1 is present in epidermal and follicular keratinocytes, melanocytes, and dermal fibroblasts, whereas sebocytes express types 1 and 2 [4, 19], the melanocortin receptors (MCR), among them MCR1 which presents high affinity for ·MSH and ACTH and is expressed in epidermal and follicular keratinocytes, epidermal and follicular melanocytes, sebocytes, sweat gland cells, endothelial cells, Langerhans cells, monocytes, macrophages, lymphocytes and dermal fibroblasts, MCR2 which is specific for ACTH and is expressed in epidermal melanocytes and adipocytes, and MCR5 which shows affinity for ·-MSH and ACTH and is present in sebocytes, sweat gland cells and adipocytes [4, 12, 35, 36], the Ì-opiate receptors which bind with high affinity ß-endorphin and are expressed in epidermal and outer root sheat keratinocytes, undifferentiated sebocytes and cells of the sweat gland secretory portion [4], the vasoactive intestinal popypeptide (VIP) receptors which are expressed in epidermal keratinocytes, sebocytes, sweat gland cells, endothelial cells, mononuclear cells and dermal nerve fibers [37–39], the neuropeptide Y receptor which is present in sebocytes [38], and the calcitonin gene-related peptide (CGRP) receptor which is expressed in sebocytes and Langerhans cells [38, 40]. The second group includes the single-transmembrane domain receptors that harbor intrinsic tyrosine kinase activity. This includes the insulin/IGF-I receptor and the epidermal growth factor receptor which are expressed in epidermal keratinocytes [8, 41]. The third group, which is functionally similar to the second group, is characterized by a large extracellular binding domain followed by a single membrane spanning segment and a cytoplasmic tail. These receptors do not possess intrinsic tyrosine kinase activity but appear to function through interaction with soluble transducer molecules which do possess such activity. In human skin, they are represented by the GH receptor which is present in melanocytes and dermal fibroblasts, epidermal and follicular keratinocytes of the outer root sheath, especially the basal ones, sebocytes, cells of the eccrine sweat gland secretory duct, hair matrix cells of the dermal papillae, endothelial cells, Schwann cells of peripheral nerve fascicles, and adipocytes of the dermis [8, 42, 43].

Steroid Hormone and Thyroid Hormone Receptors The nuclear receptors differ from the receptors of the cell membrane in that they are soluble receptors with a proclivity for employing transcriptional regulation as a means of promoting their biological effects. Thus, though some receptors are compartmentalized in the cytoplasm while others are defined to the nucleus, they all operate within the nucleus chromatin to initiate the signaling cascade. They associate in the nucleus with DNA sequences bearing a specific recognition element called ‘hormone response element’. Hormone response elements have different canonical sequences for each hormone. These receptors are expressed in human skin and can be grouped into two major subtypes based on shared structural and functional properties. The first group, the steroid receptor family, includes the glucocorticoid receptor which is mainly expressed in basal keratinocytes, Langerhans cells and dermal fibroblasts [44, 45], the androgen receptor which is present in epidermal and follicular keratinocytes, sebocytes, sweat gland cells, dermal papilla cells, dermal fibroblasts, endothelial cells, and genital melanocytes [2, 46–48], and the progesterone receptor which is expressed in basal epidermal keratinocytes only [49]. The glucocorticoid receptor is down-regulated by its ligands in dermal fibroblasts but is not affected by aging [50, 51]. Steroid receptors under basal conditions exist as cytoplasmic, multimeric complexes that include the heat shock proteins hsp 90, hsp 70, and hsp 56. Association of the steroid ligand with the receptor results in dissociation of the heat shock proteins. This, in turn, exposes a nuclear translocation signal previously buried in the receptor structure and initiates transport of the receptor to the nucleus. The second group, the thyroid receptor family, includes the thyroid hormone receptors (isotypes ·1 and ß1), whereas the isotype ß1 is present in epidermal keratinocytes, outer root sheat cells, cebocytes, dermal papilla cells, and dermal fibroblasts [6, 52, 53], the estrogen receptor-ß (but not the estrogen receptor-·) which is expressed in dermal papilla cells and dermal fibroblasts, sebocytes, adipocytes, melanocytes, and keratinocytes of the outer root sheath [48, 54–56], the retinoic acid receptors (RAR; isotypes · and Á) and retinoid X receptors (RXR; isotypes ·, ß, Á) which are expressed in epidermal keratinocytes of the stratum granulosum, follicular keratinocytes, sebocytes, and endothelial cells, while only the RXR· isotype is present in melanocytes, fibroblasts, and inflammatory cells [57–61], the vitamin D receptor which is present in keratinocytes of all epidermal layers except those of the stratum corneum, epithelial cells of the epidermal appendages, mela-

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nocytes, Langerhans cells, CD11b+ macrophages and CD3+ T-lymphocytes [62, 63], and the peroxisome proliferator-related receptors (PPAR) which are expressed in epidermal and follicular keratinocytes, sebocytes, sweat gland cells, endothelial cells, and adipocytes (isotype Á), whereas isotypes · and ‰ are also expressed in keratinocytes and sebocytes [64]. The members of the thyroid receptor family share a high degree of homology to the proto-oncogene c-erbA and high affinity for a common DNA recognition site. With the exception of the estrogen receptor they do not associate with the heat shock proteins and they are constitutively bound to chromatin in the nucleus. The estrogen receptor, though demonstrating an association with heat shock proteins, is largely confined to the nuclear compartment. The estrogen receptor binds to its regulatory element as a homodimer, while the other receptors prefer binding as heterodimers together with a RXR molecule. The latter amplifies both the DNA binding and the functional activity of the receptor.

Activation and Inactivation of Hormones in Human Skin

In addition to its capacity to produce hormones and express receptors for binding of distant, paracrine, juxtacrine, autocrine, and intracrine hormones, the human skin is able to metabolize hormones in order to activate and inactivate them. These steps are overtaken in most cases by different skin cell populations in a coordinated way indicating the endocrine autonomy of the skin. Characteristic examples for this kind of endocrine skin function are the metabolic pathways of the CRH/POMC axis, sex steroids, vitamin D, and retinoids. The CRH/POMC Axis The skin is strategically located as a barrier between the external and internal environments being permanently exposed to noxious stressors. To effectively deal with such damaging signals the skin exhibits a highly organized CRH/POMC system which is analogous to the hypothalamus/pituitary/adrenal axis [4]. Activation of this pathway by stress-sensoring cutaneous signals, mainly proinflammatory cytokines, proceeds through the production and release of CRH from keratinocytes, melanocytes, endothelial cells, and dermal nerves which stimulates skin cell CRH receptors in paracrine and autocrine manners. CRH synthesis in melanocytes is up-regulated by ultraviolet radiation B and down-regulated by dexamethasone [4]. Interestingly, CRH receptors in human sebocytes can be

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regulated by several downstream hormones, mainly by testosterone, estrogens, and GH [19]. CRH enhances the production and secretion of the POMC peptides ·-MSH, ACTH, and ß-endorphin, especially in keratinocytes, melanocytes, endothelial cells and cutaneous nerves [12, 13, 15] by a complex multistep process that requires POMC processing by prohormone convertases [4]. These enzymes are expressed in keratinocytes, melanocytes, and endothelial cells. Production of ·-MSH and ACTH can be significantly up-regulated by ultraviolet light and interleukin (IL)-1 and down-regulated by tumor growth factor-ß and dexamethasone. ACTH activates the steroidogenic acute regulatory protein and thereof the MCR inducing thereby the production and secretion of cortisol [65], a powerful natural anti-inflammatory factor that counteracts the effect of stress signals and buffers tissue damage. Steroidogenesis Human sebocytes and keratinocytes express the steroidogenic acute regulatory protein which is essential for cholesterol translocation from the outer to the inner mitochondrial membrane and thus the initiation of steroidogenesis [22] (fig. 3). They also express the P450 side chain cleavage enzyme which catalyses the conversion of cholesterol into pregnenolone, the cytochrome P450 17-hydroxylase that leads to precursors of cortisol and DHEA, and the steroidogenic factor-1 which maintains these reactions. DHEA can be further converted into androstenedione and the tissue potent androgen testosterone by sebocytes only, since only sebocytes express 3ß-hydroxysteroid dehydrogenase-¢5-4 isomerase [2]. Further activation of testosterone by its conversion into 5·-DHT is catalyzed by 5·-reductase type 1 which is expressed in almost all skin cells but especially in sebocytes [66], while fibroblasts and dermal papilla cells also express 5·-reductase type 2 [48]. Sebocytes are also able to regulate the balance of testosterone and androstenedione bidirectionally by expressing the 17ß-hydroxysteroid dehydrogenase isotypes 2 and 3 [2]. Androgen conversion to estrogens in the skin takes place in dermal fibroblasts which express the responsible enzyme cytochrome P450 19 (aromatase) and androgen inactivation to androsterone or 3·-androstanediol in epidermal keratinocytes which strongly express the responsible enzyme 3·-hydroxysteroid dehydrogenase [2, 67]. In contrast to this skin-related pathway, conversion of the adrenal DHEA sulfate – which reaches the skin through the circulation – to DHEA only occurs with the assistance of monocytes which exhibit steroid sulfatase activity [68]. Therefore, the skin is a steroidogenic tissue and different skin cell types overtake distinct duties in the

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Fig. 3. Steroidogenesis in human skin. Left panel: The complete pathway of sex hormone synthesis from cholesterine. StAR = steroidogenic acute regulatory protein, P450scc = cytochrome P450 side chain cleavage enzyme, 5·-DHT = 5·-dihydrotestosterone, ER = estrogen receptor. Middle panel: Sebocytes (S) but neither keratinocytes (K) nor melanocytes (M) express 3ß-hydroxysteroid dehydrogenase-¢5–4-isomerase (¢5–3ß-HSD), the enzyme converting dehydroepiandrosterone and androstenedione to testosterone at the mRNA level (RT-PCR). Right pannel: Sebocytes but not keratinocytes are able to metabolize 3H-dehydroepiandrosterone ([3H-]DHEA) to downstream androgen compounds.

synthesis of tissue active androgens and their inactivation leading to androgen and estrogen homeostasis. Adrenal androgens may only be activated in the skin in pathologic conditions which require the presence of inflammatory cells in the skin. In addition, evaluation of skin layer-specific prednicarbate biotransformation has shown that epidermal keratinocytes can hydrolyze the double ester prednicarbate at position 21 to the monoester prednisolone 17-ethylcarbonate which nonenzymatically transforms to prednisolone 21-ethylcarbonate. This metabolite is enzymatically cleaved to prednisolone, the main biotransformation corticosteroid product. Fibroblasts show a distinctively lower enzyme activity [23]. Prednicarbate, prednisolone 17ethylcarbonate and prednisolone 21-ethylcarbonate are hydrolyzed to a minor extent only. Therefore, epidermal keratinocytes are likely to be responsible for the conversion of potent corticosteroids to less potent ones in human skin, while dermal fibroblasts are barely able to metabolize the steroids.

The Vitamin D Pathway Skin is the unique site of vitamin D3 production and liver is thought to be the main site of conversion to 25(OH)D3. Skin is further capable of activating 25(OH)D3 via 1·-hydroxylation and the resulting 1·,25(OH)2D3 (calcitriol) plays a role in epidermal homeostasis in normal and diseased skin. Human keratinocytes have been shown to substantially but slowly convert 3H-D to 3H-25(OH)-D [24]. In addition, they were 3 3 found to slowly but constantly form calcitriol from a large reservoir of D3. Interestingly, physiological doses of ultraviolet light B radiation at 300 nm induce the conversion of 7-dehydrocholesterol via pre-D3 and D3 into calcitriol in the picomolar range in epidermal keratinocytes [25]. Skin can further degrade vitamin D3: Cytochrome P450 27 in epidermis completes the set of essential vitamin D3 hydroxylases [24]. Thus, by orchestrating the entire system of production, activation and inactivation, skin is an autonomous source of hormonally active calcitriol.

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The Retinoid Pathway Epidermal keratinocytes in vivo regulate the levels of the intracellularly active all-trans retinoic acid (atRA) by induction of retinoic acid 4-hydroxylase [69]. atRA inactivation by 4-hydroxylation prevents endogenous and exogenous atRA accumulation in the epidermis. In contrast to atRA, retinol, retinaldehyde, 9-cis retinoic acid, and 13-cis retinoic acid are not able to regulate their own hydroxylation. On the other hand, human keratinocytes in vitro rapidly take up and initially convert retinol to retinyl esters and then with time to low amounts of the intracellularly active metabolite atRA [27–29]. 3,4-Didehydro-retinol can also be detected [27, 70]. However, ester formation, especially of retinyl oleate (18:1) and retinyl palmitate (16:0), remains the main root by which excess retinol is also handled by human keratinocytes in vitro [27–29, 70]. Retinoid metabolism in human skin is likely to be a cell-specific event, since sebocytes exhibit a distinct metabolic pattern compared to epidermal keratinocytes [60].

Biological Activity of Hormones in Human Skin

GH and IGF-I The effects of the GH/IGF-I axis are addressed towards a homeostatic regulation of cell proliferation and differentiation. GH activity is mainly mediated by the IGFs but GH has also direct effects on human skin cells [6]. GH enhances androgen effects on hair growth and is likely to be involved in sebaceous gland development. It stimulates sebocyte differentiation and also augments the effect of 5·-DHT on sebaceous lipid synthesis [71]. On the other hand, GH does not affect keratinocyte or sebocyte proliferation but it enhances the proliferation of dermal fibroblasts in vitro [8, 71]. IGF molecules circulate mostly bound to IGFBPs. GH and IGF-I induce increases in skin IGFBP-3 mRNA abundance [11], with a magnitude dependent on the presence of Ca2+. IGF-I at physiological levels is essential for hair follicle growth by preventing them from entering the catagen phase [72]. IGF-I and insulin have been shown to significantly stimulate sebocyte proliferation but also influence sebocyte differentiation, especially in combination with GH, in vitro [71, 73]. Insulin may act as an IGF-I surrogate as it exhibits marked homology to the IGFs and binds the IGF-I receptor at high concentrations. IGF-I was also shown to promote clonal proliferation of cultured keratinocytes [8] and to upregulate hyaluroran synthesis in dermal fibroblasts exhibiting a similar effect to basal fibroblast growth

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factors [74]. The IGF-I/IGF-I receptor loop was found to be critically involved in maintenance of human skin organ cultures ex vivo [41]; IGF-I locally produced by dermal fibroblasts interacted in a paracrine manner with its receptor, predominantly expressed in basal keratinocytes, to maintain tissue homeostasis. The GH/IGF-I axis shows an age-related decreased hormone production concomitant with symptoms similar to those of GH-deficient adults [75]. At last, GH is able to switch the predominant CRH receptor-1 mRNA expression to a sole CRH receptor-2 expression in human sebocytes [19] indicating a possible interaction of the GH/IGF-I axis with the hypothalamus/pituitary-like axis in human skin. Neuropeptides POMC peptides are likely to play a major role in the regulation of skin pigmentary system [3, 76] and of cutaneous inflammation [12, 13]. ACTH and ·-MSH exhibit the most significant melanogenic activity via cAMPdependent pathways and tyrosinase activation, which is enhanced by ultraviolet light [4]. Melanogenesis is a highly regulated process modified by postranslational, translational, or transcriptional mechanisms. In addition, dendrite formation and stimulation of melanocyte proliferation by POMC peptides have been reported. ·-MSH can also stimulate attachment of melanocytes to laminin and fibronectin and inhibit tumor necrosis factor (TNF)-·stimulated expression of the intracellular adhesion molecule-1. In keratinocytes, ·-MSH stimulates cell proliferation and down-regulates expression of hsp 70 [77] and modulates cytokine production with up-regulation of IL10 and inhibition of the IL-1-induced production and secretion of IL-8 [12, 13]. The latter effect was also detected in sebocytes and fibroblasts, where it may be mediated by NF-kB and AP-1 [35, 78]. ß-Endorphin was shown to stimulate cytokeratin 16 expression and downregulate Ì-opiate receptor expression in human epidermis [79]. VIP, in the presence of lethally treated 3T3 fibroblast feeder cells and epidermal growth factor, stimulated proliferation of keratinocytes, whereas substance P and CGRP were ineffective. VIP stimulated adenylate cyclase activity in membranes obtained from cultured keratinocytes, indicating an involvement of cAMP as second messenger in this reaction [80]. On the other hand, it is likely that overproduction of ACTH may prolong the anagen phase of hair cycle [4]. ·-MSH also stimulates synthesis and activity of collagenase/matrix metalloproteinase-1 in dermal fibroblasts [81]. TNF-· addition resulted in increased ß-endorphin and ACTH levels [14]. In contrast, tumor growth factor-ß-stimulated fibroblasts showed no

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alteration in ß-endorphin and ·-MSH levels, whereas ACTH release was significantly enhanced. ·-MSH may play a crutial role on endothelial cell function by decreasing the adherence and transmigration of inflammatory cells, a prerequisite for immune and inflammatory reactions [4]. The POMC peptides have strong immunomodulatory potential resulting in an overall immunosuppressive effect with ·-MSH presenting the widest spectrum of activities [12], such as suppression of the contact hypersensitivity reaction to nickel by systemic or topical application [4]. Both ·-MSH and ß-endorphin induced histamine release from human foreskin mast cells in vitro [14]. Sex Steroids The local formation of sex steroids provides autonomous control to human skin which is thus able to adjust the formation and metabolism of sex steroids according to local needs [2, 82]. The situation of a high secretion rate of adrenal precursor sex steroids in men and women is completely different from the animal models used in the laboratory (except monkeys) where the secretion of sex steroids takes place exclusively in the gonads. In these lower animal species, no significant amounts of androgens or estrogens are made outside the testes or ovaries and no sex steroid is left after castration. Sex steroids in human skin are activated intracellularly and exert their action on the cells themselves without release in the extracellular space and in the general circulation (intracrine function). The rate of formation of each sex steroid thus depends upon the level of expression of each of the specific androgenand estrogen-synthesizing enzymes in each cell type. Sebaceous glands and sweat glands account for the vast majority of androgen metabolism in skin [2, 6]. The biological activity of testosterone on the skin is effected in large part by its conversion to 5·-DHT by 5·reductase [83]. Testosterone and 5·-DHT, being the tissue active androgens, stimulate 5·-reductase mRNA and 5·-reductase activity, and their effects are mediated through their binding to the androgen receptor. They stimulate proliferation of target cells, such as sebocytes and dermal papilla cells [84–87]. In addition, there is evidence that the effect of androgens on human sebocyte proliferation depends on the area of skin from which the sebaceous glands are obtained; facial sebocytes are mostly affected [84, 88]. Androgens have also been shown to stimulate sebocyte differentiation [89] which is augmented by co-incubation with PPARÁ ligands [90]. Dermal papilla cells mediate the growth-stimulating signals of androgens by releasing growth factors that act in a para-

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crine fashion on the other cells of the follicle [6, 87]. Excessive amounts of tissue active androgens were shown to induce apoptosis of dermal papilla cells through the bcl-2 pathway [91]. In aged skin, lower serum levels of testosterone and gradual decline in DHEA and DHEA sulfate are detected, at least in males [75]. Estrogens prolong the growth period of scalp hair by increasing cell proliferation rates and postponing the anagen-telogen transition [87]. On the other hand, they directly suppress an enhanced sebaceous gland function [4, 89]. Estradiol has also been shown to increase proliferation of melanocytes but decrease both the melanin content and the tyrosinase activity [56]. Inhibition of 5·reductase and of androgen receptor activity resulted in a great stimulation of vascular endothelial growth factor (VEGF) and aromatase expression in dermal papilla cells. Strong stimulation of VEGF protein and gene expression was also observed in the presence of 17ß-estradiol [48]. Both testosterone and estradiol are able to regulate CRH receptor mRNA levels, whereby in an opposite way [19]. Glucocorticoids Glucocorticoids induce hair growth [92], stimulate sebocyte proliferation [73], and induce skin atrophy probably due to an effect on dermal fibroblasts [23]. The aggravation of sebaceous gland diseases by glucocorticoids may be due to their stimulatory effects on proliferation and differentiation in the presence of other growth factors [4]. Glucocorticoids can regulate keratinocyte differentiation by repressing the expression of the basal cell specific keratins K5 and K14 and disease-associated keratins K6, K16, and K17, an effect induced directly, through interactions of keratin response elements with glucocorticoids and indirectly, by blocking the AP-1 induction of keratin gene expression [93]. Retinoids Retinoic acids exhibit earlier and stronger biological effects on the keratinocytes than retinol, probably due to their early high cellular accumulation and their less rapid metabolism [29, 94]. These findings support the assumption that the intensity of retinoid signaling is dependent, in part, on the quantity of cellular retinoic acid. This assumption is supported by the tight autoregulatory mechanism in human keratinocytes offering protection against excessive accumulation of cellular retinoic acid [58]. atRA binds to and induces cellular retinoic acidbinding protein II (CRABP II) as well as binds to and activates nuclear RARs [95]. Most actions of atRA are now recognized to be mediated through activation of RARs,

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whereas, in epithelial skin cells RAR modulate cell proliferation and RXR rather influence cell differentiation [60]. Retinoids regulate proliferation and differentiation of skin epithelial cells towards an homeostatic status [94], especially inhibit enhanced proliferation and lipogenesis in human sebocytes but are also able to enhance them under vitamin A deficient conditions [96, 97]. Vitamin D Calcitriol, like retinoids, rapidly up-regulates the major vitamin D3 metabolizing enzyme 24-hydroxylase at the mRNA level, which is an established indicator for calcitriol presence [24]. It enhances the growth-promoting activity of autocrine epidermal growth factor receptor ligands in keratinocytes [98] and can also rapidly increase the activity of 17ß-hydroxysteroid dehydrogenase (isotype 2), which leads predominantly in conversion of estradiol to estrone [99]. This estradiol inactivation increases with increased calcitriol levels, especially those who exhibit antiproliferative effects on keratinocytes. In addition, keratinocytes produce abundant PTHrP which is down-regulated by calcitriol suggesting a physiological role [100]. The antiproliferative and anti-inflammatory effects of calcitriol on the skin were shown to be mediated, at least in part, by a complex tumor growth factor-ß regulation in dermal fibroblasts [101]. Thyroid Hormones Hypothyroidism causes disturbances of skin quality and hair character and growth with an increased telogen rate and diffuse alopecia [6, 7]. Replacement reestablishes the normal anagen/telogen ratio. L-Triiodothyronine was shown to stimulate proliferation of outer root sheath keratinocytes and dermal papilla cells [102]. PTHrP Regulation of the PTH/PTHrP receptor on dermal fibroblasts increases the membrane-associated protein kinase C activity modulating proliferation of epidermal keratinocytes in a paracrine manner [32]. PPAR Ligands PPARs are pleiotropic regulators of growth and differentiation of many cell types, including skin cells. PPAR· seems to contribute to skin barrier function and to regulation of inflammation, PPARÁ is necessary for sebocyte differentiation, and PPAR‰ can ameliorate inflammatory responses in the skin [64]. PPAR‰ is the predominant subtype in human keratinocytes and is highly expressed in basal and suprabasal cells [103, 104]. Induction of PPAR·

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and PPARÁ expression is linked to differentiation, and accordingly, is confined to suprabasal keratinocytes. PPAR‰ and PPARÁ inhibition resulted in a dramatic decrease in proliferation and a robust up-regulation of the expression of involucrin and transglutaminase [104, 105]. Preliminary results have shown expression of PPAR‰ and PPARÁ in the human sebaceous gland [106, 107]. Linoleic acid, a natural PPAR‰ ligand, induces accumulation of neutral lipids in undifferentiated human sebocytes and reduces spontaneous IL-8 secretion [108]. Estradiol metabolizes prostaglandin D2 to ¢12-prostaglandin J2, a natural ligand for PPARÁ [109]. Eicosanoids Proinflammatory cytokines, such as IL-1ß and TNF-·, induce cytosolic phospholipase A2 expression in keratinocytes and are able to increase the extracellular release of arachidonic acid and stimulate eicosanoid synthesis [31] (fig. 4). Enhanced keratinocyte prostaglandin synthesis after ultraviolet light injury is also due to increased phospholipase activity [30]. The major arachidonic acid metabolites after stimulation with interleukin 1ß are prostaglandin E2 and leukotriene B4 (LTB4), while TNF-· stimulates hydroxyeicosatetraenoic acid (HETE) production. IL1· expression has been detected in follicular keratinocytes and sebocytes in vivo and in vitro [73, 110–112]. Interestingly, LTB4 is a natural ligand for PPAR· [113, 114], soluble 15-HETE, which is a natural ligand for PPAR-Á [115], is synthesized in human sebaceous glands [116], and PPARs can regulate lipid and lipoprotein metabolism, cell proliferation, differentiation and apoptosis of various cell types including sebocytes [90]. The axis IL-1/LTB4/PPAR·/lipid synthesis and inflammation was confirmed by a current clinical study; treatment of acne patients with a specific 5-lipoxygenase inhibitor administered systemically led to a 70% reduction in inflammatory acne lesions at 3 months, an approximately 65% reduction in total sebum lipids as well as a substantial decrease in proinflammatory lipids [117].

Release of Skin-Produced Hormones in the Circulation

There is increasing evidence that human skin produces hormones which are released in the circulation and are important for functions of the entire organism. Major examples include sex steroids where a large proportion of androgens and estrogens in men and women are synthesized locally in peripheral target tissues from the inactive

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Fig. 4. The cascade of eicosanoid synthesis in the skin. IL-1ß = Interleukin-1ß; TNF-· = tumor necrosis factor-·; LTB4 = leucotriene B4; 15-HETE = 15-hydroxyeicosatetraenoic acid; PPAR· = peroxisome proliferator-activated receptor-·; PPARÁ = peroxisome proliferator-activated receptor-Á.

adrenal precursors DHEA and androstenedione. DHEA and androstenedione are converted to testosterone and further to 5·-DHT by the intracellular enzyme 5·-reductase in the periphery, thus making the skin responsible for large amounts of the circulating testosterone and 5·-DHT levels. Up to 50% of the total circulating testosterone is produced in the skin and in other peripheral organs [5]. The best estimate of the intracrine formation of estrogens in peripheral tissues in women is in the order of 75% before menopause and close to 100% after menopause, except for a small contribution from ovarian and/or adre-

nal testosterone and androstenedione [82]. Thus, in postmenopausal women, almost all active sex steroids are made in target tissues by an intracrine mechanism. On the other hand, IGFBP-3 message abundance is greater in the skin that in the liver and circulating IGFBP-3 concentrations are significantly increased by GH and IGFI [11]. GH has a direct function in the regulation of IGFBP3 synthesis, and the response of skin IGFBP-3 mRNA levels to both GH and IGF-I suggests that dermal fibroblasts could be more important than the liver in the regulation of circulating reservoir IGFBP-3 in certain circumstances.

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4 Slominski A, Wortman J, Luger T, Paus R, Solomon S: Corticotropin releasing hormone and propiomelanocortin involvement in the cutaneous response to stress. Physiol Rev 2000; 80:979–1020. 5 Orfanos CE, Adler YD, Zouboulis ChC: The SAHA syndrome. Horm Res 2000;54:251– 258. 6 Deplewski D, Rosenfield RL: Role of hormones in pilosebaceous unit development. Endocr Rev 2000;21:363–392.

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79 Bigliardi-Qi M, Bigliardi PL, Eberle AN, Büchner S, Rufli T: ß-Endorphin stimulates cytokeratin 16 expression and downregulates Ì-opiate receptor expression in human epidermis. J Invest Dermatol 2000;114:527–532. 80 Haegerstrand A, Jonzon B, Dalsgaard CJ, Nilsson J: Vasoactive intestinal polypeptide stimulates cell proliferation and adenylate cyclase activity of cultured human keratinocytes. Proc Natl Acad Sci USA 1989;86:5993–5996. 81 Kiss M, Wlaschek M, Brenneisen P, Michel G, Hommel C, Lange TS, Peus D, Kemeny L, Dobozy A, Scharffetter-Kochanek K, Ruzicka T: ·-Melanocyte stimulating hormone induces collagenase/matrix metalloproteinase-1 in human dermal fibroblasts. J Biol Chem 1995;376: 425–430. 82 Labrie F, Luu-The V, Labrie C, Pelletier G, ElAlfy M: Intracrinology and the skin. Horm Res 2000;54:218–229. 83 Chen W-C, Zouboulis ChC, Orfanos CE: The 5·-reductase system and its inhibitors: Recent development and its perspective in treating androgen-dependent skin disorders. Dermatology 1996;193:177–184. 84 Akamatsu H, Zouboulis ChC, Orfanos CE: Control of human sebocyte proliferation in vitro by testosterone and 5-·-dihydrotestosterone is dependent on the localization of the sebaceous glands. J Invest Dermatol 1992;99: 509–511. 85 Akamatsu H, Zouboulis ChC, Orfanos CE: Spironolactone directly inhibits proliferation of cultured human facial sebocytes and acts antagonistically to testosterone and 5-·-dihydrotestosterone in vitro. J Invest Dermatol 1993; 100:660–662. 86 Zouboulis ChC, Seltmann H, Neitzel H, Orfanos CE: Establishment and characterization of an immortalized human sebaceous gland cell line (SZ95). J Invest Dermatol 1999;113:1011– 1020. 87 Itami S, Kurata S, Sonoda T, Takayasu S: Mechanism of action of androgens in dermal papilla cells. Ann NY Acad Sci 1991;642:385– 395. 88 Zouboulis ChC, Akamatsu H, Stephanek K, Orfanos CE: Androgens affect the activity of human sebocytes in culture in a manner dependent on the localization of the sebaceous glands and their effect is antagonized by spironolactone. Skin Pharmacol 1994;7:33–40. 89 Guy R, Ridden C, Kealey T: The improved organ maintenance of the human sebaceous gland: Modeling in vitro the effects of epidermal growth factor, androgens, estrogens, 13-cis retinoic acid, and phenol red. J Invest Dermatol 1996;106:454–460. 90 Rosenfield RL, Deplewski D, Kentsis A, Ciletti N: Mechanisms of androgen induction of sebocyte differentiation. Dermatology 1998;196: 43–46. 91 Wro´bel A, Mandt N, Hossini A, Seltmann H, Zouboulis ChC, Orfanos CE, Blume-Peytavi U: 5·-Dihydrotestosterone and testosterone induce apoptosis in human dermal papilla cells by downregulation of the bcl-2 pathway. J Invest Dermatol 2000;115:581.

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The Hair Follicle: A Paradoxical Androgen Target Organ Valerie A. Randall a Nigel A. Hibberts a M. Julie Thornton a Kazuto Hamada b Alison E. Merrick a Shoji Kato c Tracey J. Jenner a Isobel De Oliveira a Andrew G. Messenger d a Department

of Biomedical Sciences, University of Bradford, UK, b now at Kanebo Cosmetics Laboratory, Odawara City, Japan, c now at Department of Dermatology, University of Tokushima, Japan, d Department of Dermatology, Royal Hallamshire Hospital, Sheffield, UK

Key Words Balding W Beard W Dermal papilla cells W Growth factors W Hair follicle W Human W Stem cell factor

Abstract Androgens are the main regulator of normal human hair growth. After puberty, they promote transformation of vellus follicles, producing tiny, unpigmented hairs, to terminal ones, forming larger pigmented hairs, in many areas, e.g. the axilla. However, they have no apparent effect on the eyelashes, but can cause the opposite transformation on the scalp leading to the replacement of terminal hairs by vellus ones and the gradual onset of androgenetic alopecia. This paradox appears to be an unique hormonal effect. Hair follicles are mainly epithelial tissues, continuous with the epidermis, which project into the dermis. A mesenchyme-derived dermal papilla enclosed within the hair bulb at the base controls many aspects of follicle function. In the current hypothesis for androgen regulation, the dermal papilla is also considered the main site of androgen action with androgens from the blood binding to receptors in dermal papilla cells of androgen-sensitive follicles and causing an alter-

ABC

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ation of their production of paracrine factors for target cells e.g. keratinocytes. Studies of cultured dermal papilla cells from sites with different responses to androgens in vivo have confirmed the paradoxical responses. All dermal papilla cells from androgen-sensitive sites contain low capacity, high affinity androgen receptors. However, only some cells formed 5·-dihydrotestosterone, e.g. beard but not axillary cells, in line with hair growth in 5·-reductase deficiency. Incubation with androgens also stimulated the mitogenic capacity of beard cell media, but inhibited that produced by scalp cells. This suggests that the paradoxical differences are due to differential gene expression within hair follicles, presumably caused during embryogenesis. Copyright © 2001 S. Karger AG, Basel

Androgens Stimulate and Inhibit Human Hair Growth

Although the terminal hair (long, thick, frequently pigmented) present in childhood e.g. head hair, eyelashes and eyebrows, has mainly protective functions (reviewed in [1]), many human hairs are secondary sexual character-

Prof. VA Randall Department of Biomedical Sciences University of Bradford Bradford, BD7 1DP, UK Tel. +44 1274 233560, Fax +44 1274 309742, E-Mail [email protected]

Fig. 1. Different growth patterns between androgen-potentiated hair follicles on the face and axilla. Growth of terminal hair on both the face and axilla is stimulated by androgens during puberty, but while beard growth is maintained until a man is elderly (a), axillary hair growth in both sexes starts to reduce in the thirties (b). This occurs in both Caucasian (solid line and circles) and Japanese (dotted line, open circles) men. Data is redrawn from Hamilton [6] and modified from Randall [7].

istics. These fall into two groups: those which distinguish the sexually mature adult of both sexes from a child, i.e. hair in the axillae and the lower pubic triangle, and those which are only characteristic of a sexually mature man, e.g. the beard, hair on the chest and the upper pubic diamond [2, 3]. These obvious terminal hairs start to be produced for the first time at puberty in response to the rise in plasma androgens occurring in both sexes, but reaching higher levels in males [4–7]. Therefore, hair follicles in these regions are androgen target organs requiring andro-

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gens to form terminal hairs, unlike the eyelash and scalp follicles. Previously the hair follicles in these regions were producing tiny, pale, vellus hairs which were difficult to see. The follicles enlarge gradually over several hair cycles to enable these dramatic size changes to occur (fig. 1 upper diagram). Although many terminal hairs have been formed by the end of puberty, androgens continue to increase hair growth in several areas in men for many years e.g. terminal hair on the chest is still developing in the thirties and in the ear canal after fifty [6, 8]. Although this site specific difference between whether follicles need androgen or not to produce a large terminal hair is intriguing and very unusual in an endocrine target organ, the response of human hair follicles to androgens is even more complex. In certain areas of the scalp, in genetically susceptible individuals, androgens may cause the transformation of terminal follicles to vellus ones producing such tiny, pale hairs that the area appears bald. This causes male pattern baldness in men or androgenetic alopecia in both sexes and macaque monkeys [8–13]. The completely opposite effect of androgens on one type of organ, often within a few inches of the face in the same individual, is a biological paradox which appears to be unique in endocrinology. Since the follicles are responding completely differently to the same circulating hormones within an individual the specific effect produced must be dependent on factors within the individual hair follicles themselves. The precise mechanisms of these responses within the hair follicle are not yet known though understanding the factors involved in hair growth is currently the subject of much research [14]. Even within the androgen-potentiated follicles there are paradoxical differences. Axillary hair is produced in both sexes before the beard grows [2, 3] presumably in response to androgens from the adrenals (adrenache) [4, 5]. However, although both beard and axillary hair growth is high in the twenties, axillary hair growth then falls steadily while beard growth remains high until death [6] (fig. 1).

Paradoxical Hair Growth in 5·-Reductase Deficient Men

Yet another paradox of the androgenic responses of hair follicles is seen when the role of the enzyme 5·-reductase type 2 is examined as demonstrated by hair growth in patients with 5·-reductase deficiency. These patients have an insufficiency in 5·-reductase type 2 which prevents them from metabolising testosterone to 5·-dihydro-

Randall et al.

testosterone by this enzyme, the form found in the secondary sexual tissues such as the prostate (reviewed [15, 16]). In many androgen target organs, such as the prostate, testosterone is metabolised intracellularly to 5·-dihydrotestosterone which then binds to the androgen receptor and activates the appropriate gene expression. In other tissues, such as skeletal muscle, testosterone itself is the active intracellular form binding to the same receptor (reviewed in [16]). Men with 5·-reductase deficiency are frequently thought to be girls at birth, but become more virilised at puberty developing a masculinised body shape and musculature and a small penis. However, they only develop female patterns of hair growth with axillary and female pattern pubic hair, but very little, if any, beard or chest hair. This suggests that in the follicles that are androgen sensitive in both sexes the intracellular metabolism of testosterone by 5·-reductase type 2 is not essential, whereas it is required by follicles which distinguish the adult male.

Investigations into Androgen Action in Human Hair Follicles Using Dermal Papilla Cells

These paradoxes have been the focus of attention in our group for many years. We have been investigating the mechanism of androgen action in hair follicles with differing responses to androgens in vivo concentrating in our earlier studies on comparing androgen-stimulated beard follicles with relatively androgen-independent non-balding scalp follicles. More recently, we have expanded our comparisons to encompass androgen-inhibited balding scalp follicles. Working on the basis of Randall’s hypothesis that androgens act on hair follicles via the mesenchyme-derived dermal papilla situated at the base of the follicle [1, 17], we have focussed particularly on cultured dermal papilla cells. Dermal papilla cells offer a useful model system as they can be cultured from small, full depth samples of human skin [18, 19] and cultured cells retain their hair inducing properties when reimplanted in vivo [20].

receptors in the dermal papilla cells. This would alter gene expression regulating the production of a paracrine factor or factors which would alter the activity of the cells in the follicle appropriately. These paracrine factors could be soluble mitogenic factors such as growth factors e.g. IGF-I [21] and/or extracellular matrix components. Extracellular matrix components are also likely to be involved because the size of the hair produced by the follicle is related to dermal papilla size which is formed mainly of the extracellular matrix which is in close contact with the other cellular components of the follicle [22, 23]. Potential targets for androgens in the follicle include: the dermal papilla cells, since the dermal papilla size must be altered to change the hair size [22, 23]; the follicular keratinocytes, which divide and mature to form the hair and its various sheaths; the melanocytes, which alter the amount of pigment produced under different endocrine conditions; and the follicular endothelial cells as the larger the follicle the greater the blood supply will be required. In this hypothesis the actions of androgens would be interpreted by the dermal papilla cells and transmitted to the other target cells via paracrine factors. This model has received a great deal of experimental support [reviewed 7, 24] and is now well established. Androgen Receptors in Cultured Dermal Papilla Cells from Follicles with Differing Responses to Androgens in vivo Investigations into the mechanism of androgen action in cultured dermal papilla cells from human follicles with various responses to androgens in vivo have revealed the same paradoxes in vitro as seen in vivo. Both androgenstimulated beard [25] and androgen-inhibited balding scalp [26] dermal papilla cells contained higher levels of high affinity, low capacity androgen receptors than control non-balding scalp. In beard, non balding scalp and balding scalp cells the specificity of the binding for a range of steroids was similar confirming that androgens were acting through the same receptor to have their paradoxical effects (fig. 2).

Model for Androgen Action in Hair Follicles The hypothesis for androgen action was based on the important regulatory role of the dermal papilla which determines many aspects of hair follicle activity including the type of hair produced [reviewed in 20] and parallels between the hair follicle and the classic androgen target tissue, the prostate [reviewed in 1, 17]. In this model of androgen action [7], androgens would enter the dermal papilla via its own blood capillaries and then bind to

Testosterone Metabolism in vitro Reflects Hair Growth in 5·-Reductase Deficient Men When the metabolism of testosterone by dermal papilla cells in culture was examined interesting differences were seen. Initial experiments on beard cells demonstrated that they contained 5·-reductase activity and rapidly metabolised testosterone to 5·-dihydrotestosterone [27, 28] which was retained intracellularly [28]. This is what would be predicted from the lack of beards in men

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Fig. 2. Androgen receptors from non-balding scalp (a) and balding scalp dermal papilla cells (b) show similar binding characteristics for a range of steroids confirming that androgenetic alopecia does not involve a different form of the androgen receptor. The effectiveness of various androgenic steroids, the antiandrogen cyproterone acetate, and other types of steroid hormones at 100 nM concentration to compete with the binding of the non-metabolisable synthetic androgen, mibolerone, at 1 nM by dermal papilla cells was assessed by incubation for 2 h in serum-free medium at 37 ° C. The competition studies revealed that androgens plus the antiandrogen competed strongly for

the binding in all cell types with 17ß-oestradiol (Oest) also having some effect. Progesterone (Prog), triamicinolone acetonide (TMA) with corticosteroid and progestational activity and hydrocortisol (HC) had virtually no effects. EtOH: ethanol vehicle alone; DHT: 5·-dihydrotestosterone; Mib: mibolerone; Test: testosterone; Cyp: cyproterone acetate. Results are the mean B SEM from 3 primary cell lines of non-balding, and 4 of balding scalp, dermal papilla cells with each point assayed in triplicate. Results from Hibberts et al. [26].

with 5·-reductase type 2 deficiency [15, 16]. In marked contrast neither axillary nor pubic cells [29] contained significant amounts of 5·-dihydrotestosterone intracellularly even when cultured for 24 h (fig. 3). This again reflects hair growth in men with 5·-reductase deficiency where the female patterns of pubic and axillary hair are evident. It is possible that these follicles are so much more sensitive to androgens than beard and other male sexual characteristic hair follicles that there is sufficient 5·-dihydrotestosterone circulating as a result of 5·-reductase type 1 activity to stimulate pubic and axillary hair growth. However, this seems unlikely and would also raise the question of why one system depended on extracellular 5·-dihydrotestosterone and the other on making it intracellularly. Since there was virtually no intracellular metabolism of testosterone to 5·-dihydrotestosterone in pubic and axil-

lary cells, testosterone does not appear to be being metabolised to 5·-dihydrotestosterone by the isoenzyme 5·reductase type 1 either, although this enzyme also appears to be present in beard dermal papilla cells [27]. It seems more likely that testosterone itself is binding to the androgen receptor and stimulating the gene expression as occurs in some other tissues such as skeletal muscle [reviewed in 16]. In the beard cells, like the prostate and other secondary sexual tissues, 5·-dihydrotestosterone is formed intracellularly and will be recognised and bound preferentially by the androgen receptor [16]. This would be a distinction in the mechanism of androgen action which reflected that seen between androgen actions which occur in both sexes e.g. skeletal muscle and only males e.g. secondary sexual characteristics. However, it still leaves the paradox that essentially the same androgen effect, the transformation

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The metabolism of testosterone by 5·-reductase type 2 also appears to play an important role in androgenetic alopecia. Men with 5·-reductase type 2 deficiency are also reported not to go bald [15], although this is quite difficult to assess as many come from groups where androgenetic alopecia is less prevalent. The metabolism of testosterone in androgenetic alopecia dermal papilla cells differed from both that in beard and pubic cells with testosterone being metabolised to a range of steroids including 5·dihydrotestosterone, androstenedione and androstanedione [30]. Recent treatment of men with androgenetic alopecia with the 5·-reductase type 2 inhibitor, finasteride, has inhibited the progression of balding on the scalp and in many cases caused hair re-growth [31, 32]. This suggests that 5·-dihydrotestosterone, formed via the 5·reductase type 2 isoenzyme, is the active intracellular androgen in androgenetic alopecia in men. However, since plasma 5·-dihydrotestosterone also falls significantly it is not completely established whether intracellular metabolism is required [31]. Nevertheless, this means that the paradoxical androgenic inhibition of hair growth on the scalp and stimulation on the face both involve the same type of receptor binding to the same form of intracellular androgen, 5·-dihydrotestosterone; the specificity must lie at some post metabolism/receptor event.

of a vellus follicle to a large terminal one, is working through the same androgen receptor but by combination with a different form of the androgenic steroid. Since this presumably involves regulation of many of the same genes, the system seems to need another factor. There may be availability of additional transcription factors in the axillary and pubic cells which enables the weaker androgen to be as effective as 5·-dihydrotestosterone in the beard cells in the hormone receptor-hormone response element complexes.

Paracrine Factors Produced by Dermal Papilla Cells The obvious difference between any two cell types is the genes which are expressed within each type. Since the dermal papilla plays such a regulatory role in the hair follicle [20] and is also believed central in androgen action [1, 7], there is a great deal of interest in identifying paracrine factors involved in hair growth and produced by dermal papilla cells. Space constraints preclude major discussion here but they have recently been reviewed elsewhere [33– 36]. Our studies are focussed on investigating factors produced by dermal papilla cells whose expression is altered by androgens [reviewed in more detail in 24]. Androgens stimulated the production of mitogenic factors for primary lines of outer root sheath keratinocytes [21] and beard dermal papilla cells [37] and also transformed epidermal keratinocytes [38]. However, in contrast androgens inhibited the production of mitogens by androgenetic alopecia dermal papilla cells from both human [39] and macaque [13] scalps. This is an intriguing mirror in vitro of the biological effects in vivo supporting the original hypothesis of androgen action and confirming that dermal papilla cells from follicles with differing responses to androgens in vivo offer a useful model for studies of androgen action in vitro.

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Fig. 3. Dermal papilla cells from pubic hair follicles produce little

5·-dihydrotestosterone in culture in marked contrast to beard cells. Dermal papilla cells derived from beard and pubic hair follicles were incubated with 5 nM 3H-testosterone for up to 24 h. The medium was collected, cells washed, the intracellular steroids extracted and after the addition of marker 14C-steroids, the steroid content analysed by thin layer chromatography and recrystallisation [29]. Beard cells rapidly metabolised the 3H-testosterone to 5·-dihydrotestosterone (a), but pubic cells contained very little even after 24 h (b). Results from Hamada et al. [29].

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Fig. 4. Differential expression of the para-

crine factor, stem cell factor (SCF) in beard (a) and scalp dermal papilla cells (b). The amounts of SCF in medium conditioned by dermal papilla cells under various conditions were assayed by ELISA. No medium conditioned (i.e. serum-free medium in which cells were grown) by any type of cell contained SCF after 15 min, therefore any detected after 24 h had been secreted by the cells during the experiment. Testosterone (10 nM) in vitro had no effect on SCF levels in either cell type. However, both beard (6 primary cell lines) and non-balding scalp (7 primary cell lines) cells produced SCF with beard cells secreting significantly more (p ! 0.001). Results (mean B SEM with each point assayed in duplicate) are from Hibberts et al. [41].

Analysis of specific factors produced by dermal papilla cells by Japanese scientists led by Takayasu and Itami identified androgen-stimulation of IGF-I gene expression by beard dermal papilla cells causing a mitogenic response by outer root sheath cells [21]. In our studies also using RT-PCR the expression of hepatocyte growth factor (HGF), or scatter factor, was much greater in beard than non-balding scalp cells and much less in balding scalp [40]. This differential expression may indicate that HGF may be important in maintaining large follicles and that its levels in androgen-dependent follicles may be altered by exposure to androgens in vivo. This would be the sort of differential expression that would explain the paradoxical responses to androgens, but this particular observation would require another mechanism to explain the paradox of one particular gene’s expression being increased by testosterone in one type of follicle and inhibited in another! Since androgens also alter hair colour, we determined that dermal papilla cells secreted stem cell factor (SCF), also known as c-kit ligand, steel factor or mast cell growth factor, by analysing conditioned media from dermal papilla cells by ELISA [41]. Since SCF is known to play an important role in the development of epidermal [42, 43] and hair pigmentation [44, 45], it is feasible that the dermal papilla is a local source of SCF for the adult hair follicle melanocytes [41]. Adult human scalp follicle melanocytes do express c-kit giving them the ability to respond

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to SCF from the papilla. Interestingly, although androgen in vitro had no effect on the secretion of SCF in any cell type, beard cells, i.e. those which had responded in vivo to androgens by producing darker hairs, did secrete significantly higher levels of SCF than non-balding scalp cells [41] (fig. 4). These are probably only the first examples of differential gene/protein expression by dermal papilla cells from follicles with varying responses to androgens in vivo. Further investigations should increase our understanding of these biological paradoxes.

Conclusions

The Paradoxical Effects of Androgens on Human Hair Follicles Appear to Be due to Intrinsic Differential Gene Expression within Individual Cells All these studies on the mechanism of androgen action in dermal papilla cells from follicles with varying androgenic responses support the view that intrinsic differences within the follicles are responsible for the various responses to androgens seen at different body sites. Since the androgen receptors appear to be the same in androgen stimulated beard and inhibited balding follicles this is unlikely to be the source of the different reactions. Differences in the amounts of intracellular 5·-reductase type 2 and hence the ability to metabolise circulating androgens

Randall et al.

to the most potent form, 5·-dihydrotestosterone, are found between the follicles characteristic of both sexes of adults e.g. the axilla and those normally restricted to men e.g. beard. Since both respond similarly to androgens, presumably involving the regulation of at least some of the same genes, there may be specific differences at the post receptor level between the two types of androgen stimulated follicles. Since the marked paradox of androgen effects on balding and beard growth both appear to involve androgen receptors and 5·-reductase type 2, the genes available for expression within each cell type seem to be the mechanisms by which the paradox occurs. Presumably, gene availability in each cell type will be determined during the embryonic patterning mechanisms. Further studies on the paracrine factors produced by dermal papilla cells, partic-

ularly those whose expression is altered by androgens, should provide greater understanding of this endocrine paradox and, hopefully, lead to better forms of treatment for androgen potentiated hair disorders.

Acknowledgements The help of Mr Chris Bowers with the figures and Mrs Christine Dove with the preparation of the manuscript is gratefully acknowledged. The research in this paper was supported by grants to Prof. Randall and Dr. Messenger from the Medical Research Council (G 8610976 SB; G 9108798 SB) and to Professor Randall from Kanebo Ltd., Japan and the Trimill Trust. Dr. Kato and Mrs. de Oliveria were visiting research scientists in Professor Randall’s laboratory supported by the Japanese Ministry and Health and CAPES, Brazil, respectively.

References 1 Randall VA: Androgens and human hair growth. Clin Endocrinol 1994;40:439–457. 2 Marshall WA, Tanner JM: Variations in pattern of pubertal change in girls. Arch Dis Child 1969;44:291–303. 3 Marshall WA, Tanner JM: Variations in the pattern of pubertal changes in boys. Arch Dis Child 1970;45:13–23. 4 Winter JSD, Faiman C: Pituitary-gonadal relations in male children and adolescents. Paed Res 1972;6:125–135. 5 Winter JSD, Faiman C: Pituitary-gonadal relations in female children and adolescents. Paed Res 1973;7:948–953. 6 Hamilton JB: Age, sex and genetic factors in the regulation of hair growth in man: A comparison of Caucasian and Japanese populations; in Montagna W, Ellis RA (eds): The Biology of Hair Growth. New York, Academic Press, 1958, pp 399–433. 7 Randall VA: Androgens: The main regulator of human hair growth; in Camacho FM, Randall VA, Price VH (eds): Hair and Its Disorders: Biology, Pathology and Management. London, Martin Dunitz, 2000, pp 69–82. 8 Hamilton JB: A secondary sexual character that develops in men but not in women upon ageing of an organ present in both sexes. Anat Record 1946;94:466–467. 9 Hamilton JB: Patterned loss of hair in man; types and incidence. Ann NY Acad Sci 1951; 53:708–728. 10 Hamilton JB: Male hormone stimulation is a prerequisite and an incitant in common baldness. Amer J Anat 1942;71:451–480. 11 Hamilton JB: Effect of castration in adolescent and young adult males upon further changes in the proportions of bare and hairy scalp. J Clin Endocrinol Metabol 1960;20:1309–1318.

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12 Randall VA: The biology of androgenetic alopecia; in Camacho FM, Randall VA, Price VH (eds): Hair and Its Disorders: Biology, Pathology and Management. London, Martin Dunitz, 2000, pp 123–136. 13 Uno H, Imamura K, Pan Heui-ju: Androgenetic alopecia in the stump-tailed macaque: An important model for investigating the pathology and antiandrogenic therapy of male pattern baldness; in: Camacho FM, Randall VA, Price VH (eds): Hair and Its Disorders: Biology, Pathology and Management. London, Martin Dunitz, 2000, pp 137–151. 14 Camacho FM, Randall VA, Price VH (eds): Hair and Its Disorders: Biology, Pathology and Management. London, Martin Dunitz, 2000. 15 Wilson JD, Griffin JE, Russell DW: Steroid 5·reductase 2 deficiency. Endocr Rev 1993;14: 577–593. 16 Randall VA: The role of 5·-reductase in health and disease; in Sheppard M, Stewart P (eds): Hormones, Enzymes and Receptors. Baillières Clin Endo Metabol 1994;8:405–431. 17 Randall VA, Thornton MJ, Hamada K, Redfern CPF, Nutbrown M, Ebling FJG, Messenger AG: Androgens and the hair follicle: Cultured human dermal papilla cells as a model system. Ann NY Acad Sci 1991;642:355–375. 18 Messenger AG: The culture of dermal papilla cells from human hair follicles. Br J Dermatol 1984;110:685–689. 19 Randall VA, Hibberts NA, Hamada K: A comparison of the culture and growth of dermal papilla cells derived from normal and balding (androgentic alopecia) scalp. Br J Dermatol 1996;134:437–444.

20 Jahoda CAB, Reynolds AJ: Dermal-epidermal interactions; adult follicle – derived cell populations and hair growth; in Whiting DA (ed): Dermatol Clin 14. Update on hair disorders. Saunders, Philadelphia, 1996, pp 573–583. 21 Itami S, Kurata S, Takayasu S: Androgen induction of follicular epithelial cell growth is mediated via insulin-like growth factor I from dermal papilla cells. Biochem Biophys Res Commun 1995;212:988–994. 22 Van Scott EJ, Ekel TM: Geometric relationships between the matrix of the hair bulb and its dermal papilla in normal and alopecic scalp. J Invest Dermatol 1958;31:281–287. 23 Elliot K, Stephenson TJ, Messenger AG: Differences in hair follicle dermal papilla volume are due to extracellular matrix volume and cell number: Implications for the control of hair follicle size and androgen responses. J Invest Dermatol 1999;113:873–877. 24 Randall VA, Hibberts NA, Thornton MJ, Merrick AE, Hamada K, Kato S, Jenner TJ, De Oliveira I, Messenger AG: Do androgens influence hair growth by altering the paracrine factors secreted by dermal papilla cells? Eur J Dermatol 2001; in press. 25 Randall VA, Thornton MJ, Messenger AG: Cultured dermal papilla cells from androgendependent human follicles (e.g. beard) contain more androgen receptors than those from nonbalding areas. J Endocrinol 1992;133:141– 147. 26 Hibberts NA, Howell AE, Randall VA: Dermal papilla cells from human balding scalp hair follicles contain higher levels of androgen receptors than those from non-balding scalp. J Endocrinol 1998;156:59–65.

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27 Itami S, Kurata S, Takayasu S: 5·-reductase activity in cultured human dermal papilla cells from beard compared with reticular dermal fibroblasts. J Invest Dermatol 1990;94:150– 152. 28 Thornton MJ, Liang I, Hamada K, Messenger AG, Randall VA: Differences in testosterone metabolism by beard and scalp hair follicle dermal papilla cells. Clin Endocrinol 1993;39: 633–639. 29 Hamada K, Thornton MJ, Liang I, Messenger AG, Randall VA: The metabolism of testosterone by dermal papilla cells cultured from human pubic and axillary hair follicles concurs with hair growth in 5·-reductase deficiency. J Invest Dermatol 1996;106:1017–1022. 30 Hamada K, Randall VA: The metabolism of testosterone by dermal papilla cells cultured from scalp follicles of men with androgenetic alopecia. J Invest Derm 1993;101:440. 31 Kaufman KD, Olsen EA, Whiting D, Savi R, De Villez R, Bergfeld W and the Finasteride Male Pattern Hair Loss Study Group: Finasteride in the treatment of men with androgenetic alopecia. J Am Acad Dermatol 1988;39:578– 589. 32 Price VH: Clinical trials of oral finasteride; in Camacho FM, Randall VA, Price VH (eds): Hair and Its Disorders: Biology, Pathology and Management. London, Martin Dunitz, 2000, pp 159–166.

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33 Stenn KS, Combates NJ, Eilertson KJ, Gordon JS, Poardinas JR, Parimoo S, Prouty S: Hair follicle growth controls; in Whiting DA (ed): Dermatol Clin 14. Update on hair disorders. Saunders, Philadelphia, 1996, pp 543–558. 34 Blume-Peytavi, U Mandt N. Signalling molecules in human hair follicle cell populations; in Camacho FM, Randall VA, Price VH (eds): Hair and Its Disorders: Biology, Pathology and Management. London, Martin Dunitz, 2000, pp 95–101. 35 Philpott M: The roles of growth factors in hair follicles: investigations using cultured hair follicles; in Camacho FM, Randall VA, Price VH (eds): Hair and Its Disorders: Biology, Pathology and Management. London, Martin Dunitz, 2000, pp 103–113. 36 Paus R, Müller-Röver S, McKay I: Control of the hair follicle growth cycle; in Camacho FM, Randall VA, Price VH (eds): Hair and its Disorders: Biology, Pathology and Management. London, Martin Dunitz, 2000 pp 83–94. 37 Thornton MJ, Hamada K, Messenger AG, Randall VA: Beard, but not scalp, dermal papilla cells secrete autocrine growth factors in response to testosterone in vitro. J Invest Dermatol 1998;111:727–732. 38 Hibberts NA, Quick JR, Messenger AG, Randall VA: Only androgen-dependent cultured dermal papilla cells secrete additional proteinaceous factors mitogenic for keratinocytes in response to testosterone. Br J Dermatol 1994; 131:427.

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39 Hibberts NA, Sato K, Messenger AG, Randall VA: Dermal papilla cells from human hair follicles secrete factors (e.g. VEGF) mitrogenic for endothelial cells. J Invest Dermatol 1996;106: 341. 40 Merrick AE, Hibberts NA, Messenger AG, Thornton MJ, Randall VA: Balding dermal papilla cells from human hair follicles express less hepatocyte growth factor (HGF) than normal scalp and beard cells. 7th European Hair Res Soc 2000. 41 Hibberts NA, Messenger AG, Randall VA: Dermal papilla cells derived from beard hair follicles secrete more stem cell factor (SCF) in culture than scalp cells or dermal fibroblasts. Biochem Biophys Res Commun 1996;222: 401–405. 42 Williams DE, de Vries P, Namen AE, Widmer MB, Lyman SD: The steel factor. Dev Biol 1992;151:368–376. 43 Grichnik JM, Burch JA, Burchette J, Shea CR: The SCF/KIT pathway plays a critical role in the control of normal human melanocyte homeostasis. J Invest Dermatol 1998;111:233– 238. 44 Geissler EN, Cheng SV, Gusella JF, Housmann D: The dominant-white spotting (W) locus of the mouse encodes the c-kit proto-oncogene. Cell 1988;55:185–192. 45 Fleischman RA, Saltman DL, Stastry V, Zneimer S: Deletion of the c-kit proto-oncogene in the human developmental defect piebald trait. Proc Natl Acad Sci 1991;88:10885– 10889.

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The SAHA Syndrome Constantin E. Orfanos YaeI D. Adler Christos C. Zouboulis Department of Dermatology, University Medical Center Benjamin Franklin, The Free University of Berlin, Berlin, Germany

Key Words SAHA W Seborrhoea W Acne W Hirsutism W Androgenetic alopecia

Abstract The presence of seborrhoea, acne, hirsutism and alopecia in women has first been summarized as SAHA syndrome in 1982 and can be associated with polycystic ovary syndrome, cystic mastitis, obesity and infertility. In 1994, the association of these androgen-dependent cutaneous signs, was classified according to their etiology into four types: (1) idiopathic, (2) ovarian, (3) adrenal, and (4) hyperprolactinemic SAHA. The HAIRAN syndrome has been currently described as a fifth variant with polyendocrinopathy. The SAHA syndrome generally occurs in young to middle-aged women and involves either the presence of elevated blood levels of androgens or increased androgen-driven peripheral response with normal circulating androgen levels. Peripheral metabolism of androgens takes place in various areas within the pilosebaceous unit, as indicated by local differences in the activities of aromatase, 5·-reductase as well as of the presence of the androgen receptors. In cases of SAHA syndrome, careful diagnostic and clinical evaluation has to be performed in order to identify the cause for peripheral hyperandrogenism and to exclude androgen-producing tumors. Treatment will target the etiology, whereas the management in idiopathic cases will aim to improve the clinical features of SAHA.

Introduction

The SAHA syndrome summarizes the major cutaneous features indicating peripheral hyperandrogenism in young females. This term, introduced by us in 1982 [1], stands for seborrhea, acne, hirsutism on face, trunk and extremities and androgenetic alopecia of the scalp. Although all four signs of SAHA syndrome are only present in approx. 20% of the patients [2], its knowledge is important for recognizing hormonal disorders involving androgen metabolism. In skin, the formation of excessive active androgen metabolites in the pilosebaceous unit is responsible for the appearance of cutaneous hyperandrogenism [3, 4], however, the characteristic clinical phenotype does not always correlate with elevated blood levels of androgens [5]. In cases of systemic virilization, additional signs are present, such as deepening of the voice, increased muscle bulk, clitoris hypertrophy, loss of smooth skin contours or obesity, irregularities of the menstrual cycle and infertility. Many clinical characteristics of the SAHA syndrome are common with those seen in cases with polycystic ovaries (PCO) and similar disorders [6].

Pathogenetic Background

Women produce androgens in the ovaries, the adrenal glands and also in peripheral organs, especially the skin, the skeletal muscles and the liver [7]. The ovaries secrete

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Prof. Dr. Christos C. Zouboulis Department of Dermatology, University Medical Center Benjamin Franklin The Free University of Berlin Fabeckstrasse 60–62, D–14195 Berlin (Germany) Tel. +49 30 84456910, Fax +49 30 84456908, E-Mail [email protected]

Fig. 1. Synthesis of the two major circulating

androgens, androstenedione and testosterone [from ref. 23].

20% of the circulating testosterone, 20–30% of dehydroepiandrosterone (DHEA), less than 10% of DHEA sulfate (DHEA-s) and 60% of ¢4-androstenedione (A-dione) (in the midcycle 70%). Free testosterone makes only 1–3% of the total testosterone levels. The rest circulates bound to the sexual hormone-binding globuline (SHBG) (78%), albumin (20%) and cortisol (1%) [8, 9]. The adrenal glands are responsible for 30% of testosterone, 70–80% of DHEA, 90% of DHEA-s and 40% of A-dione (in the midcycle 25%). Androgen production of ovaries and adrenal glands is influenced by the pituitary gland through its regulatory hormones FSH, LH and ACTH. The human skin also plays an important role in the development of peripheral hyperandrogenism (fig. 1); Adione and DHEA are converted to testosterone and further to 5·-dihydrotestosterone (5·-DHT) by the intracellular enzyme 5·-reductase in the periphery [10], thus making the skin responsible for large amounts of the circulating testosterone and 5·-DHT levels [3, 4]. Up to 50% of the total circulating testosterone is produced in the skin and in other peripheral organs. On the other hand, the function of the pilosebaceous unit in the skin appears strongly dependent on biological-

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ly active androgens. Signs of SAHA syndrome do not occur until the process of puberty begins [11]. DHEA, Adione and testosterone stimulate sebum secretion in humans [12, 13]. 5·-DHT seems to be necessary for male beard growth and scalp hair recession, while testosterone alone is sufficient for stimulation of axillary and pubic hair growth (reviewed in [14]). The effect of active androgens is mediated by binding to nuclear androgen receptors [15, 16]. Lack of functional androgen receptors, e.g. in the total androgen insensitivity syndrome, prevents the action of androgens on skin appendages [17]. Peripheral hyperandrogenism can develop in one or more different steps [18]: (1) excess androgen synthesis by adrenals and ovaries [19], (2) reduced clearance of androgens in the liver, (3) induction of metabolism and activation of androgens in the skin [3, 20], (4) disturbance of metabolic evolution of androgens according to age, (5) reduction of SHBG levels in serum [21], (6) increased amounts of nuclear androgen receptors [15, 16], and (7) reduction of conversion rates of androgens to estrogens by low activity of peripheral aromatase [22]. Therefore, peripheral hyperandrogenism occurs either on the basis of high levels of circulating free androgens

Orfanos/Adler/Zouboulis

caused by a series of disorders or due to the capacity of the pilosebaceous apparatus to respond with increased sensitivity to normal circulating androgen levels [23]. Interestingly, although the skin is a complex organ, a large number of its structures are influenced or closely controlled by hormones, especially by androgens [3, 24]. Since the pilosebaceous apparatus is the main locus of conversion of testosterone to its more potent metabolite 5·-DHT, it is of major importance for the development of idiopathic peripheral hyperandrogenism [24], whereas total testosterone, DHEA, and A-dione usually remain within normal limits in female patients. In contrast, 5·androstanediol glucuronide was found increased and SHBG decreased in women with hair loss [24, 25] and also in bald males [26]; for assessing peripheral hyperandrogenism, therefore, the ratio of total androgens to SHBG seems to be valuable [27]. On the other hand, in case that increased androgen blood levels have to be considered, six serological parameters are relevant: DHEA-s, A-dione, prolactin, free testosterone, SHBG, and 3·androstanediol glucoronide. Despite the fact that the development and function of the pilosebaceous unit depends on androgens, other signal transduction pathways, such as peroxisome proliferatoractivated receptors, growth hormone, insulin-like growth factor, insulin, other peptides, glucocorticoids, estrogens, cytokines, angiokines, and neuropeptides, seem to contribute [24]. There is apparently a close communication system between dermal papilla cells, follicular keratinocytes and sebaceous gland cells.

Effects of Peripheral Androgens on Hair Follicles and Sebaceous Glands

Having recognized the key effects of biologically active androgens on the skin and its appendages [11–14], their local synthesis and degradation have gained special interest. It is known that the precursors of active androgens in tissue, such as DHEA-s, DHEA and A-dione, are mostly derived from glandular secretion, while testosterone in women and 5·-DHT in men and women are mainly synthesized in the periphery. Therefore, an association between a possible local overproduction of active androgens with skin disorders, such as acne and androgenetic alopecia in males, has been suggested by several groups. Skin in both acne [28] and androgenetic alopecia [29] produces higher rates of testosterone and 5·-DHT from blood precursors than in healthy individuals. Intracellular conversion of testosterone to 5·-DHT was found to be 2–30

The SAHA Syndrome

times greater in acne-bearing skin than in normal skin. In addition, elevated plasma levels of 5·-DHT and 3·androstanediol glucuronide have been found in female patients with acne and androgenetic alopecia, while DHEA-s, A-dione and testosterone were normal [30]. Five major enzymes are involved in the intracellular activation and deactivation of androgens [3, 31, 32]: In a first step, 3ß-hydroxysteroid dehydrogenase/¢5-4-isomerase (¢5-3ß-HSD) converts DHEA to A-dione. Two isoforms of the enzyme have been described [33]. Human skin seems to express exclusively the type 1 isoform with approximately 5- to 8-fold higher substrate affinity compared to type 2 [3, 34]. Cutaneous ¢5-3ß-HSD is practically located in the sebaceous glands only [3, 34, 35]. In a second step, A-dione is further activated to testosterone through the 17ß-hydroxysteroid dehydrogenase (17ßHSD). Seven isoforms of this enzyme have been identified, whereby isoforms 2, 3 and 5 catalyze the conversion of testosterone to A-dione and vice versa, respectively [36]. In a third step, 5·-reductase irreversibly converts testosterone to 5·-DHT, the most potent naturally occurring androgen. For this enzyme, two isoforms have been described [37], whereby type 1 is dominating in the skin in vivo and in vitro [10, 38]. Immunohistochemical and enzyme activity studies suggested predominant expression of the enzyme in sebaceous glands, but also in sweat glands, epidermal cells and in hair follicles [39, 40]. Finally, 3·-hydroxysteroid dehydrogenase (3·-HSD), an enzyme existing in three isoforms [41], catabolizes active androgens to compounds which do not bind the androgen receptor. After glucuronidation, water-soluble compounds, such as 3·-androstanediol glucuronide, are eliminated through the kidney [42]. Alternatively, aromatase can convert testosterone and androstenedione to estrogens in certain cell types [43]. Another important factor that plays a role in the cellular response to androgens is the variance in number, type, and affinity of the androgen receptor proteins [44, 45].

Cutaneous Changes Induced by Peripheral Hyperandrogenism

Seborrhoea and Acne Seborrhoea occurs in varying degrees on body areas with high sebaceous gland density, such as face, breast, back and scalp. In such disorders as the constitutional seborrhoeic skin in women, size and number of lobules per sebaceous gland are increased. A coincidence of seborrhoea with follicular hyperkeratinization, microbial colo-

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253

Table 1. Laboratory values for evaluating

androgenetic alopecia in females

Hormonal values

Male pattern androgenetic alopecia

Female pattern androgenetic alopecia

Female pattern androgenetic alopecia with hirsutism

DHEA Testosterone SHBG Testosterone/SHBG 3·-Androstanediol glucuronide/SHBG

elevated elevated decreased/normal elevated

normal/elevated normal normal normal

normal/elevated normal/elevated decreased/normal elevated

elevated

elevated

elevated

nization and inflammatory processes lead to acne [46]. Seborrhoea and acne which are the two most frequent signs in SAHA are induced by androgen activity. Seborrhoea is a result of hypersensitivity of the follicular target organ to androgens. Acne alone, even comedonal acne, may be induced due to androgen excess, thus, minor or mild acne can be the sole manifestation of peripheral hyperandrogenism [47]. In the bald scalp the sebaceous glands rapidly transform DHEA-s through the action of ¢5-3ß-HSD into androstenedione which is rapidly transformed into testosterone due to an increased activity of 17ß-HSD, a fact that does not occur in non-bald scalp [48]. Interestingly, eunuchs never suffer from acne, but after injection of testosterone they may develop acne lesions and possibly male androgenetic alopecia, if they have an adequate family history [49]. A majority of women with acne (60–70%) note worsening of their lesions a few days before menstruation [50]. Increased levels of DHEA-s [46], enhanced androgen activation in the skin [3], and low numbers of CAG repeats in the androgen receptor gene [51] have been accused as the responsible factors, however, there is still no androgen marker for the presence of acne in SAHA. Hirsutism Hirsutism is typically defined as excessive male pattern hair growth in women. This definition distinguishes hirsutism from hypertrichosis, which is a term that describes the androgen-independent, often familial growth of vellus body hair in non-sexual areas or hair growth, usually associated with metabolic disorders or particular medications [24]. The degree of hirsutism has been classified by Ferriman and Galway [52]. Mild hirsutism alone or in combination with the other three major signs of SAHA shows a great variability in serum androgen levels with 50% of the cases clearly exhibiting increased free testosterone levels [47]. However, women with marked hirsutism alone are usually hyperandrogenic.

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Androgenetic Alopecia Androgenetic alopecia is a result of marked telogen effluvium presenting a decreased anagen/telogen ratio in trichogram examinations in the frontoparietal region of the scalp. Androgens cause a progressive conversion of terminal hair to intermediate and vellus hair by reducing the follicular anagen period, leading to ‘miniaturization’ of the follicles [24, 53]. Female alopecia usually begins in puberty and later develops to a diffuse thinning of hair in the ‘crown’ area with the frontal hair remaining intact. This picture, described by Ludwig [54], is divided into 3 degrees of severity, whereas increased androgen levels result in a male pattern of androgenetic alopecia in females [55] (table 1). The female pattern androgenetic alopecia is associated with increased peripheral androgenetic activities; the circulating testosterone levels remain into normal limits, the DHEA-s levels are normal or increased and the levels of 3·-androstanediol glucuronide are increased. The biochemical marker for this pattern of hair loss is the 3·-androstanediol glucuronide/SHBG ratio [5]. Another factor mediating androgen activity is the androgen receptor [45].

Clinical Classification of SAHA Syndrome

The pattern of the syndrome can markedly vary regarding the expressiveness and the severity of the clinical signs from only cosmetical discomfort to serious illness. Each of the four major signs can be slightly or intensively expressed and can represent the leading clinical feature of the disease. Seborrhoea is always present, whereas androgenetic alopecia only occurs in 21% of the cases, acne in 10%, and hirsutism in 6% as isolated signs [18]. Etiologically, SAHA syndrome is a response of the pilosebaceous unit to androgenic stimulation due to peripheral familial androgen hypersensitivity or due to hormonal disorders. Many females suffering from SAHA with or without men-

Orfanos/Adler/Zouboulis

strual irregularities are obese. Weight loss may result in improvement of hyperandrogenism signs and in regulation of menses. Familial SAHA Familial SAHA is also known as ‘ethnic hyperandrogenism’, being common p.e. in Southern Europe, particularly in women from the Mediterranean area, representing the familial occurrence of clinical signs of peripheral hyperandrogenism. Although there is usually no alteration of the hormonal levels in plasma, biochemical examination of blood androgen levels has to be performed. The women involved usually present lateral facial hirsutism accompanied by mild inflammatory acne located on the mid-facial area. A genetically determined increased androgen receptor sensitivity and/or enhanced androgen metabolism in skin have been postulated as the molecular basis of the disorder [3]. Ovarian SAHA It represents the most common cause of hyperandrogenism, often a PCO syndrome [56]. Ovarian SAHA is characterized by functional ovarian hyperandrogenism, whereby ovarian changes are macroscopically inconspicuous, particularly in mild cases. Histopathology reveals multiple subcapsular follicular cysts, cortical thickening and increased stromal tissue. In severe cases, enlarged ovaries with hyperthecosis and large persistent follicular cysts can be detected by sonography. The patients are usually young women with significant seborrhea, inflammatory scarring acne, mammary, lateral facial and central body hirsutism, and androgenetic alopecia. Young females can be obese or have a tendency to obesity with regular menses or suffer from oligo- or amenorrhoea and virilization [57]. The suggested molecular basis of ovarian SAHA is an autosomal dominant trait leading to increased synthesis of adrenal androgens that provide an extra ovarian source of estrogens by peripheral metabolism and increased LH secretion by the pituitary gland [58]. The LH/FSH ratio is characteristically increased in these patients; in addition, slightly increased serum Adione and free testosterone levels, decreased SHBG, and increased 3·-androstanediol glucuronide can be detected. Adrenal SAHA Adrenal SAHA occurs due to anatomical or only functional adrenal hyperplasia. Clinically, significant seborrhoea and severe nodulocystic acne with scar formation in the face and on the back, female androgenetic alopecia,

The SAHA Syndrome

and slight to moderate central as well as lateral hirsutism may be present. Linear extension of the pubic triangle over the abdomen up to the middle breast is often found. The patients are usually thin and seem constantly stressed; their menstrual cycles are usually longer than 30 days, sometimes skipping a cycle, and their menstruation is long-lasting and painful [57]. Biochemically, increased DHEA-s and A-dione levels, and normal prolactin, SHBG, and testosterone levels are found. In severe cases, plasma cortisol levels may be increased [18]. Hyperprolactinemic SAHA The clinical manifestations in hyperprolactinemic SAHA are similar to those of adrenal SAHA. Nodulocystic acne and central hirsutism are the major signs, although galactorrhoea may occasionally be present. The major biochemical finding is increased serum prolactin. The HAIRAN Syndrome: SAHA with Polyendocrinopathy Hyperandrogenism, insulin resistance and acanthosis nigricans constitute the so called HAIRAN syndrome that may be classified as a variant of SAHA syndrome with polyendocrinopathy [59]. Clinically, patients are young and obese, with seborrhoea, mild inflammatory acne, moderate to severe hirsutism, androgenetic alopecia, acanthosis nigricans in the skin faults, and insulin resistant diabetes mellitus [60]. Insulin resistance is caused by a receptor or post-receptor defect. The combination of insulin resistance and acanthosis nigricans occurs in up to 5% of women with hyperandrogenism, in whose development insulin may play a central role. In vitro studies have shown that insulin exerts a stimulatory effect on ovarian production of androgens and that it inhibits the synthesis of SHBG in the liver [61]. Insulin acts through binding to insulin-like growth factor receptors which are present on multiple organs, so in the ovaries and the skin, and may stimulate ovarian testosterone synthesis. In skin, acanthosis nigricans results from stimulation of epidermal keratinocyte proliferation [62]. Additionally, gonadotropinreleasing hormone and consequently LH secretion is promoted. Stimulation of insulin-like growth factor receptors leads to hyperlipidemia and obesity. lt is still unclear whether hyperinsulinemia and insulin resistance are primary phenomena or secondary to obesity. Biochemically, insulin, glucose, cortisol, progesterone, and several androgens can be found increased in serum.

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Fig. 2. Peripheral hyperandrogenism in females and its differential diagnosis (androgenetic vs. androgenic alopecia) [from ref. 23].

Diagnostic Procedures

Diagnosis and classification of the SAHA syndrome is based on: (a) the characteristic clinical picture of peripheral hyperandrogenism as well as virilization signs (changes of the voice, increased muscle masses, clitorimegaly and amenorrhoea) and defeminization signs (breast atrophy, decreased rugosity of the vaginal columns, menstrual alterations and sterility), (b) the hereditary predisposition, (c) the typical trichogram examination showing telogen effluvium, particularly in the frontal or frontoparietal area of the scalp, (d) biochemical screening (fig. 2) including DHEA-s, A-dione, free testosterone, SHBG, prolactin, 3·-androstanediol glucuronide. In certain cases FSH, LH, 17-hydroxyprogesterone, ferritin, thyroxin and antimicrosomal antibodies can be requested. In exceptional cases glucose, insulin, cortisol, 11-desoxycortisol, and 17ß-estradiol are required. The dexamethasone suppression test, the ACTH stimulation test and imaging techniques may be necessary, in addition.

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Treatment

Any treatment measures will depend on the particular type of SAHA syndrome present in each individual case. In idiopathic SAHA local (androgenetic alopecia) or systemic (seborrhoea, acne, hirsutism) treatment may be introduced according to the severity of the clinical signs [24, 46, 63–68]. As a rule, 2–3 months may be needed to achieve a beneficial effect on seborrhoea and acne and 9– 12 months for influencing hair growth, at least to some extent. In ovarian SAHA with elevated androgen blood levels an antiandrogen (progestin)-estrogen combination may be seen as the first-line treatment. Two years oral medication with cyproterone acetate 10–50 mg during the first 6 months from the 5th to the 15th day of cycle and 2 mg for the rest 18 months from the 5th to the 26th day of cycle and ethinylestradiol 35 mg/day from the 5th to the 26th day of cycle is a standard regimen. In older females estradiol valerate (4 mg/day) may also be administered. In

Orfanos/Adler/Zouboulis

cases of intolerance to oral contraceptives estradiol valerate 10 mg i.m. at the 5th and 15th days of cycle or medroxyprogesterone acetate (10 mg p.o.) from the 1st to the 12th day of cycle can be introduced. Norgestagens and levonogestagens should be avoided as they exhibit a rest androgenic activity [46]. Adrenal SAHA has to be treated by adrenal suppression with moderate doses of glucocorticoids together with antiandrogens in order to avoid binding of 5·-DHT to the transporting proteins. Glucocorticosteroids reduce synthesis of DHEA-s, A-dione and testosterone. A 6-month treatment with 2-month regimens of 10 mg (0-5-5), 5 mg (0-0-5) and finally 2.5 mg (0-0-2.5) prednisolone seems appropriate. In addition, cyproterone acetate (50–100 mg/day) from the 5th to the 15th day of the cycle, spironolactone (50–200 mg/day) and flutamide (250–375 mg/

day) can be used. As cyproterone acetate usually causes menstrual alterations, 35 Ìg estradiol have to be added from the 5th to the 20th day of cycle. Treatment of hyperprolactinemic SAHA is performed by a combination of corticosteroids, at dose levels similar to those in adrenal disease, and bromocriptine (2.5–7.5 mg/day), a dopaminergic agonist, in order to achieve normalization of the prolactin levels within 3–5 months. The treatment of HAIRAN syndrome focuses on the significant reduction of insulin levels. Weight loss is important; metformin, thiazolidinediones, and D-chiroinositol may be administered also reducing androgen levels to a greater or lesser degree [24]. The extent to which these effects correlate with clinical improvement of SAHA remains to be determined [69].

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12 Pochi PE, Strauss JS: Sebaceous gland response in man to the administration of testosterone, ¢4-androstenedione, and dehydroisoandrosterone. J Invest Dermatol 1969;52:32–36. 13 Diamond P, Cusan L, Gomez JL, Bélanger A, Labrie F: Metabolic effects of 12-month percutaneous dehydroepiandrosterone replacement therapy in postmenopausal women. J Endocrinol 1996;150:S43–S50. 14 Messenger AG: The control of hair growth: An overview. J Invest Dermatol 1993;101:4S–9S. 15 Zouboulis CC, Akamatsu H, Stephanek K, Orfanos CE: Androgens affect the activity of human sebocytes in culture in a manner dependent on the localization of the sebaceous glands and their effect is antagonized by spironolactone. Skin Pharmacol 1994;7:33–40. 16 Sawaya ME: Purification of androgen receptors in human sebocytes and hair. J Invest Dermatol 1992;98:92S–96S. 17 Imperato-McGinley J, Gautier T, Cai L-Q, Yee B, Epstein J, Pochi P: The androgen control of sebum production. Studies of subjects with dihydrotestosterone deficiency and complete androgen insensitivity. J Clin Endocr Metab 1983;76:524–528. 18 Camacho F: SAHA Syndrome; in: Camacho F, Montagna W (eds): Trichology. Diseases of the pilosebaceus follicle. Madrid, Aula Medica, 1997; pp 673–690. 19 Lookingbill DP, Horton R, Demers LM, Egan N, Marks JG Jr, Santen RJ: Tissue production of androgens in women with acne. J Am Acad Dermatol 1985;12:481–487. 20 Thiboutot D, Harris G, Iles V, Cimis G, Gilliland K, Hagari: Activity of the type 1 5·-reductase exhibits regional differences in isolated sebaceous glands and whole skin. J Invest Dermatol 1995;105:209–214.

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30 Gilliland JM, Kirk J, Smeaton TC: Normalized androgen ratio: Its application in clinical dermatology. Clin Exp Dermatol 1982;6:349– 353. 31 Hay JB, Hodgins MB: Distribution of androgen metabolizing enzymes in isolated tissues of human forehead and axillary skin. J Endocrinol 1978;79:29–39. 32 Sawaya ME, Penneys NS: Immunohistochemical distribution of aromatase and 3ß-hydroxysteroid dehydrogenase in human hair follicle and sebaceous gland. J Cutan Pathol 1992;19: 309–314. 33 Labrie F, Simard J, Luu-The V, Bélanger A, Pelletier G: Structure, function and tissue-specific gene expression of 3ß-hydroxysteroid dehydrogenase/5-ene-4-ene isomerase enzymes in classical and peripheral intracrine steroidogenic tissues. J Steroid Biochem Molec Biol 1992; 43:805–826. 34 Dumont M, Luu-The V, Dupont E, Pelletier G, Labrie F: Characterization, expression, and immunohistochemical localisation of 3ß-hydroxysteroid dehydrogenase/¢5-¢4 isomerase in human skin. J Invest Dermatol 1992;99: 415–421. 35 Courchay G, Boyera N, Bernard BA, Mahe Y: Messenger RNA expression of steroidogenesis enzyme subtypes in the human pilosebaceous unit. Skin Pharmacol 1996;9:169–176. 36 Labrie F, Luu-The V, Lin S-X, Labrie C, Simard J, Breton R, Bélanger A: The key role of 17ß-hydroxysteroid dehydrogenases in sex steroid biology. Steroids 1997;62:148–158. 37 Russell DW, Wilson JD: Steroid 5·-reductases: Two genes/two enzymes. Annu Rev Biochem 1994;63:25–61. 38 Chen W-C, Zouboulis CC, Fritsch M, BlumePeytavi U, Kordelja V, Goerdt S, Orfanos CE: Evidence of heterogeneity and quantitative differences of the type 1 5·-reductase expression in cultured human skin cells – first evidence of its presence in melanocytes. J Invest Dermatol 1998;110:84–89. 39 Luu-The V, Sugimoto Y, Puy L, Labrie Y, Lopez Solache I, Singh M, Labrie F: Characterization, expression, and immunohistochemical localization of 5·-reductase in human skin. J Invest Dermatol 1994;102:221–226. 40 Eicheler W, Dreher M, Hoffmann R, Happle R, Aumüller G: Immunhistochemical evidence for differential distribution of 5·-reductase isoenzymes in human skin. Br J Dermatol 1995;133:371–376. 41 Khanna M, Qin K-N, Wang RW, Cheng K-C: Substrate specificity, gene structure, and tissuespecific distribution of multiple human 3·hydroxysteroid dehydrogenases. J Biol Chem 1995;270:20162–20168.

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42 Sperling LC, Heimer WL: Androgen biology as a basis for the diagnosis and treatment of androgenic disorders in women. J Am Acad Dermatol 1993;28:669–683. 43 Berkovitz GD, Brown TR, Fujimoto M: Aromatase activity in human skin fibroblasts grown in cell culture. Steroids 1987;50:281– 295. 44 Sawaya ME, Hordinsky MK: Advances in alopecia areata and androgenetic alopecia. Adv Dermatol 1992;7:211–227. 45 Poujol N, Wurtz JM, Tahiri B, Lumbroso S, Nicolas JC, Moras D, Sultan C: Specific recognition of androgens by their nuclear receptor. A structure-function study. J Biol Chem 2000; 275:24022–24031. 46 Zouboulis CC: Acne: Current aspects on pathology and treatment. Dermatol Experiences 1999;1:6–37. 47 Reingold SB, Rosenfield RL: The relationship of mild hirsutism or acne in women to androgens. Arch Dermatol 1987;123:209–212. 48 Sperling LC, Heimer WL: Androgen biology as a basis for the diagnosis and treatment of androgenic disorders in women. J Am Acad Dermatol 1993;28:669–683. 49 Cunliffe WJ, Shuster S: The rate of sebum excretion in man. Br J Dermatol 1969;83:697– 704. 50 Cunliffe WJ, Clayden AD, Gould D, Simpson NB: Acne vulgaris. Its aetiology and treatment. A review. Clin Exp Dermatol 1981;6:461–469. 51 Sawaya ME, Shalita AR: Androgen receptor polymorphisms (CAG repeat lengths) in androgenetic alopecia, hirsutism, and acne. J Cutan Med Surg 1998;3:9–15. 52 Ferriman D, Gallwey JD: Clinical assessment of body hair growth in women. J Clin Endocr Metab 1961;21:1440–1447. 53 Stenn KS, Paus R: Controls of hair follicle cycling. Physiol Rev 2001;81:449–494. 54 Ludwig E: Classification of the types of androgenic alopecia (common baldness). Occurrence in the female sex. Br J Dermatol 1977;97:249– 254. 55 Kasick JM, Bergfeld WF, Steck WD, Gupta MK: Adrenal androgenic female pattern alopecia: Sex hormone and the balding woman. Cleve Clin Q 1983;50:111–122. 56 Stein IF, Leventhal ML: Amenorrhea associated with bilateral polycystic ovaries. Am J Obstet Gynecol 1925;29:181–185. 57 Camacho F, Sa´nchez-Pedreño P: Sı´ndrome SAHA. Piel 1991;6:272–286.

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58 Bunker CB, Newton JA, Conway GS, Jacobs HS, Greaves MW: The hormonal profile of women with acne and polycystic ovaries. Clin Exp Dermatol 1991;16:420–423. 59 Adler YD, Orfanos CE, Zouboulis CC: HAIRAN syndrome: A fifth variant of SAHA syndrome. Horm Res 2000;53:94. 60 Dunaif A, Green G, Phelps RG, Lebwohl M, Futterweit W, Lewy L: Acanthosis nigricans, insulin action, and hyperandrogenism: Clinical, histological, and biochemical findings. J Clin Endocr Metab 1991;73:590–595. 61 Plymate SR, Jones RE, Matej LA, Friedl KE: Regulation of sex hormone binding globulin (SHBG) production in Hep G2 cells by insulin. Steroids 1988;52:339–340. 62 Tavakkol A, Varani J, Elder JT, Zouboulis CC: Maintenance of human skin in organ culture: Role for insulin-like growth factor-1 receptor and epidermal growth factor receptor. Arch Dermatol Res 1999;291:643–651. 63 Fiedler VC, Camara CR: Topical hair growth promoters in androgenetic alopecia. Dermatol Ther 1998;8:34–41. 64 Orfanos CE, Vogels L: Lokaltherapie der Alopecia androgenetica mit 17·-Östradiol. Eine kontrollierte, randomisierte Doppelblindstudie. Dermatologica 1980;161:124–132. 65 Orfanos CE, Zouboulis CC: Oral retinoids in the treatment of seborrhoea and acne. Dermatology 1998;196:140–147. 66 Moghetti P, Tosi F, Tosti A, Negri C, Misciali C, Perrone F, Caputo M, Muggeo M, Castello R: Comparison of spironolactone, flutamide and finasteride efficacy in the treatment of hirsutism: A randomized double blind placebocontrolled trial. J Clin Endocr Metab 2000;85: 89–94. 67 Azziz R, Ochoa TM, Brandley EL, Potter HD, Boots LR: Leuprolide and estrogen versus oral contraceptive pills for the treatment of hirsutism: A prospective randomized study. J Clin Endocr Metab 1995;80:3406–3411. 68 Heiner JS, Greendale GA, Kawakami AK, Fisher LM, Young D, Judd HL: Comparison of a gonadotropin-releasing hormone agonist and low dose oral contraceptive given alone or together in the treatment of hirsutism. J Clin Endocr Metab 1995;80:3412–3418. 69 Azziz R, Ehrmann D, Legro RS, Whitcomb RW, Hanley R, Fereshetian AG, O’Keefe M, Ghazzi MN: Troglitazone improves ovulation and hirsutism in the polycystic ovary syndrome: A multicenter, double blind, placebocontrolled trial. J Clin Endocr Metab 2001;86: 1626–1632.

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Horm Res 2000;54:259–262

Oestrogen Receptor Beta Is Not Present in the Pilosebaceous Unit of Red Deer Skin during the Non-Breeding Season M. Julie Thornton a Anthony H. Taylor b Kellie Mulligan b Farook Al-Azzawi b a Department

of Biomedical Sciences, University of Bradford, Bradford, UK and b Department of Obstetrics and Gynaecology, Leicester Royal Infirmary, Leicester, UK

Key Words Oestrogen receptor beta W Androgen receptor W Skin W Hair follicle W Sebaceous gland

Abstract Androgens and oestrogens both have roles in skin physiology. Recently a second oestrogen receptor (ERß) has been identified in androgen-dependent tissues. The red deer grows a breeding season, androgen-dependent mane when plasma testosterone rises; this is replaced with small neck hairs during the non-breeding season. In non-breeding season deer skin, ERß was localised to the blood vessels and arrector pili muscle, but in contrast to human skin, not in the pilosebaceous unit or epidermis. The androgen receptor was not expressed. Further studies with breeding season skin may help to elucidate whether serum androgens or androgen receptor expression can modulate the expression of ERß in skin. Copyright © 2001 S. Karger AG, Basel

Function of the Hair Follicle

Many hair follicles produce different types of hair in response to environmental changes or to the age or sex of the mammal. The hairs formed may significantly change in thickness, length and colour. This allows many mam-

ABC

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Accessible online at: www.karger.com/journals/hre

mals to adjust to seasonal changes in the climate by altering the thermal insulating properties of their coat. It also allows the alteration of coat colour to retain camouflage when the seasons change, as is seen in some arctic mammals [1]. This transforming ability of the hair follicle also allows ageing hair changes, which allow obvious distinctions between young animals and adults, and between the adult sexes [2].

Oestrogen Receptor Beta

Since the isolation and cloning of the oestrogen receptor from uterus in 1986, the consensus was that only one oestrogen receptor existed. In 1996, a new member of this nuclear receptor superfamily was cloned from rat [3] and human [4] tissue that specifically binds oestrogens. This new receptor is called oestrogen receptor beta (ERß) to distinguish it from the classical oestrogen receptor, now termed oestrogen receptor alpha (ER·). Both receptors bind 17ß-oestradiol with high affinity [5] and bind to classical oestrogen response elements in a similar, if not identical fashion [6]. ERß mRNA and protein have been detected in a wide range of tissues including the vasculature, bone, brain, heart, the gonads and genital tracts of both males and females [7]. However, there are major differences between ERß and ER· with respect to their tissue distribution [8, 9], the phenotype of the corresponding

Dr M.J. Thornton Department of Biomedical Sciences University of Bradford Bradford, West Yorkshire, BD7 1DP UK Tel. +44 1274 235517, Fax +44 1274 309742, E-Mail [email protected]

knock-out mice and their transcriptional activities [6]. The recent identification of a second oestrogen receptor has caused considerable interest amongst endocrinologists and is leading to a re-evaluation of oestrogen signalling pathways and oestrogen-dependent physiology.

Oestrogen and Skin

That oestrogen has an important role in the maintenance of the skin can be seen in postmenopausal women, whose skin undergoes profound changes, including a decrease in dermal collagen and reduced skin thickness [10]. Recently, it has been shown that the rate and quality of wound healing is oestrogen-dependent [11] and the delay in wound healing in elderly patients of both sexes can be significantly reduced by topical oestrogen [12]. Although the role of oestrogens in skin has not been studied as extensively as the role of androgens, it is apparent that oestrogens play a significant role in the regulation of both the hair follicle and the sebaceous gland. For some time it has been known that oestrogens slow the moult cycle in rat skin [13]. Recent studies indicate that topical treatment of mouse skin with oestradiol maintains the hair follicle in the resting phase, blocking its transition into the growing phase, resulting in the inhibition of hair growth in both sexes [14]. In addition, oestrogens are powerful suppressors of androgen-stimulated sebaceous gland activity in doses markedly lower than those required for anti-androgens [16]. However, the mechanism of action of oestradiol in the sebaceous gland is distinct from that of an antiandrogen. It appears that oestradiol does not decrease mitosis, but may act by inhibiting intracellular lipid synthesis both in vivo [17] and in vitro [18,19]. Immunohistochemical studies have localised the ER· isoform to the dermal papilla of telogen follicles in the mouse [15] while molecular biological studies have identified the presence only of ER· and not ERß transcripts [14]. However, we have recently, described the localisation of ERß in human male skin by immunohistochemistry [20], suggesting that oestrogen action in the mouse and human skin are likely to differ.

Complementary Actions of Androgens and Oestrogens

Immunohistochemical staining for the androgen receptor has also shown it to be present in both the sebaceous gland and the hair follicle dermal papilla of many

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species. The presence of androgen and oestrogen receptors in human skin suggests that androgens and oestrogens may act separately, combined or synergistically, or even in an opposing manner to regulate human follicular physiology. Indeed, both hormones control the type of hair produced by mammalian follicles [1, 2], but the precise mechanisms are unclear. Androgens are the most obvious regulators of human hair growth in some body sites, with increasing evidence that androgens exert their effects via the mesenchyme-derived dermal papilla at the base of the hair follicle [21, 22]. The presence of ERß in prostate [3, 7], in the sebaceous gland and hair follicle dermal papilla of human skin [20], is of great interest because these are traditionally considered classical androgen target-tissues. However, it is now becoming apparent that tissues thought to be responsive to one class of steroids also contain receptors for other classes. For example, human breast and prostate contain receptors for oestrogens, androgens and progesterone, and there is growing evidence that steroid receptors can cross talk with one another [23]. Clinically, human prostate cancer often responds favourably to oestrogen treatment, presumably through ERß and not the androgen receptor. Recently, studies in male and female hamsters have shown that oestrogen receptor expression in white adipose tissue is modulated by androgen status; the levels of testosterone in the male up-regulating oestrogen receptor levels [24]. Furthermore, the expression of aromatase in genital skin is androgen-dependent, with much lower levels of aromatase expression in cells from patients with androgen insensitivity syndromes [25]. Additionally, aromatase activity in cultured genital skin fibroblasts can be blocked by an anti-androgen [26], demonstrating that the response is mediated via the androgen receptor. However, aromatase converts androgen into oestrogen, which in turn could activate oestrogen receptors in a paracrine manner. Obviously, these data suggest that the simple model of androgen activation of hair cycle growth may be more complex than previously thought.

The Red Deer

The red deer (Cervus elaphus) is an accessible animal model for hair growth studies since it has a highly synchronised seasonal coat growth cycle, with two distinct and separate pelage types each year. Furthermore, the adult male also grows an androgen-dependent mane, only during the winter breeding season, when circulating testosterone levels are high. Short neck hairs subsequently replace the mane during the summer non-breeding sea-

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son, when circulating testosterone levels are low [27]. This biannual change in the type of hair produced in the neck region, corresponding with dramatic fluctuations of plasma testosterone, provides an excellent model in which to investigate the hormonal regulation of hair growth. To establish whether in vivo androgens can modulate ERß expression, we decided to investigate the distribution of ERß by immunohistochemistry in red deer stag skin at different times of the year, when plasma testosterone levels change. This study describes the expression of ERß during the non-breeding season when plasma testosterone levels are at their lowest.

Table 1. Immunohistological distribution of

ERß and AR in non-breeding red deer skin Tissue component

ERß

AR

Blood vessel Arrector pili muscle Nerve capsule sheath Epidermis Hair follicle Sebaceous gland

+ + + – – –

– – – – – –

(+) Immunoreactive staining observed; (–) no immunoreactive staining.

Experimental Design

Skin was taken from the neck and flank of six adult red deer stags during the non-breeding season when in vivo testosterone is low. Small pieces of skin were fixed in formol saline for 24 h before processing into paraffin wax. Sections (4 Ìm) were incubated with a polyclonal rabbit anti-rat ERß antibody (1:25; Upstate Biotechnology, USA), as described previously [8] or a monoclonal antibody for the androgen receptor (1:40; Novocastra, UK) according to the manufacturer’s instructions. Briefly, sections were dewaxed, rehydrated through graded alcohol to water. Next, endogenous peroxide, biotin and non-specific sites were blocked with standard procedures before incubation with primary antibody at 4 ° C for 18 h. After secondary anti-rabbit IgG (for ERß) and anti-mouse (for AR) IgG incubation, antibody-antigen complex was amplified using HRP-complexed avidin-biotin (Vector Laboratories, Peterborough, UK). Visualisation of antibody complexes was achieved with Cu2+ enhanced diaminobenzidine and light haematoxylin counterstaining, before mounting in XAM mounting medium (BDH, Poole, Dorset, UK). Additionally, to control for non-specific effects, sections were produced in the absence of the primary antibody, or with substitution of molar equivalents of rabbit IgG (for ERß) or mouse IgG (for AR). Human prostate and testis acted as positive controls for ERß and AR, respectively.

The distribution of ERß in the non-breeding red deer skin contrasts markedly with that of human skin, as we described previously [20]. The only skin components, which stained in both species, were the endothelial cells of

the blood vessels and the arrector pili muscle cells. The intensity of staining in the arrector pili muscle was much less intense in human skin compared to red deer skin. In contrast to red deer skin, ERß in human male scalp skin was localised to the epidermal keratinocytes, to the outer root sheath, epithelial matrix and dermal papilla cells of the hair follicle, and to the partially differentiated sebocytes of the sebaceous gland [20]. This observation is noteworthy since it may mean that ERß has a unique role in human skin that is not shared by other mammals. This notion is supported further by the identification of ER· but not ERß in mouse hair follicles [14]. Additionally, the expression of ERß protein differs markedly in other human tissues when compared to the mouse or rat [8, 9]. Alternatively, the distribution of ERß in red deer skin may be linked to either plasma androgen levels or the expression of the androgen receptor in the same tissue. Binding studies in male and female hamsters have shown that oestrogen receptor levels are modulated by androgen [24]. The red deer skin used in this study was taken from the neck of a stag during the non-breeding season when serum androgen levels are low [27] and when the androgen receptor is not expressed in the hair follicle cells [22]. If the expression of oestrogen receptors is modulated by in vivo androgens, then the expression of ERß in red deer skin, at this time of the year, provides a baseline measurement of ERß expression prior to the autumn testosterone surge. If ERß expression is linked to in vivo levels of androgens, then following the large surge in circulating testosterone in the male that accompanies the breeding season, we would expect to see an alteration in the expression of ERß. During the breeding season, the significant increase in serum testosterone is accompanied by the replacement of

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Discussion and Results (table 1)

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the neck hairs by long, thick androgen-dependent hairs, producing a mane for sexual attraction [27]. This suggests that the primary signal for hair growth in the stag mane is androgen. This hypothesis is supported by studies assessing androgen receptor expression in cultured dermal papilla cells derived from mane follicles [22]. Those studies show that specific androgen receptors are expressed in mane follicles but not present in the corresponding flank hairs taken simultaneously or from the short neck hairs taken during the non-breeding season when serum testosterone is low [22]. Indeed the expression of ERß may not

only be modulated by serum testosterone, but also the expression of the androgen receptor in either the same cells, or neighbouring cells. This idea needs further clarification by comparing skin from the non-breeding season when in vivo androgens are low, and the breeding season when in vivo androgens are high. Furthermore, since the androgen receptor is only expressed in mane and not flank dermal papilla cells during the breeding season, comparisons of hair follicles between these two sites may help us to understand further the role of androgen and oestrogen receptors in the same tissues.

References 1 Ebling FG, Hale PA, Randall VA: Hormones and hair growth; in Goldsmith LA (ed): Biochemistry and Physiology of the Skin, ed 2. New York, Oxford University Press, 1991, pp 660–696. 2 Randall VA: Androgens and human hair growth. Clin Endocrinol 1994;40:439–457. 3 Kuiper GG, Enmark E, Pelto-Huikko M, Nilsson S, Gustafsson JA: Cloning of a novel receptor expressed in rat prostate and ovary. Proc Natl Acad Sci USA 1996;93:5925–5930. 4 Mosselman S, Polman J, Dijkema R: ERß: Identification and characterisation of a novel human estrogen receptor. FEBS Lett 1996;392: 49–53. 5 Kuiper GG, Carlsson B, Grandien K, Enmark E, Haggblad J, Nilsson S, Gustafsson JA: Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors · and ß. Endocrinol 1997;138:863–870. 6 Dechering K, Boersma C, Mosselman S: Estrogen receptors alpha and beta: Two of a kind? Curr Med Chem 2000;7:561–76. 7 Saunders PT: Oestrogen receptor beta (ER beta). Rev Reprod 1998;3:164–171. 8 Taylor AH, Al-Azzawi F: Immunolocalisation of oestrogen receptor beta in human tissues. J Mol Endocrinol 2000;24:145–155. 9 Saunders PTK, Maguire SM, Gaughan J, Millar MR: Expression of oestrogen receptor beta (ERbeta) in multiple rat tissues visualised by immunohistochemistry. J Endocrinol 1998; 154:R13–R16. 10 Bolognia JL: Aging Skin. Am J Med 1995;98: 99s–103s. 11 Ashcroft GE, Dodsworth J, van Boxtel E, Tarnuzzer RW, Horan MA, Schultz GS, Ferguson MW: Estrogen accelerates cutaneous wound healing associated with an increase in TGFbeta1 levels. Nat Med 1997;3:1209–1215.

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12 Ashcroft GE, Greenwell-Wild T, Horan MA, Wahl SM, Ferguson MW: Topical estrogen accelerates cutaneous wound healing in aged humans associated with an altered inflammatory response, Am J Path 1998;155:1137–1146. 13 Ebling FJ: Hair. J Invest Dermatol 1976;67: 98–105. 14 Chanda S, Roninette CL, Couse JF, Smart RC: 17beta-estradiol and ICI-182780 regulate the hair follicle cycle in mice through an estrogen receptor-alpha pathway, Am J Physiol Endocrinol Metab 2000;278:E202–210. 15 Oh HS, Smart RC: An estrogen receptor pathway regulates the telogen-anagen hair follicel transition and influences epidermal cell proliferation. Proc Natl Acad Sci USA 1996;93: 12525–12530. 16 Ebling FJ: The effects of cyproterone acetate and oestradiol upon testosterone stimulates sebaceous gland activity in the rat. Acta Endocrinol 1973;72:361–365. 17 Ebling FJ: Hormonal control and methods of measuring sebaceous gland activity. J Invest Dermatol 1974;62:161–171. 18 Guy R, Ridden C, Kealey T: The improved organ maintenance of the human sebaceous gland: modeling in vitro the effects of epidermal growth factor, androgens, estrogens, 13-cis retinoic acid, and phenol red. J Invest Dermatol 1996;106:454–460. 19 Guy R, Kealey T: The organ-maintained human sebaceous gland. Dermatology 1998;196: 16–20. 20 Thornton MJ, Taylor AH, Mulligan K, AlAzzawi F: Estrogen receptor beta is present in the hair follicle and sebaceous gland in male human skin. J Invest Dermatol 2000;114:abstract 351.

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21 Randall VA, Thornton MJ, Messenger AG: Cultured dermal papilla cells from androgendependent human hair follicles (e.g. beard) contain more androgen receptors than those from non-balding areas of scalp. J Endocrinol 1992;133:141–147. 22 Thornton MJ, Hibberts NA, Street T, Brinklow BR, Loudon ASI, Randall VA: Androgen receptors are only present in mesenchyme-derived dermal papillla cells of red deer (Cervus elaphus) neck follicles when raised androgens induce a mane in the breeding season. J Endocrinol 2001;168:401–408. 23 Migliaccio A, Piccolo D, Castoria G, Di Domenico M, Bilancio A, Lombardi M, Gong W, Beato M, Auricchio F: Activation of the Src/ p21ras/Erk pathway by progesterone receptor via cross-talk with estrogen receptor. EMBO J 1998;17:2008–2018. 24 Jaubert AM, Pecquery R, Dieudonne MN, Giudicelli Y: Estrogen binding sites in hamster white adipose tissue: Sex- and site-related variations: Modulation by testosterone. Gen Comp Endocrinol 1995;100:179–187. 25 Stillman SC, Evans BA, Hughes IA: Aromatase activity in genital skin fibroblasts from normal and androgen-insensitive individuals. J Endocrinol 1990;127:177–183. 26 Stillman SC, Evans BA, Hughes IA: Androgen dependent stimulation of aromatase activity in genital skin fibroblasts from normals and patients with androgen insensitivity. Clin Endocrinol 1991;35:533–538. 27 Linclon G: Puberty in a seasonally breeding male, the red deer stag (Cervus elaphus L.). J Reprod Fertil 1971;25:41–54.

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Nuclear Hormone Receptors and Mouse Skin Homeostasis: Implication of PPARß Liliane Michalik a Béatrice Desvergne a Sharmila Basu-Modak a, b Nguan Soon Tan a Walter Wahli a a Institut

de Biologie Animale, Université de Lausanne, Bâtiment de Biologie, Lausanne, Switzerland; of Pharmacy and Pharmacology, University of Bath, UK

b Department

Key Words PPAR gene expression W Mouse epidermis W Keratinocyte proliferation W Skin wound healing

Abstract PPARß is expressed in the mouse epidermis during fetal development, and progressively disappears from the interfollicular epidermis after birth. Interestingly, its expression is strongly reactivated in the adult epidermis in conditions where keratinocyte proliferation is induced and during wound healing. Data obtained on PPARß heterozygous mice reveal that PPARß is implicated in the control of keratinocyte proliferation and is necessary for rapid healing of a skin wound. Copyright © 2001 S. Karger AG, Basel

Introduction

The epidermis serves as a protective barrier against dehydration and microbial, mechanical, chemical agression. It is composed of four histologically and molecular well defined layers, each corresponding to a distinct maturation stage of the keratinocytes. Keratinocytes arise from the proliferating cells in the basal layer, and move through a series of differentiation events as they migrate from the

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basal to the outermost layer, until they are finally sloughed into the environment. The protective function of the skin mainly resides in the outermost layer, the stratum corneum, which is composed of dead cornified keratinocytes embedded in a complex lipid matrix [1, 2]. Many nuclear hormone receptors have been implicated in skin maturation and development. More recently, PPAR· ligands were shown to accelerate epidermal development of fetal rat skin [3], and the expression of PPARß was shown to be associated with keratinocyte proliferation [4] and/or differentiation [5]. These results show that PPARs belong to the group of nuclear hormone receptors participating in the epidermal homeostasis. Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors of the superfamily of nuclear hormone receptors. They bind to fatty acids and their derivatives as well as hypolipidemic and antidiabetic agents and play important roles in energy homeostasis. Three PPAR isotypes have been identified (PPAR·, ß/‰ or FAAR or NUC1, and Á) (NR1C1, NR1C2, NR1C3, respectively) [6], in various species (Xenopus laevis, rodents, human), each of them showing a specific pattern of expression [7]. They are expressed in both rat skin [8, 9] and human [10] keratinocytes. We show here that PPARß is involved in the control of keratinocyte proliferation and is crucial for normal healing of a skin wound.

Walter Wahli Institut de Biologie Animale, Université de Lausanne Bâtiment de Biologie CH–1015 Lausanne (Switzerland) Tel. +41 21 692 41 10, Fax +41 21 692 41 15, E-Mail [email protected]

Fig. 1. PPARß expression in SV129 adult

mouse epidermis after topical application of TPA. Vehicle (a–c) or TPA treated (d–f) dorsal skin. Hematoxylin/eosin (HE) staining (a, d); Ki67 immunolabeling (b, e); in situ hybridization with PPARß antisense probe (c, f). Bars: 20 Ìm. Similar results were observed in 6 SV129 mice, from independent litters.

Results

The experimental procedures are described in reference 11. PPARß Is expressed during Epidermal Development and upon Keratinocyte Proliferation PPARß expression in mouse skin was studied by in situ hybridization with a PPARß digoxigenin-labeled specific probe, on embryonic, newborn, postnatal stages and adult skin sections [11]. PPARß was expressed in the differentiating epidermis during fetal development, from embryonic day 13.5 on to the end of gestation. After birth, PPARß expression decreased after a few days of postnatal development, and PPARß was found to be absent from the interfollicular adult epidermis. This pattern of expression suggests that the presence of PPARß in the keratinocytes is more related to proliferation and/or differentiation in the developing skin, rather than to the adult epidermis renewal. If the PPARß presence in the epidermis is linked to keratinocyte proliferation, its expression might be reactivated in the adult epidermis unpon keratinocyte proliferation stimuli. In order to address this hypothesis, we studied PPARß expression in the adult epidermis after stimu-

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lation of keratinocyte proliferation, either by topical application of TPA or hair plucking. Histological staining showed that both the TPA application (fig. 1) [11] and the hair plucking [11] induced the expected keratinocyte stratification compared to the corresponding control samples (ethanol treated and unplucked skin). As a keratinocyte proliferation marker, we used the expression of Ki67 nuclear antigen. Consistent with the epidermal thickening observed after histological staining, the number of Ki67 positive cells in the basal layer of the epidermis was strongly increased in both the TPA treated (fig. 1) [11] and the plucked skin [11], confirming that both treatment efficiently induced keratinocyte proliferation. In situ hybridization with PPARß specific antisense probe revealed that PPARß expression was significantly upregulated upon both proliferation stimuli, whereas PPARß labeling remained negative in the control samples (fig. 1) [11]. The marked increase of PPARß expression in the keratinocytes under conditions inducing keratinocyte proliferation strongly suggests that PPARß might be implicated in this process. Generation of PPARß Mutant Mice In order to check PPARß implication in the keratinocyte proliferation control in vivo, we generated PPARß

Michalik/Desvergne/Basu-Modak/Tan/ Wahli

Fig. 2. Induced proliferation of keratinocytes on PPARß+/– and wild type mice after TPA topical application. PPARß+/+ vehicle (a, b) or TPA treated (e, f) dorsal epidermis, hematoxylin/eosin staining (HE) (a, e), and after Ki67 immunostaining (b, f). PPARß+/– vehicle (c, d) or TPA treated (g, h) dorsal epidermis, hematoxylin/eosin staining (HE) (c, g), and after Ki67 immunostaining (d, h).

mutant mice. Due to the high penetrance of a lethal phenotype, also recently reported by others [4], a PPARß null mouse line could not be obtained so far. We therefore used PPARß+/– mice in our experiments. The PPARß+/– mice expressed approximately 50% less PPARß compared to the wild type control mice, both at the RNA and protein levels. Both PPAR· and PPARÁ expression remained identical in the PPARß+/– and the wild type mice, indicating that the decrease in PPARß expression was not compensated by any of the two other PPAR isotypes.

Keratinocyte Proliferative Response in PPARß Mutant Mice If PPARß expression is linked to keratinocyte proliferation, as strongly suggested by the above mentioned results, the latter might be affected in the PPARß+/– mice after keratinocyte proliferation stimuli. We therefore performed TPA and hair plucking keratinocyte proliferation stimulation on dorsal skin of PPARß+/– and wild type mice. As shown in figure 2, TPA treatment induced a more pronounced hyperplasia on the PPARß heterozy-

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265

Fig. 3. Expression of PPARß in adult mouse

epidermis during cutaneous wound closure. Cryosections of mouse skin from day 1 to day 10 (+ 1 to + 10) after the excision of a full thickness dorsal skin biopsy were hematoxylin/eosin stained (HE) (a–d) or hybridized with specific antisense digoxigenin labeled PPARß riboprobe (e–h). Bar: 20 Ìm. A similar pattern of expression was observed for each time point in 6 different mice, from independent litters.

gous mice compared to their wild type couterparts. Consistent with this histological observation, the increase of the number of Ki67 positive cells was higher as well in the PPARß mutant mice than in the wild type animals (fig. 2). Similar results were obtained after hair plucking [11]. Quantitative analysis of the epidermal thickening and of the number of Ki67 positive cells showed that the proliferative response of the keratinocytes was significantly higher in the PPARß+/– mice than in the wild type animals in both proliferation assays [11]. These data provide further evidence for the implication of PPARß in the control of keratinocyte proliferation.

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PPARß Heterozygous Mice Exhibit Altered Skin Wound Healing Because of PPARß expression pattern in the epidermis on the one hand, and its likely involvement in the control of keratinocyte proliferation on the other hand, we decided to study the consequences of an impaired PPARß expression on the efficiency of skin wound healing. Wound healing is an extreme situation in which the adult skin has to regenerate a neo-epidermis. The regeneration of the neo-epidermis includes both proliferation of the keratinocytes and differentiation of a new epithelium upon closure of the wound [12]. Skin wound healing might therefore be impaired in the PPARß+/– mice. PPARß expression in the adult epidermis during wound healing was first examined by in situ hybridization

Michalik/Desvergne/Basu-Modak/Tan/ Wahli

Fig. 4. Wound healing kinetics of PPARß mutant mice compared to their respective wild type counterparts. After excision of a full thickness skin biopsy, the surfaces of the healing wounds were measured over time on wild type (+/+) and heterozygous (+/–) PPARß mice. The surfaces are plotted as a percentage of the surface of the wound at day zero (B SEM, n = 5). Asterisks indicate that the difference is statistically significant (* p ! 0.05). Arrows indicate the mean time for complete healing of the wild type control mice (black lozenge) or mutant mice (grey square).

on wild type mice. In situ hybridization showed that PPARß expression is strongly upregulated in the epidermis of the wound edges as early as 24 h after the injury, and remains expressed in the neo-epidermis during the whole healing process (fig. 3) [11], suggesting that indeed, PPARß is involved in some of the events leading to the closure of a skin wound. We then measured the healing efficiency of a skin wound in PPARß+/– mice, compared to wild type control animals. A dorsal full thickness skin biopsy was excised on adult animals, the surfaces of the wounds were measured until complete healing, and the efficiency of the healing was addressed by comparing the wound healing kinetics in PPARß+/– and wild type mice. Compared to their wild type littermates, PPARß+/– mice showed a significant delay in wound healing during the whole process, and the final closure of the wound was postponed by 2–3 days (fig. 4) [11].

Discussion

We show here that PPARß expression, which is undetectable in the unchallenged mouse adult interfollicular epidermis, is significantly upregulated upon various conditions that result in keratinocyte proliferation, and during skin wound healing. Additionally, using mice with a mutated PPARß gene, we show that a reduced PPARß

Nuclear Hormone Receptors and Mouse Skin Homeostasis

expression leads to an impaired control of cell proliferation in the epidermis, characterized by a hyperproliferative reaction in response to TPA or hair plucking stimuli. Moreover, these mice cannot sustain normal cutaneous wound healing, indicating that PPARß is crucial for the rapid closure of a skin wound. Our data provide strong evidence for the involvement of PPARß in the control of keratinocyte proliferation. A role of PPARß in the control of epithelial cell proliferation has also been recently reported in colon cancer [13]. Consistent with our observation, a role of PPARß in the control of keratinocyte proliferation was also reported in PPARß null mice [4], whereas PPARß has been associated to keratinocyte differentiation in human keratinocytes [5]. During skin wound healing, where keratinocyte proliferation is one of the major events, an impaired control of epithelial cell proliferation due to an insufficient PPARß upregulation is likely to result in an impaired closure of the wound. However, the re-epithelialization of a wound also includes the migration of the keratinocytes from the edges of the wound, and following proliferation, their stratification and differentiation to form the neoepidermis. Whilst we cannot exclude a PPARß implication in some other mechanisms involved in wound healing, the data reported here provide evidences for the necessity of an increased PPARß expression to control a well balanced proliferation/differentiation process required for normal cutaneous healing.

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Acknowledgements We thank Nathalie Deriaz for assistance. We are grateful to Jozsef Zakany and Denis Duboule for their contribution to obtain the PPARß mutant mice.

This work was supported by the Swiss National Science Foundation (grant to Walter Wahli and to Béatrice Desvergne), by the Etat de Vaud, by the Human Frontier Science Program Organization and by Parke-Davis Pharmaceutical Research.

References 1 Downing DT: Lipid and protein structures in the permeability barrier of mammalian epidermis. J Lipid Res 1992;33:301–313. 2 Roop D: Defects in the barrier [comment]. Science 1995;267:474–475. 3 Hanley K, Jiang Y, Crumrine D, Bass NM, Appel R, Elias PM, Williams ML, Feingold KR: Activators of the nuclear hormone receptors PPARalpha and FXR accelerate the development of the fetal epidermal permeability barrier. J Clin Invest 1997;100:705–712. 4 Peters JM, Lee SS, Li W, Ward JM, Gavrilova O, Everett C, Reitman ML, Hudson LD, Gonzalez FJ: Growth, adipose, brain, and skin alterations resulting from targeted disruption of the mouse peroxisome proliferator-activated receptor beta(delta). Mol Cell Biol 2000;20: 5119–5128.

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5 Matsuura H, Adachi H, Smart RC, Xu X, Arata J, Jetten AM: Correlation between expression of peroxisome proliferator-activated receptor beta and squamous differentiation in epidermal and tracheobronchial epithelial cells. Mol Cell Endocrinol 1999;147:85–92. 6 Nuclear Receptors Nomenclature Committee: A unified nomenclature system for the nuclear receptor superfamily. Cell 1999;97:161–163. 7 Desvergne B, Wahli W: Peroxisome proliferator-activated receptors: Nuclear control of metabolism. Endocr Rev 1999;20:649–688. 8 Braissant O, Foufelle F, Scotto C, Dauca M, Wahli W: Differential expression of peroxisome proliferator-activated receptors (PPARs): Tissue distribution of PPAR-alpha, -beta, and -gamma in the adult rat. Endocrinology 1996; 137:354–366. 9 Braissant O, Wahli W: Differential expression of peroxisome proliferator-activated receptoralpha, -beta, and -gamma during rat embryonic development. Endocrinology 1998;139:2748– 2754.

10 Rivier M, Safonova I, Lebrun P, Griffiths CE, Ailhaud G, Michel S: Differential expression of peroxisome proliferator-activated receptor subtypes during the differentiation of human keratinocytes. J Invest Dermatol 1998;111: 1116–1121. 11 Michalik L, Desvergne B, Tan NS, Basu-Modak S, Escher P, Rieusset J, Peters JM, Kaya G, Gonzalez FJ, Zakany J, Metzger D, Chambon P, Duboule D, Wahli W: Impaired skin wound healing in PPAR· and PPARß mutant mice. J Cell Biol 2001;154:799–814. 12 Woodley DT: Reepithelialization; in Clark RAF (ed): The Molecular and Cellular Biology of Wound Repair. New York, Plenum Press, 1996, pp 339–354. 13 He TC, Chan TA, Vogelstein B, Kinzler KW: PPARdelta is an APC-regulated target of nonsteroidal anti-inflammatory drugs. Cell 1999; 99:335–345.

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Peroxisome Proliferator-Activated Receptors and Skin Development Robert L. Rosenfield a, b Dianne Deplewski a Marianne E. Greene c a Departments of Pediatrics, b Medicine and The Committee on Developmental Biology, The University of Chicago Pritzker School of Medicine and c Department of Pathology, Northwestern University Medical School, Chicago, Ill., USA

Key Words Epidermal water barrier W Sebaceous glands

Abstract PPARs are nuclear hormone receptors. PPAR subtypes (·, Á, ‰, the latter a xPPARß homologue) were initially investigated in skin because of their known role in regulating lipid metabolism. Studies adding specific PPAR ligand activators to cultured skin or skin cells are compatible with the concepts that PPAR· activation mediates early lipogenic steps common to the function of both skin epidermal cells (keratinocytes) and sebaceous cells (sebocytes), PPARÁ activation plays a unique role in stimulating sebocyte lipogenesis, and PPAR‰ activation may contribute to lipid biosynthesis in both sebocytes and keratinocytes under certain circumstances. Epidermal keratinocytes appear to express small amounts of PPAR· and PPAR‰ mRNA and a trace of PPARÁ mRNA which is up-regulated with differentiation. Sebocytes express all subtypes; PPARÁ gene expression excedes that in epidermis. The emerging data on PPAR protein expression suggests that epidermis normally expresses predominantly PPAR·, while sebocytes express more PPARÁ than PPAR·. These expression patterns may change during hyperplasia, differentiation and inflammation. Gene disruption studies in mice are compatible with a contribution of PPAR· to skin barrier function, suggest that

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PPARÁ is necessary for sebocyte differentiation, and indicate that PPAR‰ can ameliorate inflammatory responses in skin. PPARs appear to play a role in keratinocyte synthesis of the lipids that they export to the intercellular space to form the skin permeability barrier. They also appear to be important for sebocyte formation of the intracellular fused lipid droplets that constitute the holocrine secretion of the sebaceous gland. In addition, they may play roles in keratinocyte growth and differentiation and the inhibition of skin inflammation by diverse mechanisms not necessarily related to fat metabolism. Copyright © 2001 S. Karger AG, Basel

Introduction

Peroxisome proliferator-related receptors (PPARs) are a subfamily of former ‘orphan receptors’ within the nonsteroid receptor family of nuclear hormone receptors [1]. There are three subtypes (·, Á, ‰); rat and human PPAR‰ are homologous to xPPARß [2–4]. The clofibrate analogue WY-14643 (WY) is a specific PPAR· ligand-activator at low dosage [5, 6]. A natural prostaglandin J2 metabolite [6, 7] and insulin-sensitizing thiazolidinediones such as BRL-49653 (BRL, rosiglitazone) and troglitazone [8] are specific PPARÁ ligand-activators at low doses [5, 7, 9, 10]. The prostacyclin analogue carbaprostacyclin and the C18:2 essential fatty acid linoleic acid are among the most

Robert L. Rosenfield University of Chicago Children’s Hospital 5841 S. Maryland Ave. (MC 5053) Chicago, IL 60637-1470 (USA) Tel. +1 773 702 6432, Fax +1 773 702 0443, E-Mail [email protected]

potent known ligand-activators of PPAR‰; both activate PPAR‰ and PPAR· to a comparable extent and severalfold more than PPARÁ [2, 5, 9]. Activated PPARs act through PPAR response elements to up-regulate multiple genes encoding enzymes involved in peroxisomal, microsomal, and mitochondrial ß-oxidation, fatty acid ˆ-hydroxylation, ketone body formation, and fatty acid synthesis, as well as fatty acid binding proteins, lipoprotein lipase, and apolipoproteins [11– 13]. Expression and activation of a PPARÁ isoform, specifically Á2, is necessary and sufficient to induce preadipocyte cell lines to form fat and thus undergo terminal differentiation to become adipocytes [14].

Effects of PPAR Activators on Cultured Epidermal and Sebaceous Cell Lipid Formation

PPARs were initially investigated in skin because of their potential role in regulating lipid metabolism. Extracellular lipids in the outermost layer of the epidermis, the stratum corneum, provide the hydrophobic seal between the internal milieu and the outside environment which creates the epidermal water barrier. Ceramides and cholesterol derivatives are important structural components of this water barrier. Epidermal epithelial cells (keratinocytes) synthesize and secrete these lipids into the interstitial space as lamellar bodies, where they are reorganized into the continuous matrix of lamellar unit structures which prevents transdermal water loss. The PPAR· activator clofibric acid and the PPAR·,‰ activator linoleic acid have been found to accelerate the development of the epidermal water barrier in rat fetal skin explants [15]. Electron microscopy demonstrated that this advancement of barrier function was accompanied by early development of mature lipid lamellar units. Studies of a human skin equivalent system yielded similar results [16]: PPAR· activation stimulated increased expression of several genes for the synthesis of ceramides and cholesterol derivatives and increased formation of lamellar bodies. PPARÁ activators did not stimulate barrier formation in skin [15]. Fused lipid droplet formation was not discernable in cultured keratinocytes in response to PPAR· and/ or PPARÁ activator treatments, but was in response to linoleic acid or carbaprostacyclin treatment, suggesting that PPAR‰ activation can stimulate epidermal lipid synthesis [17]. Sebaceous cells (sebocytes) are specialized epithelial cells which terminally differentiate by accumulating neutral fat droplets until they burst, giving rise to the lipid-

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rich holocrine secretion, sebum. PPAR· and PPARÁ activators were found to stimulate lipid droplet accumulation in cultured immature sebocytes, but not in keratinocytes, as assessed histochemically using Oil Red O staining in cells cultured under high calcium conditions [17, 18]. PPARÁ activation induced a greater degree of sebocyte colony differentiation than PPAR· activation and significantly enhanced the differentiative response of sebocytes to androgen. The combination of PPAR· and PPARÁ activators tended to be more effective than either alone in stimulating sebocyte differentiation. The PPAR·,‰ activator carbaprostacyclin stimulated late sebocyte differentiation moreso than the combination of PPAR· and PPAR‰ activators. Linoleic acid, which has a profile of PPAR activation similar to carbaprostacyclin though 100-fold less potent, at the high fatty acid concentrations physiologic for sebum stimulated more lipid accumulation in sebocytes than any other treatment; it was also more effective in sebocytes than keratinocytes. The latter finding suggests that the linoleic effect is not merely due to passive fatty acid uptake by these cells and that PPAR‰ is potentially involved in sebocyte lipid formation. PPAR· and PPARÁ ligand-activators induced a different pattern of lipid droplet accumulation in preputial gland sebocyte colonies than did PPAR·,‰ activators: in response to PPAR· and PPARÁ activation, advanced sebocyte differentiation was seen at the center of colonies, where the older sebocytes reside, whereas in response to PPAR·,‰ activation, advanced sebocyte differentiation was seen throughout colonies, even at the edges of colonies where the younger cells are located. In summary, these studies suggest that PPAR· mediates early lipogenic steps common to both keratinocyte and sebocyte function, and PPARÁ plays a unique role in stimulating sebocyte lipogenesis. PPAR‰ may contribute to lipid biosynthesis in both sebocytes and keratinocytes under certain circumstances, as in response to the high concentrations of fatty acids found in sebum. These results led to new insights into retinoid action in sebaceous cells. PPARs are known to act in conjunction with the retinoid X receptor (RXR). This is a class of retinoid receptor distinct from the retinoic acid receptor (RAR) [19]. The natural ligands of RARs and RXRs have been identified as all-trans retinoic acid and 9-cis retinoic acid, respectively [20–22]. RXR is distinctive in having the capability to form heterodimers with PPARs, as well as other ligand-regulated receptors [1, 23]. Gene reporter systems have shown that a ligand of either receptor alone can activate the RXR-PPAR heterodimer and that the ligands of the two receptors cooperate in activating the

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heterodimer [24–27]. This led to the findings that RXR agonists are stimulatory to sebocyte differentiation and proliferation in culture, effects opposite to those of RAR agonists [28]. RXR activation also leads to sebocyte proliferation, again opposite to that of RAR. Whereas the RXR differentiative effect is enhanced by PPAR ligandactivators, the RXR agonist proliferative effect is not. Thus, the cooperation between PPAR and RXR activation observed in gene reporter systems appears to be limited to the induction of differentiation in cultured sebocytes [29].

PPAR Gene Expression in Normal Skin

The evidence for roles of PPARs in skin and sebaceous development has spurred research into the localization of expression of PPAR genes in order to understand their exact roles in development. PPAR· has been detected from day E15.5 and PPAR‰(ß) from day E13.5 of embryonic life, but not in adult life, in rat epidermis according to in situ hybridization [30, 31]. PPARÁ was not detectable by this method. In both freshly dispersed (relatively mature) and cultured (relatively immature) adult rat epidermal cells, PPAR‰ mRNA was more highly expressed than PPAR· mRNA (Deplewski, unpublished data), and PPARÁ mRNA was not detected by RNase protection assay [17, 18]. Parallel results have been been obtained in cultured human keratinocytes [32]. PPAR‰ mRNA was similarly expressed in non-differentiated and differentiated keratinocytes according to Northern analysis. PPAR· expression was not detectable by this method, but was detectable by reverse transcriptase-polymerase chain reaction (RT-PCR) over time in culture. In addition, PPARÁ (both Á1 and Á2) could be detected by RTPCR after inducing keratinocyte differentiation by shifting to a high calcium medium, a finding which has been confirmed [33]. Remarkably, the specific ligand BRL did not cause reporter gene transactivation of endogenous PPARÁ; the possibility that the culture medium was not optimal for keratinocyte differentiation during this experiment cannot be ruled out from the data. Adult rat preputial sebocytes express mRNAs for PPAR·, PPARÁ1, and PPAR‰ according to RNase protection assay [17, 18] (Deplewski, unpublished data), although PPARÁ expression could not be detected in skin sebaceous glands by in situ hybridization [30]. PPAR‰ expression was similarly abundant in freshly dispersed (relatively mature) and cultured (relatively immature) sebocytes, but PPAR· and PPARÁ1 were expressed to a

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much lesser extent in cultured cells. In human sebaceous glands, a preliminary report has appeared of PPAR‰ being detectable by RT-PCR [34], but no other such studies have appeared. Data on the expression of PPAR proteins are relatively sparse due to the paucity of antisera for immunodetection. Our preliminary immunocytological studies of PPARs in freshly dispersed skin and preputial gland cells of adult rats indicate that PPAR· is expressed predominantly in immature keratinocytes and sebocytes; PPARÁ expression is not detected in keratinocytes, but is abundant in sebocytes throughout development; and PPAR‰ protein has been detected in both types of cells by Western analysis. The latter finding is surprising in view of the abundance of PPAR‰ mRNA. Reports are beginning to appear of the immunodetection of PPARs in human skin. PPARÁ immunofluorescence was found in human keratinocytes cultured under differentiating conditions (high calcium medium) [33]. One preliminary study found immunohistochemical expression of all the PPAR subtypes in facial skin sections [34]. Immunoreactivity for PPARÁ was the most intense, with staining observed in epidermis, hair follicles, and eccrine glands as well as sebaceous glands. The specificity of sebaceous gland PPARÁ immunoreactivity was confirmed by Western blotting. A preliminary study using a monoclonal antibody to PPARÁ1 found it to be expressed in basal and early differentiated sebaceous gland cells and the sebokeratinocytes of the pilosebaceous duct, but not in epidermis [35]. Our preliminary data, using an affinity purified antiserum to PPARÁ [36] in the setting of nonspecific chronic superficial skin inflammation, show PPARÁ immunoreactivity in the nuclei of human basal and maturing keratinocytes, hair follicles, sweat glands, and endothelial cells. In summary, studies in the rat indicate that epidermis expresses predominantly mRNA for PPAR· and PPAR‰. A small amount of PPARÁ expresssion has been detected by RT-PCR and immunohistochemistry in human keratinocytes. Immunocytochemically, PPAR· predominates in epidermis, with only small amounts of PPARÁ protein detectable. Sebocytes appear to express all subtypes. PPARÁ1 mRNA and protein expression are notably greater than in epidermis. PPAR· is prominent at both the mRNA and immunocytochemical level, and PPAR‰ mRNA is abundant. Preliminary immunohistochemical evidence for PPARÁ expression in the human pilosebaceous duct and hair follicle exists.

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Gene Disruption Studies

Targeted disruption of PPAR· has led to no reported skin phenotype, although the responses to clofibrate are blocked and the mice develop hepatomegaly and fatty livers in response to fasting [37, 38]. However, responses to clofibrate are also blocked in a mouse model of decreased skin barrier function which has been created by targeted expression of a mutant RAR· that exhibits a broad spectrum of dominant-negative activity because of its ability to form inactive heterodimers with RXR which sequester it [39]. PPAR‰ null mice were found to have hyperplastic responses of epidermis, as well as inflammatory responses to phorbol ester application that were resistant to nonsteroidal antiinflammatory drug (NSAID) treatment [40]. In neither PPAR· nor PPAR‰ knockout models have epidermal and sebaceous gland function been closely examined. In contrast, there is evidence from gene inactivation studies for PPARÁ being necessary for sebocyte differentiation [41]. The role of PPARÁ in development has been difficult to ascertain because homozygous PPARÁ null mice do not survive past the midembryonic stage. This problem was circumvented by the preparation of mice chimeric for wild-type and PPARÁ null genotypes to determine the relative contribution of PPARÁ positive and negative cells to various tissues. In such mice, neither the adipocytes of fat nor sebaceous glands contained PPARÁ null cells. This contrasts with the situation in other tissues, which were comprised of equal amounts of wild type and PPARÁ null cells. This suggests that PPARÁ null cells cannot develop into either adipocytes or sebocytes. In summary, gene disruption studies in mice do not rule out a contribution of PPAR· to skin barrier function, suggest that PPAR‰ can ameliorate inflammatory responses in skin, and indicate that PPARÁ is necessary for sebocyte differentiation.

Psoriatic skin lesions were reported to underexpress PPAR· and PPARÁ mRNA [32]. However, in nonspecifically inflamed epidermis our preliminary studies show abundant PPARÁ immunostaining. Further investigation of the role of PPARÁ in epidermis was prompted by the serendipitous finding that thiazolidinediones improve psoriasis and the discovery that thiazolidinediones can inhibit proliferation of a variety of malignant and nonmalignant tissues [33]. PPARÁ ligands were found to inhibit the proliferation of cultured normal and psoriatic human keratinocytes, as well as to reduce epidermal hyperplasia in organ culture and skin transplant models of psoriasis. Furthermore, the PPAR activator troglitazone has been found to improve psoriasis. PPAR‰ mRNA overexpression is found in hyperplastic [40] and psoriatic skin [32]. Since down-regulation of PPAR‰ appears to mediate the prevention of colorectal cancer growth by the adenoma polyposis coli (APC) tumor suppressor gene [43], a general role for PPAR‰ in the regulation of epithelial cell growth has been proposed. The possibility that the PPAR‰ role is for the purpose of generating energy for mitosis cannot be excluded. In addition, PPARs may be involved in modulating skin inflammation through their effects on antiinflammatory cells. Leukotriene B4, a potent chemotactic factor, is an activating ligand for PPAR· [44], and PPARÁ is a negative regulator of macrophage activation [45]. PPAR‰ has been proposed to play a role in mediating the anitinflammatory responses to NSAIDs: these agents are known to down-regulate the transcriptional regulation of PPAR‰ by APC [43] and the exaggerated inflammatory response of PPAR‰ null mice is resistant to NSAID treatment [40], observations which suggests that PPAR‰ mediates the NSAID effect. In summary, PPARs have diverse antiproliferative and prodifferentiative effects on skin, as well as antiinflammatory effects. These processes may be mediated by transcriptional mechanisms and interplay with signalling pathways other than those resulting from PPAR effects on the target genes involved in lipid metabolism.

Evidence for PPAR Effects on Proliferative and Inflammatory Processes in Skin Summary

PPAR· activation has been shown to stimulate facets of keratinocyte differentiation that bear no obvious relationship to lipid metabolism. For example, PPAR· activation by clofibrate stimulated cornified envelope formation and inhibited the growth of cultured keratinocytes [42]. This suggests a role of PPAR· in the regulation of keratinocyte growth and differentiation.

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PPARs appear to play a role in the synthesis of the lipids that are exocytosed into the intercellular space by keratinocytes to form the skin permeability barrier. They also appear to be important for formation of the intracellular fused lipid droplets that constitute the differentiated holocrine secretion of sebocytes. They may also play roles in

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inhibiting epidermal cell growth, promoting epidermal terminal differentiation, and inhibiting skin inflammation by diverse mechanisms not necessarily related to fat metabolism.

Acknowledgements These studies were supported in part by USPHS grants HD06308 (RLR, DD) and KO8-DK02309 (MG) and the Children’s Research Foundation (DD, MG).

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23 Zhang XK, Lehmann J, Hoffmann B, Dawson MI, Cameron J, Graupner G, Hermann T, Tran P, Pfahl M: Homodimer formation of retinoid X receptor induced by 9-cis retinoic acid. Nature 1992;358:587–591. 24 Kliewer SA, Umesono K, Noonan DJ, Heyman RA, Evans RM: Convergence of 9-cis retinoic acid and peroxisome proliferator signalling pathways through heterodimer formation of their receptors. Nature 1992;358:771–774. 25 Gearing K, Gottlicher M, Teboul M, Widmark E, Gustafsson J-A: Interaction of the peroxisome-proliferator-activated receptor and retinoid X receptor. Proc Natl Acad Sci 1993;90: 1440–1444. 26 Mukherjee R, Jow L, Croston GE, Paterniti JR Jr: Identification, characterization, and tissue distribution of human peroxisome proliferatoractivated receptor (PPAR) isoforms PPARgamma2 versus PPARgamma1 and activation with retinoid X receptor agonists and antagonists. J Biol Chem 1997;272:8071–8076. 27 Tontonoz P, Singer S, Forman BM, Sarraf P, Fletcher JA, Fletcher CD, Brun RP, Mueller E, Altiok S, Oppenheim H: Terminal differentiation of human liposarcoma cells induced by ligands for peroxisome proliferator-activated receptor gamma and the retinoid X receptor. Proc Natl Acad Sci USA 1997;94:237–241. 28 Kim MJ, Ciletti N, Michel S, Reichert U, Rosenfield RL: The role of specific retinoid receptors in sebocyte growth and differentiation in culture. J Invest Dermatol 2000;114: 349–353. 29 Kim MJ, Ciletti N, Michel S, Reichert U, Rosenfield RL: Limited cooperation between peroxisome proliferator-activated receptors and retinoid X receptors in induction of sebocyte differentiation and proliferation. Pediatric Research 2000;47:72A (abstr 424). 30 Braissant O, Foufelle F, Scotto C, Dauca M, Wahli W: Differential expression of peroxisome proliferator-activated receptors (PPARs): Tissue distribution of PPAR-alpha, -beta, and -gamma in the adult rat. Endocrinol 1996;137: 354–366. 31 Braissant O, Wahli W: Differential expression of peroxisome proliferator-activated receptoralpha, -beta, and -gamma during rat embryonic development. Endocrinology 1998;139:2748– 2754. 32 Rivier M, Safonova I, Lebrun P, Griffiths CE, Ailhaud G, Michel S: Differential expression of peroxisome proliferator-activated receptor subtypes during the differentiation of human keratinocytes. J Invest Dermatol 1998;111: 1116–1121.

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Regulation of Macrophage Gene Expression by the Peroxisome Proliferator-Activated Receptor-Á Mercedes Ricote a John S. Welch a Christopher K. Glass b Department of a Cellular and Molecular Medicine and b Medicine, University of California at San Diego, La Jolla, Calif., USA

Key Words Nuclear Receptors W Macrophages W PPAR-Á W IL-4 W STAT-6 W CD36 W iNOS

Abstract The peroxisome proliferator-activated receptor-Á (PPARÁ), which is a member of the nuclear hormone receptor superfamily and was originally shown to play an important role in adipocyte differentiation and glucose homeostasis, is now known to regulate cellular proliferation and inflammatory responses. A range of synthetic and naturally occurring substances activates PPAR-Á, however the identities of endogenous ligands for PPAR-Á and their means of production in vivo have not been well established. In monocytes and macrophages, interleukin-4 (IL4) increases the expression of 12/15-lipoxygenase and thus13-HODE and 15-HETE production. We show that IL4 induces the expression of PPAR-Á and provide evidence that the coordinate induction of PPAR-Á and 12/ 15-lipoxygenase mediates IL-4 dependent transcription of the CD36 gene and down-regulation of iNOS in macrophages. These findings suggest that PPAR-Á activity may play an important role in mediating macrophage gene expression signaled by IL-4.

Introduction

The macrophage plays an important role in both the innate and acquired immune responses. As such, macrophage activity is precisely and potently regulated by both host produced cytokines and pathogen specific inflammatory signals; such as bacteria cell wall products [1]. We and others have recently become interested in the potential of nuclear receptors, such as the peroxisome proliferator-activated receptor-Á (PPAR-Á), to modulate macrophage function and activity. PPAR-Á is expressed in diverse tissues, including adipose tissue, spleen, adrenal gland, endothelium and hematopoetic tissue [2, 3]. Synthetic and natural PPAR-Á ligands have been found to potently alter macrophage gene expression and function [4]. They have been found to inhibit the induction of genes whose expression is triggered by inflammatory signals such as lipopolysaccaride (LPS), interferon-Á (IFN-Á) or the phorbol ester (TPA). Such genes include the inducible nitric oxide synthate (iNOS), gelatinase B, the scavenger receptor A (SR-A), IL-2, IL-6 and TNF· [5, 6]. Second, treatment of monocytes and THP-1 monocytic leukemia cell lines with PPAR-Á resulted in their conversion to foam cells [7, 8]. The ability of PPAR-Á activity to abrogate the induction of macrophage inflammatory media-

Copyright © 2001 S. Karger AG, Basel

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Accessible online at: www.karger.com/journals/hre

Mercedes Ricote Department of Cellular and Molecular Medicine University of California, San Diego 9500 Gilman Drive, La Jolla, CA 92093-0651 (USA) Tel. +1 858 534 8867, Fax +1 858 822 2127, E-Mail [email protected]

Fig. 1. Regulation of PPAR-Á expression by IL-4. a IL-4 induced PPAR-Á protein expression in resident macrophages. Resident macrophages were treated with IL-4 (10 ng/ml) for 48 h prior to analysis by Western blot. The arrowhead indicates the specific 55-kDa PPAR-Á band. The asterisk denotes a nonspecific band. b Regulation of PPAR-Á mRNA expression in thioglycollate-elicited macrophages from wild-type and STAT-6 knockout mice. Elicited macrophages were treated with IL-4 (10 ng/ml) and rosiglitazone (Ro) (10 ÌM) for 24 h prior to isolation of total RNA.

tors and to induce foam cell formation suggests that PPAR-Á may play a key role in regulating macrophage activity and function. Recent studies have shown that PPAR-Á ligands mediate a direct inhibitory role on T cell dependent immune responses, suggesting that PPAR-Á may regulate cellular function and activity in several distinct hematopoietic cell types [9, 10]. Here we demonstrate the induction of PPAR-Á expression in macrophages by IL-4 and show that this induction is dependent on STAT-6 mediated signal transduction. We further suggest that IL-4 may mediate a portion of its anti-inflammatory effects and gene expression program by coordinately inducing the expression of PPAR-Á and providing ligands 13-HODE and 15-HETE via the expression and activity of the 12/15-lipoxygenase (12/15-LO).

Material and Methods Cell Culture Thioglycollate-elicited peritoneal macrophages were isolated and culture as described [5]. RAW 264.7 (ATCC) cells were cultured in DMEM (Gibco) supplemented with 10% fetal calf serum (Gemini Biological Products, Calabasas, Calif.). Western Blot Analysis Western blot analysis was performed by using standard procedures [11]. Total protein was extracted in SDS sample buffer, resolved by SDS-PAGE and transferred to nitrocellulose filters. To detect the 55K PPAR-Á protein, incubation with a guinea-pig antiserum against full length murine PPAR-Á fused to glutathione-S-transferase was performed at 1:500 dilution overnight at 4 ° C. We used a guinea-pig antiserum against the recombinant extracellular domain of murine CD36 at 1:1,000 dilution. The secondary antibody (rabbit anti-guinea pig from Dako) was diluted at 1:10,000 and incubated at room temperature for 1 h. Detection was performed by using chemiluminescence (Pierce). Protein levels were determined to be similar in each sample by using an antibody against ß-actin (Sigma).

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Northern Blot Analysis Total RNA was prepared by standard protocols (RNAzolTMB, Leedo Pharmaceutical Laboratories) and samples were hybridized with cDNA probes for mouse PPAR-Á, iNOS and GAPDH. Transient Transfection RAW 264.7 (ATCC) cells were transiently transfected using lipofectamin (Gibco) as described previously [12]. The iNOS promoterluciferase construct, PPAR-Á dependent reporter construct (AOX)3TK-luciferase, PPARÁ expression vector pcMX-PPARÁ and pcDNA3-15-LO expression vector have been described previously [13]. Cells were allowed to rest overnight in medium containing 0.5% FBS, followed by treatment with the indicated compounds for 24 h and luciferase activity were determined.

Results

In order to study the role of PPAR-Á in macrophage biology, we carried out a series of experiments searching for potential regulatory molecules that would control its expression in macrophages. We found that IL-4 induced the expression of PPAR-Á in human monocytes [13] and peritoneal macrophages (fig. 1). IL-4 plays a central role in the regulation of immune responses and uses the STAT-6 pathway to mediate much of its signal transduction. Recent studies from STAT6-deficient mice have revealed the essential role of STAT-6 in IL-4 mediated biological actions [14]. To examine the possibility that IL4 affects PPAR-Á expression by activation of the STAT-6 pathway, we examined PPAR-Á expression in thioglycollate-elicited peritoneal macrophages obtained from wild type and STAT-6 deficient mice. IL-4 induction of PPAR-Á was significantly reduced in macrophages from STAT-6 knockout mice, compared to wild-type mice (fig. 1b).

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Fig. 2. 12/15-lipoxygenase products of ara-

chidonic (AA) and linoleic acid (LA) metabolism potentiate transcriptional activation of PPAR-Á. a RAW 264.7 cells were transfected with a PPAR-Á expression plasmid and a luciferase reporter gene (AOX)3-TKluc. Cells were treated with the indicated concentrations of AA, rosiglitazone, 15HETE and 13-HODE and analyzed for luciferase activity 24 h later. b, c RAW 264.7 cells were transfected with the iNOS promoter reporter gene and a PPAR-Á expression plasmid. Cells were treated with combinations of LPS (1 Ìg/ml), IFN-Á (100 U/ml) and the indicated PPAR-Á ligands. Cells were analyzed for luciferase activity 24 h later. Error bars represent SD.

IL-4 also regulates the expression of 12/15-LO, an enzyme that catalyzes the synthesis of the PPAR-Á ligands 12- and 15-HETE from arachidonic acid and 13-HODE from linoleic acid [15]. These observations raised the possibility that IL-4 might regulate gene expression in the macrophage in part by inducing the expression of 12/15LO. We have tested this hypothesis first in the macrophage-like cell RAW 264.7 by cotransfecting a reporter plasmid with a multimerized PPAR-Á response element driving luciferase and expression plasmids for PPAR-Á and 12/15-LO. We found that either 13-HODE or 15HETE could induce luciferase activity, but that this required cotransfection of the PPAR-Á expression plas-

mid. Furthermore, we found that arachidonid acid could induce reporter activity, but only in the presence of both the PPAR-Á and the 12/15-LO expression plasmids (fig. 2a). We also found that products of the 12/15-LO pathway were able to stimulate transrepression activity of PPAR-Á. PPAR-Á ligands inhibited the induction of iNOS by LPS and IFN-Á [5]. The observation that IL-4 can inhibit induction of iNOS in primary mouse macrophages [16], raised the possibility that IL-4 may inhibit iNOS by inducing PPAR-Á and its ligands. Consistent with this hypothesis, 15-HETE and 13-HODE inhibited induction of the iNOS promoter in a PPAR-Á dependent manner in

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4 Fig. 4. Model for IL-4 regulation of PPAR-Á function: IL-4 induces and represses

target gene expression by coordinately enhancing PPAR-Á levels and the production of activating ligands through 12/15-LO.

Fig. 3. IL-4 affects iNOS and CD36 gene expression by coordinate induction of PPAR-Á and 12/15-LO in elicited macrophages. a IL-4 and PPAR-Á ligands inhibit induction of iNOS mRNA following LPS (1 Ìg/ml) treatment. b CD36 protein levels is increased by IL-4, 13-HODE and troglitazone (TGZ). c Induction of CD36

expression by IL-4 is defective in 12/15-LO –/– macrophages.

3

RAW 264.7 cells, and linoleic acid inhibited the iNOS promoter in the presence of co-expressed 12/15-LO and PPAR-Á (fig. 2b and c). Consistent with these findings, IL4 and rosiglitazone inhibited LPS-activation of iNOS mRNA in thioglycollate-elicited macrophages (fig. 3a). Finally, to test the hypothesis that IL-4 affects macrophage gene expression by coordinate induction of PPARÁ and 12/15-LO, we studied the potential roles of these proteins in mediating IL-4 stimulation of the PPAR-Á target gene CD36 (fig. 3b). Treatment with increasing concentration of 13-HODE or IL-4 significantly induced CD36 expression. Furthermore, the ability of IL-4 to induce CD36 expression in macrophages was significantly impaired in macrophages derived from mice deficient

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for 12/15-LO (12/15-LO –/–) (fig. 3c). Taken together these results suggest that IL-4 coordinately induce and 12/15LO expression, resulting in production of PPAR-Á ligands, activation of PPAR-Á target genes and repression of pro-inflammatory genes (fig. 4)

Discussion

PPAR-Á is activated by arachidonic acid metabolites derived from the cyclooxygenase and lipoxygenase pathways such as 15-deoxy-¢12, 14prostaglandin and 15-HETE. In addition, fatty acid derived compounds of oxidized LDL, such as 13-HODE and 9-HODE, activate PPAR-Á

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[7, 17, 18]. Furthermore, the antidiabetic thiazolidinediones, which improve insulin resistance in patients with type 2 diabetes mellitus [19], are high-affinity ligands for PPAR-Á, and non-steroidal anti-inflammatory drugs (NSAIDs) function as ligands for PPAR-Á [20]. PPAR-Á activates target genes through direct interactions with specific elements as a heterodimer with RXR [2]. In addition, PPAR-Á inhibits inflammatory response genes by antagonizing the activities of AP-1, NF-ÎB and STAT1 transcription factors [5, 21]. The possibility that PPAR-Á could influence macrophage gene expression was initially suggested because PPAR-Á was expressed in spleen [22]. As the spleen is a macrophage rich organ, we quantified PPAR-Á mRNA levels in various macrophage populations and observed that PPAR-Á was highly expressed in macrophages isolated from inflammatory peritoneal exudates, while significantly lower levels were found in bone marrow-derived and resting macrophage populations [5, 12]. In order to further characterize the expression of PPAR-Á in the macrophage we searched for potential regulatory molecules that would control its expression in these cells. This search lead to the identification of IL-4 as a significant inducer of PPAR-Á expression in monocytes and peritoneal macrophages. IL-4 is a prototype type 2 cytokine than can suppress the production of many macrophage pro-inflammatory mediators such as COX-2, IL10, IL-6, iNOS, TNF-·, Rantes and IL-12 [16, 23]. Interestingly some of these genes have been shown by our group and other groups to be inhibited by PPAR-Á [5, 6]. In addition, IL-4 also regulates the expression of enzymes that could potentially be required for the production of endogenous ligands for PPAR-Á. IL-4 inhibits the production of cyclooxygenase-2 (COX-2), which would be required for the production of pro-inflammatory prostaglandins such as PGE2 and PGJ2 [23]. In contrast, in monocytes IL-4 induces the expression of the 12/15-LO, an enzyme capable of catalyzing 12- and 15-HETE production from arachidonic, and 13-HODE from linoleic acid [15]. Each of these compounds is capable of activating PPAR-Á [7, 13]. These observations raised the possibility that IL-4 might regulate gene expression in the macrophage in part by coordinately inducing the expression of PPAR-Á and the expression of 12/15-LO. In the present study, we describe evidence supporting this hypothesis. We found that IL-4 induces the expression of PPAR-Á through the STAT6 pathway, and we also demonstrated that CD36 and iNOS, genes that are respectively positively and negatively regulated by PPAR-Á, were similarly regulated by 12/15-LO products or IL-4

treatment. Furthermore, this regulation by IL-4 appeared to be 12/15-LO dependent. The present iNOS data confirm and extend previous results from our laboratory. The inhibition of LPS- induced mRNAs of iNOS by IL-4 and PPAR-Á ligands reflects its anti-inflammatory effect. In agreement with these results, Kitamura et al. [24] have shown similar results in isolated microglial cultures. The finding that disruption of the 12/15-LO gene significantly reduced CD36 expression supports an important role for the structurally related 12/15-LO products, including 15HETE and 13-HODE, as endogenous regulators of PPARÁ in the macrophage. Finally, the coordinate regulation of both 12/15-LO and PPAR-Á by IL-4 suggests a new paradigm for the regulation of nuclear receptor function by cytokines. Additional studies will be required to determine whether or not PPAR-Á plays a more general role in mediating IL-4 response in the macrophage. It is also likely that PPAR-Á will have additional roles, as PPAR-Á can be highly expressed in macrophages in a manner that is independent of IL-4 (unpublished observations). The application of microarray technologies and the ability of mice selectively lacking PPAR-Á in the macrophage should be helpful in further defining the biological role of PPAR-Á in this cell type.

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Acknowledgements We thank Dr Colin D. Funk for providing the 12/15-LO knockout mice and Amy J. Mottahedeh for assistance with manuscript preparation. M.R. was supported by a Postdoctoral Fellowship from the American Heart Association. J.S.W. was supported by a Predoctoral Fellowship from the American Heart Association Association. These studies were also supported by NIH grant (5 RO1 HL5969403) to C.K.G.

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18 Kliewer SA, Lenhard JM, Willson TM, Patel I, Morris DC, Lehmann JM: A prostaglandin J2 metabolite binds peroxisome proliferator-activated receptorÁ and promotes adipocyte differentiation. Cell 1995;83:813–819. 19 Willson TM, Cobb JE, Cowan DJ, Wiethe RW, Correa ID, Prakash SR, Beck KD, Moore LB, Kliewer SA, Lehmann JM: The structure-activity relationship between peroxisome proliferator-activated receptor gamma agonism and the antihyperglycemic activity of thiazolidinediones. J Med Chem 1996;39:665–668. 20 Lehmann JM, Lenhard JM, Oliver BB, Ringhold GM, Kliewer SA: Peroxisome proliferator-activated receptors · and Á are activated by indomethacin and other non-steroidal antiinflammatory drugs. J Biol Chem 1997;272: 3406–3410. 21 Chinetti G, Griglio S, Antonucci M, Torra IP, Delerive P, Majd Z, Fruchart J-C, Chapman J, Najib J, Staels B: Activation of proliferatoractivated receptors · and Á induces apoptosis of human monocyte-derived macrophages. J Biol Chem 1998;273:25573–25580. 22 Kliewer SA, Forman BM, Blumberg B, Ong ES, Borgmeyer U, Mangelsdorf DJ, Umesono K, Evans RM: Differential expression and activation of a family of murine peroxisome proliferator-activated receptors. Proc Natl Acad Sci USA 1994;91:7355–7359. 23 Dworski R, Sheller JR: Differential sensitivities of human blood monocytes and alveolar macrophages to the inhibition of protaglandin endoperoxide synthase-2 by interleukin-4. Prostaglandins 1997;53:237–251. 24 Kitamura Y, Taniguchi T, Kimura H, Nomura Y, Gebicke-Haerter PJ: Interleukin-4-inhibited mRNA expression in mixed rat glial and in isolated microglial cultures. J Neuroimmunol 2000;106:95–104.

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Neuroimmunoregulation of Androgens in the Adrenal Gland and the Skin Salvatore Alesci Stefan R. Bornstein Pediatric and Reproductive Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Md., USA

Key Words Adrenal androgens W DHEA W Hyperandrogenism W Hirsutism W Acne W Androgenic W Alopecia W Hair loss

Abstract Human adrenals produce large quantities of androgens, especially DHEA which is the most abundant circulating hormone in the human body. Adrenal androgens are regulated by several factors, including pituitary ACTH and an intricate intraadrenal network involving immune cells, cytokines and neuroendocrine factors. The skin is a major target of androgens and androgen receptors are expressed in the epidermis, dermis, sebaceous glands and hair. In addition, the skin has the capacity to metabolize androgens into more powerful compounds. Similar to the adrenal gland, there is an intradermal neuroimmune network involving the local expression of cytokines and neuropeptides. Dysregulation of androgens in the adrenals and/or the skin is associated with acne, hirsutism and androgenic alopecia. Therefore, understanding the mechanisms of these intricate networks is of both basic and clinical relevance and may help to develop new strategies for the treatment of androgen-dependent skin disorders.

Introduction

Adrenal androgens (AA) are the major product of the adrenal cortex zona reticularis (ZR). Their precursor pregnenolone, a C-21 steroid, is synthesized from cholesterol by the cytochrome P450scc enzyme (CYPscc), which is found on the inner mitochondrial membrane. The movement of cholesterol from the outer to the inner mitochondrial membrane is regulated by the steroidogenic acute regulatory protein (StAR) [1]. Cytochrome P45017· (CYP17), a 17·-hydroxylase with 17,20-lyase activity is responsible for the conversion of pregnenolone to the C19 steroid dehydroepiandrosterone (DHEA), while a hydrosteroid sulfotransferase (DHEAST) converts DHEA to dehydroepiandrosterone sulfate (DHEAS) [2]. Pregnenolone can be also converted to progesterone by the 3ßhydroxysteroid dehydrogenase (3ßHSD), which can also convert 17·-hydroxypregnenolone to 17·-hydroxyprogesterone and DHEA to androstenedione. After a dramatic increase just before puberty (adrenarche), DHEA peaks during the the third decade of life [3, 4]. Its levels drop by the age of 50–60 (adrenopause), reaching the lowest concentrations at the age of 70–80 (20–30% of the peak) [5].

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Neuroimmunoendocrine Intraadrenal Network

The control of AA is still not entirely understood. AA production seems to be partially modulated by ACTH. However, several clinical conditions show a clear disso-

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ciation between adrenal androgen and cortisol secretion [6], suggesting that other non-ACTH mediated mechanisms must play a role in such regulation. Several factors and hormones have been proposed as ‘regulators’ of AA secretion: among these oestrogens [7–9], prolactin [10– 12], growth hormone [13, 14], insulin and IGFs [15–18]. Besides systemic factors, AA production seems to be regulated through an intricate network involving nerves and neuropeptides, cytokines and the ‘cross-talk’ between AA secreting cells of ZR, chromaffin cells and cellular components of the immune system. Adrenals receive a rich nerve supply [19, 20]. Intimate contacts between nerve endings and steroid-producing cells of the adrenal cortex have been recently described [21]; in addition, cortical cells and medullary cells were found to be highly interwoven [22]. Paracrine interactions between the ‘sympathoadrenal system’ and the adrenal cortex might be mediated by these contacts [21–24]. Normal intraadrenal glucocorticoid levels are required to keep a normal function of the adrenal medulla and increased levels of other steroids, including AA, cannot compensate for glucocorticoid deficiency [25, 26]. On the other hand the adrenomedullary system regulates AA production. Thus stimulation of the splanchnic nerves and epinephrine may induce the release of androstenedione [27]. In addition VIP, a neuropeptide occurring in nerve fibers and chromaffin cells in rat [28] and human adrenal glands [29], stimulates both the release of androstenedione and DHEA [30, 31]. Other neuropeptides of hypothalamic origin have been linked to AA regulation: among them ß-endorphin, whose levels are high in polycystic ovary syndrome (PCOS) [32] and the so called ‘joining peptide’, a peptide derived from proopiomelanocortin (POMC). This last finding however has been disputed [33–35]. More recently, the corticotropin-releasing hormone (CRH) has been reported to stimulate DHEA secretion from human fetal adrenals [36, 37] and in young men [38]. A local CRH/ACTH system has also been identified in human adrenals [39] and there is growing evidence that the CRH-induced increase of adrenal steroidogenesis can be blocked by CRH type-I receptor antagonists, suggesting a role for this receptor in the local CRH/ACTH system [40]. Cellular components of the immune system, namely macrophages, monocytes, dendritic cells, mast cells and lymphocytes have been described within the human adrenal cortex during all stages of life under normal and pathological conditions. Activated macrophages, mostly located in ZR, produce IL-1, [41] IL-6 [42] and TNF-· [43] and TGF-ß [44]. Similar to macrophages, adrenocor-

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tical cells themselves are able to synthesize several cytokines. Within the adrenal, they produce TNF-· [43], IL-1 [41], IL-6 [42]. The distribution of this expression is species specific: in rats, high amounts of cytokines have been detected in the zona glomerulosa [45, 46], whereas in humans the zona reticularis seems to be the main site of production. These cytokines may exert either stimulatory or inhibitory action. IL-6 increases the secretion of cortisol and androgens probably acting directly on the steroid cells, since these cells express IL-6 receptors [47]. However, it was demonstrated that indomethacin can inhibit the IL6-induced increase of cortisol secretion, suggesting a prostaglandin-mediated mechanism [48]. Together with IL-1 and IL-6, TNF-· is one of the most important cytokines released during an immune response. In human fetal adrenal cells, it decreased both ACTH-driven and basal cortisol production. Moreover, a shift toward androgen synthesis was observed [49, 50]. Angiotensin II [51] and ACTH [52] can induce IL-6 in the adrenal cortex, whereas TNF-· release is inhibited by ACTH [53]. TGF-ß belongs to the group of cytokines that play an important role in the modulation of immune functions. It may affect adrenocortical growth, differentiation, and hormone secretion. TGF-ß has been detected in the mouse [54] and bovine adrenal cortex [55]. TGF-ß receptors have also been found in the bovine adrenal cortex [56, 57] and are increased in number by ACTH stimulation. In humans, TGF-ß seems to reduce the synthesis of DHEAS and DHEAST mRNA [58]. The immunoregulation of AA production may also include cytokine-independent mechanisms. There is growing evidence, in fact, that differentiated adrenal steroid cells themselves have important immune functions. The adrenal androgen producing cells of the ZR express MHC class II [59] and two molecules involved in cellular immune response and apoptosis, Fas (CD95) and Fas ligand (Fas-L), which may play a key role in the ‘cross talk’ between the adrenal gland and the immune system [60]. Adrenal androgens and adrenal glucocorticoids both play an important role in T-helper cells differentiation, which is of relevance for the systemic balance of Th1 and Th2 cells [61]. Interestingly, lymphocytes have been found in the innermost cortical zone: they are CD4+ and express IL-2 receptors. Electron microscopy demonstrated direct cell-to-cell contact between adrenal cortical cells and lymphocytes through filopodia and gap junctions, which may represent a pathway for signals acting on steroidogenesis [62]. Co-cultures of human T lymphocytes and adrenocortical cells led to a 4-fold increase of DHEA levels, which

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was greater than the increase observed after the addition of maximal concentrations of ACTH. Separation of cells by semipermeable membranes abolished this effect, and transfer of leukocyte-conditioned medium had little androgen-stimulating effect [62]. These data suggest that the stimulation of androgen secretion requires cell contact in addition to paracrine factor(s).

The skin is a site of active metabolism of androgens, which are converted into more powerful ones, such as androstenedione and 5·-dihydrotestosterone [63]: for this reason it is considered a ‘peripheral endocrine gland’. More recently it became evident that the skin can also be considered an ‘immunocompetent organ’, able to modulate inflammatory and immune responses [64]. Epidermal keratinocytes produce several cytokines, including IL-1·, IL-1ß, IL-3, IL-6, IL-8, IL-10, GM-CSF, G-CSF and TNF· [65] while dermal fibroblasts produce IL-1ß, IL-6, IL-8 and G-CSF. Some evidence suggests that also the sebaceous glands, which are a major target for androgen [66], may produce immunomodulatory peptides: in fact both TNF-· [67] and IL-1· [68] were found to be highly expressed in the human sebaceous gland. These cytokines, other than acting as mediators of immune response and inflammation, are involved in the control of cell proliferation and differentiation, playing an important role in wound healing and tissue remodelling [69, 70]. Moreover, neuropeptides such as ß-endorphin and ·-MSH, a POMC-derived peptide, and their receptors have been demonstrated within the pilosebaceous unit of murine and human skin by immunostaining [71]. Several studies have demonstrated that POMC peptides can act as immunosuppressants: ·-MSH, for example can antagonize the effects of proinflammatory cytokines, such as IL-1, IL-6 and TNF-· [72], downregulate the production of IFN-Á by human lymphocytes, induce IL-10, a suppressor cytokine in monocytes [73] and downregulate MHC class antigen in these cells [74, 75]. Interestingly, receptors for ·-MSH have been found on immunocompetent cells, such as monocytes and neutrophils [72, 73]. Finally, CRH and ACTH and their receptors have been demonstrated in murine and human skin [76]. On this basis, taking into account that CRH, ACTH, POMC peptides and ß-endorphin are all stress-related hormones, the existence of a local ‘skin stress-response system’ (SSRS) has been proposed [77]. In response to local stress (e.g., wounding etc.), the skin neuroendocrine system

(SNS) produces CRH and POMC peptides. The skin immune system (SIS) modulates this response by releasing proinflammatory cytokines, which may stimulate further secretion of CRH an POMC peptides from the SNS. On the other hand, the interaction between CRH, ACTH, MSH, ß-endorphin with their skin receptors generate signals to counteract the effects caused by the local stressor. In addition, locally produced neuropeptides and/or steroids may inhibit the SIS and the SNS through a negative feedback mechanism [77]. There is evidence that the immune system may be involved in the regulation of hair growth and its cycle. The hair follicle itself can be considered as an ‘immune organ’ which may replenish injured epidermis by its reservoir of cells [78]. During the catagen phase of the hair cycle, class I MHC is expressed on the whole follicle epithelium. Moreover, immunohistochemical studies have shown the presence of perifollicular macrophages and T cells in catagen but not in anagen or telogen [79]. CD1apositive Langerhans’ cells, which function as antigen processing cells have also been found in the hair infundibulum [80, 81]. More recently, it has been reported that IL1·, IL-1ß and TNF-· are potent inhibitors of hair follicle growth in vitro, causing changes in the hair follicle morphology which result in the formation of dystrophic anagen hair follicles. These changes can be blocked by addition of IL-1 receptor antagonist (IL-1 Ra) [82]. Taken together, these data suggest that immune mechanisms can play a key role in the transition of the hair cycle from anagen to catagen and then to telogen. Hair loss is a dermatose commonly associated with hyperandrogenism, including adrenal hyperandrogenism. The local neuroimmunoendocrine cellular control in the skin might be involved in the pathogenesis of androgenic hair loss. The balance between AA and glucocorticoids regulates the differentiation of T helper cells (Th) into Th1 and Th2. Th1 cells produce IFN-Á and IL-2, while Th2 secrete different cytokines (IL-3, -4, -5, -10, -13). The Th1 response promotes cellular immunity, whereas the Th2 initiates humoral immunity and counteracts the Th1 response. Interestingly, recent data show that glucocorticoids can promote a shift of the immune response from a Th1 type to a Th2 type [61]. According to our hypothesis model (fig. 1), increased levels of AA, as during adrenal hyperandrogenism, will shift the immune response from Th2 towards Th1 within the SIS. This will cause an activation of hair perifollicular macrophages, with release of IL-1, IL-6 and TNF-·. In addition, DHEA and more potently androstenediol (AED) and androstenetriol (AET),

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Neuroimmunoendocrine Intradermal Network

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Fig. 1. Neuroimmunoendocrine network in

the skin. Hypothetical model of androgendependent hair loss. Increased levels of DHEA will cause a shift of the immune response from Th1 to Th2 within the skin. This will activate perifollicular macrophages with release of IL-1, IL-6 and TNF-·. Androstenediol (AED) and androstenetriol (AET), produced by DHEA conversion in the skin will themselves stimulate this cytokine release. At the same time, the high levels of DHEA may suppress the CRH-ACTH response of the skin, through a negative feedback, with a decrease in ·-MSH which normally antagonizes the cytokine-mediated inflammatory response. The increased cytokine levels will produce changes in hair morphology and cycle and will further stimulate adrenal DHEA production from the adrenal gland.

produced by DHEA conversion at the skin level, may themselves stimulate this cytokine production [83]. Similar to the adrenals, a direct interaction with lymphocytes may occur. The high levels of AA will also inhibit the skin CRH-ACTH response through a negative feedback, with a decrease in ·-MSH production, which normally counteracts the effect of the proinflammatory cytokines. The increase in cytokine levels will cause changing in the hair morphology and cycle, leading to hair loss. At the same time, IL-6 and TNF-· will further stimulate AA production in a vicious cycle, with an amplification of the ‘hairkilling’ process and a slow progression of most of the hair to the telogen state of the cycle. Finally, since IL-1· and IL-1ß affect the expression of vascular endothelial growth factor/vascular permeability factor in epithelial and mesenchymal cells derived from sebaceous glands [84], these cytokines may also contrib-

ute to the development of acne and other related skin disorders.

Conclusions

In conclusion, both in the adrenal gland, a classical endocrine gland, and the skin, a newly discovered endocrine active tissue, similar patterns of neuroimmunoendocrine regulation participate in local androgen metabolism. In these systems skin elements and endocrine cells ‘cross-talk’ with immune cells through a network of cytokines and neuropeptides, finely adjusting the response to local and systemic hormonal stimuli. Any abnormal stimulus, such as hyperandrogenism, can alter this delicate equilibrium, causing an abnormal skin response and ultimately loss of its integrity.

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3 Adams JB: Control of secretion and the function of C19-delta 5-steroids of the human adrenal gland. Mol Cell Endocrinol 1985;41:1–17. 4 Parker LN: Control of adrenal androgen secretion. Endocrinol Metab Clin North Am 1991; 20:401–421.

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33 Mellon SH, Shively JE , Miller WL: Human proopiomelanocortin (79–96), a proposed androgen stimulatory hormone, does not affect steroidogenesis in cultured human fetal adrenal cells. J Clin Endocrinol Metab 1991;72:19–22. 34 Penhoat A, Sanchez P, Jaillard C, Langlois D, Begeot M, Saez JM: Human proopiomelanocortin (79–96), a proposed cortical androgen stimulating hormone, does not affect steroidogenesis in cultured human adult adrenal cells. J Clin Endocrinol Metab 1991;72:23–26. 35 Robinson P, Baterman A, Mulay S, Spencer SJ, Jaffe RB, Solomon S, Bennett HP: Isolation and characterization of three forms of joining peptide from adult pituitaries: Lack of adrenal androgen stimulating activity. Endocrinology 1991;129:859–867. 36 McLean M, Bisits A, Davies J, Woods R, Lowry P, Smith R: A placental clock controlling the length of human pregnancy. Nat Med 1995; 1:460–463. 37 Smith R, Mesiano S, Chan EC, Brown S, Jaffe RB: Corticotropin-releasing hormone directly and preferentially stimulates dehydroepiandrosterone sulfate secretion by human fetal adrenal cortical cells. J Clin Endocrinol Metab 1998;83:2916–2920. 38 Ibanez L, Potau N, Marcos MV, de Zegher F: Corticotropin-releasing hormone as adrenal androgen secretagogue. Pediatr Res 1999;46: 351–353. 39 Suda T, Tomori N, Tozawa F, Demura H, Shizume K, Mouri T, Mirura Y, Sasano N : Immunoreactive corticotropin and corticotropin-releasing factor in human hypothalamus, adrenal, lung cancer and pheochromocytoma. J Clin Endocrinol Metab 1984;58:919–924. 40 Willenberg HS, Bornstein SR, Hiroi N, Path G, Goretzki PE, Scherbaum WA, Chrousos GP: Effects of a novel corticotropin-releasing-hormone receptor type I antagonist on human adrenal function. Mol Psychiatry 2000;5:137– 141. 41 Gonzalez-Hernandez JA, Bornstein SR, Ehrhart-Bornstein M, Geschwend JE, Gwosdow AR, Jirikowski GF, Scherbaum WA: Interleukin 1 is expressed in human adrenal gland in vivo. Possible role in a local immune-adrenal axis. Clin Exp Immunol 1995;99:137–141. 42 Gonzalez-Hernandez JA, Bornstein SR, Ehrhart-Bornstein M, Späth-Schwalbe E, Jirikowski GF, Scherbaum WA: Interleukin-6 mRNA expression in human adrenal gland in vivo: New clue to a paracrine or autocrine regulation of adrenal function. J Clin Endocrinol Metab 1994;79:1492–1497. 43 Gonzalez-Hernandez JA, Ehrhart-Bornstein M, Späth-Schwalbe E, Scherbaum WA, Bornstein SR: Human adrenal cells express TNF ·mRNA: Evidence for a paracrine control of adrenal function. J Clin Endocrinol Metab 1996;81:807–813. 44 Bornstein SR, Chrousos GP: Clinical review 104: Adrenocorticotropin (ACTH)- and nonACTH-mediated regulation of the adrenal cortex: Neural and immune inputs. J Clin Endocrinol Metab 1999;84:1729–1736.

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59 Marx C, Bornstein SR, Wolkersdorfer GW, Peter M, Sippell WG, Scherbaum WA: Relevance of major histocompatibility complex class II expression as a hallmark for the cellular differentiation in the human adrenal cortex. J Clin Endocrinol Metab 1997;82:3136–3140. 60 French LE, Hahne M, Viard I, Radlgruber G, Zanone R, Becker K, Muller C, Tschopp J: Fas and Fas-ligand expression in embryos and adult mice: Ligand expression in several immune-privileged tissues and coexpression in adult tissues characterized by apoptotic cell turnover. J Cell Biol 1996;133:335–343. 61 Elenkov IJ, Webster EL, Torpy DJ, Chrousos GP: Stress, corticotropin-releasing hormone, glucocorticoids, and the immune/inflammatory response: Acute and chronic effects; in Cutolo M, et al (eds): Neuroendocrine Immune Basis of the Rheumatic Diseases. Ann NY Acad Sci, 1999, v. 876. 62 Wolkersdörfer GW, Lohmann T, Marx C, Schroder S, Pfeiffer R, Stahl HD, Scherbaum WA, Chrousos GP, Bornstein SR: Lymphocytes stimulate dehydroepiandrosterone production through direct cellular contact with adrenal zona reticularis cells: A novel mechanism of immune-endocrine interaction. J Clin Endocrinol Metab 1999;84:4220–4227. 63 Verschoore M: Hyperandrogenism and pilosebaceous follicles. Rev Prat 1993;43:2363– 2369. 64 Böhm M , Luger AT : The pilosebaceous unit is part of the skin immune system. Dermatology 1998;196:75–79. 65 Nozaki S, Feliciani C, Sauder DN: Keratinocyte cytokines. Adv Dermatol 1992;7:83–100. 66 Zouboulis CC, Akamatsu H, Stephanek K, Orfanos CE: Androgens affect the activity of human sebocytes in culture in a manner dependent on the localization of the sebaeous glands and their effect is antagonized by spironolactone. Skin Pharmacol 1994;7:33–40. 67 Kolde G, Schulze-Osthoff K, Meyer H, Knop J: Immunohistological and immunoelectron microscopic identification of TNF-· in normal human and murine epidermis. Arch Dermatol Res 1992;284:154–158. 68 Zouboulis CC, Xia L, Akamatsu H, Seltmann H, Fritsch M, Hornemann S, Ruhl R, Chen W, Nau H, Orfanos CE: The human sebocyte culture model provides new insights into development and management of seborrhoea and acne. Dermatology 1998;196:21–31. 69 Whicher JT: Cytokines in disease. Clin Chem 1990;36:1269–1281. 70 Sporn MB, Roberts AB: Peptide growth factors are multifunctional. Nature 1988;332:217– 219. 71 Slominski A, Paus R, Mazurkiewicz J: Proopiomelanocortin expression in the skin during induced hair growth in mice. Experientia 1992; 48:50–54.

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72 Lipton JM, Catania A: Antiinflammatory actions of the neuroiimunomodulator ·-MSH. Immunol Today 1997;181:140–145. 73 Bhardwaj RS, Schwarz A, Becher E, Mahnke K, Aragane Y, Schwarz T, Luger TA: Proopiomelanocortin-derived peptides induce IL10 production in human monocytes. J Immunol 1996;156:2517–2521. 74 Bhardwaj R, Becher E, Mahnke K, Hartmeyer M, Schwarz T, Scholzen T, Luger TA: Evidence for the differential expression of the functional alpha-melanocyte-stimulating hormone receptor MC-1 on human monocytes. J Immunol 1997;158:3378–3384. 75 Luger TA, Kock A, Schauer E, Urbanski A, Trautinger F, Schwarz T: Cytokine neuropeptide interactions in the skin; in Van Vlotken WA, Lomabat WE (eds): Basic Mechanisms of Physiological and Aberrant Lymphoproliferation in the Skin. Plenum Press, 1994, pp 95– 102. 76 Roloff B, Fechner K, Slominski A, Furkert J, Botchkarev VA, Bulfone-Paus S, Zipper J, Krause E, Paus R: Hair cycle-dependent expression of corticotropin-releasing factor (CRF) and CRF receptors in murine skin. FASEB J 1998;12:287–297. 77 Slominski A, Botchkareva NV, Botchkarev VA, Chakraborty A, Luger T, Uenalan M, Paus R: Hair cycle-dependent production of ACTH in mouse skin. Biochim Biophys Acta 1998; 1448:147–152. 78 Jaworsky C, Gilliam AC: Immunopathology of the human hair follicle. Dermatol Clin 1999; 17:561–568. 79 Westgate GE, Craggs RI, Gibson WT: Immune privilege in hair growth. J Invest Dermatol 1991;97:417–420. 80 Jimbow K, Sato S, Kukita A: Langerhans’ cells of the normal human pilosebaceous system. An electron microscopic investigation. J Invest Dermatol 1969;52:177–180. 81 Moresi JM, Horn TD: Distribution of Langerhans cells in human hair follicle. J Cutan Pathol 1997;24:636–640. 82 Philpott MP, Sanders DA, Bowen J, Kealey T: Effects of interleukins, colony-stimulating factor and tumour necrosis factor on human hair follicle growth in vitro: a possible role for interleukin-1 and tumour necrosis factor-alpha in alopecia areata. Br J Dermatol 1996;135:942– 948. 83 Padgett DA, Loria RM: Endocrine regulation of murine macrophage function: Effects of dehydroepiandrosterone, androstenediol, and androstenetriol. J Neuroimmunol 1998;84:61– 68. 84 Kozlowska U, Blume-Peytavi U, Kodelja V, Sommer C, Goerdt S, Jablonska S, Orfanos CE: Vascular endothelial growth factor expression induced by proinflammatory cytokines (interleukin 1 alpha, beta) in cells of the human pilosebaceous unit. Dermatology 1998;196:89–92.

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The Role of Melanocortins in Skin Homeostasis Markus Böhm Thomas A. Luger Department of Dermatology and Ludwig Boltzmann Institute for Cell Biology and Immunobiology of the Skin, University of Münster, Germany

Key Words Melanocortins W Melanocortin receptors W Alpha-melanocyte-stimulating hormone

Abstract Melanocortins are structurally related bioactive peptides which are produced by many extra-neural tissues including the skin. All of the melanocortins (·, ß, and Á-melanocyte-stimulating hormone and adrenocorticotropin) have melanotropic activity but can elicit many other effects on skin cells. On the basis of in vitro and in vivo findings melanocortins have been shown to regulate immune and inflammatory responses, hair growth, exocrine gland activity and extracellular matrix composition. These effects are mediated by melanocortin receptors among which the melanocortin-1 receptor is most ubiquitously expressed by human skin cells. Simultaneous expression of melanocortins and their receptors suggest a complex autocrine and/or paracrine regulatory network whose disruption invariably affects skin homeostasis. Expression of melanocortin receptors on various skin cell types further indicates novel pharmacological targets for the treatment of skin diseases. Copyright © 2001 S. Karger AG, Basel

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In the last years there has been a rapid progress in our understanding of the melanocortins (MCs) and their receptors. Originally identified as neurohormones regulating pigmentation of the skin and steroid synthesis in the adrenal gland, MCs are now being recognized as modulators of many extra-pigmentary functions. This article is a brief review on the complex role of MCs and their receptors in skin homeostasis with emphasis on the human system. It was our intention to include novel findings but also open questions and future perspectives in this expanding research area. Due to space limitations, a review exhaustively dealing with the pro-opiomelanocortin (POMC) system is beyond the scope of this article. For this purpose the reader is referred to the recent review by Slominski et al. [1].

Biochemistry and Biosynthesis of Melanocortins

The MCs define a group of structurally related peptides which include melanocyte-stimulating hormone (·-, ß-, and Á-MSH) and adrenocorticotropic hormone (ACTH). The term MC refers to the bioactivity of these peptides, namely, the stimulatory (melanotropic) effect on pigment cells and the steroidogenic (corticotropic) effect on adrenocortical cells. MCs are derived from a single common precursor molecule called POMC. Generation of the MCs involves successive proteolysis of this 31– 36 kDa protein and additional posttranslational modifi-

Markus Böhm, MD Department of Dermatology, Westfälische Wilhelms Universität Von Esmarch-Strasse 56 D–48149 Münster (Germany) Tel. +49 251 835 8635, Fax +49 251 835 6522, E-Mail [email protected]

cations of the peptide fragments [2]. The enzymes sufficient to cleave POMC are the prohormone convertase 1 (PC1) and prohormone convertase 2 (PC2). PC1 generates ACTH and ß-lipotropic hormone (ß-LPH) whereas PC2 is critical for the production of ·-MSH and ß-endorphin (ß-ED) [3]. ·-MSH is a tricecapeptide whose sequence is contained in the amino acids 1–13 of the Nterminal portion of ACTH. N-acetylation and C-amidation is required for ·-MSH to obtain full biologic activity. All MCs share a short peptide motif, His6-Phe7-Arg8-Trp9, which is required for their melanotropic activity [4].

The Skin as a Factory of Melanocortins

MCs were originally discovered as neuropeptides produced by the pituitary gland long time ago. It is now established that the POMC gene is expressed in the skin and cleaved into POMC-derived peptides. Thody and coworkers [5] detected desacetylated, mono- and diacetylated MSH forms in isolated epidermis. In situ hybridisation on normal human skin has revealed POMC mRNA expression in epidermal melanocytes and keratinocytes, endothelial cells and perivascular cells [6]. Immunohistochemical studies have subsequently shown that MCs are expressed in several resident cell types of normal human skin. Accordingly, ·-MSH and ACTH as well as PC1 and PC2 were found to be expressed in keratinocytes, melanocytes, possibly Langerhans cells and some isolated dermal cells [7, 8]. Immunoreactivity for ACTH, ·-MSH and ß-ED was also detected in the human anagen hair follicle where POMC peptides accumulated in the outer root sheath [9]. In addition, cutaneous nerve fibers of the basal layer of the epidermis as well as those located in the upper dermis, close to Merkel cells, around the hair follicle, sweat gland and blood vessels were immunoreactive for Á3-MSH [10, 11]. Immunoreactivity for ·-, ß- and Á3MSH was also detected in the sweat gland and in pilosebaceous orifices [11]. In sebaceous glands, ·-, ß- and Á3MSH immunoreactivity were only detected in regions adjacent to a pathologic process, i.e. cutaneous melanomas suggesting a reactive phenomenon [12]. In culture, normal human keratinocytes, melanocytes, dermal microvascular endothelial cells express the POMC gene and secrete ·-MSH and ACTH into the culture medium [13–15]. These findings are supported by the detection of PC1 and PC2 (or its essential cofactor 7B2) in melanocytes and endothelial cells [15, 16]. Interestingly, PC1 and PC2 colocalized with ·-MSH in the melanosomes of human melanocytes as shown by immunostain-

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ing [16]. There is also recent evidence that normal dermal fibroblasts express POMC at the RNA level [17]. Recently, we could demonstrate that this cell type also expresses PC1 and PC2 in vitro and in situ and can generate POMC peptides in vitro [18]. Expression of MCs by skin cells in culture and their regulation by various natural and artificial stimuli has been subject to a number of studies. In fact, ultraviolet (UV) light has long been speculated to affect the amount of MCs expressed in skin since UV exposure is associated with increased levels of ·-MSH in the peripheral blood and since the human adult pituitary gland lacks the pars intermedia [19]. Among the identified stimuli of POMC gene expression and MC production in human melanocytes, keratinocytes and microvascular dermal endothelial cells are interleukin-1 (IL-1), UVB, tumor necrosis factor-· (TNF-·), and phorbol esters [13–15]. Transforming growth factor-ß supresses POMC mRNA expression [17]. Of note, not all cell types respond similarly to these stimuli [18], suggesting cell type-specific regulation of POMC expression, POMC peptide generation and secretion. In addition to transcriptional regulation of the POMC gene, up-regulators of POMC peptide production such as UVA/B and IL-1 have also been shown to affect PC1 transcription [15]. In summary, these findings indicate that many if not all resident skin cell types can express the POMC gene and are capable of generating MCs. However, there is still little known about several skin cell types such as Merkel cells or sebocytes which deserve further investigation.

The Skin as a Target Organ for Melanocortins

The MCs bind to a family of surface receptors known as the MC receptors (MC-Rs). These receptors belong to the large super-family of G-protein coupled receptors with 7 transmembrane domains and have a homology of 39 to 61% to one another on the amino acid level [20]. All MC-Rs activate adenylate cyclase, resulting in increased intracellular accumulation of cAMP. Five MC-Rs (MC-1 to MC-5) have been cloned so far. They differ in their tissue distribution and relative affinity to the MCs. The MC1R was the first MC-R to be cloned by 2 independent investigative teams from cDNAs of a mouse melanoma cell line and normal human melanocytes, and from a cDNA of a human melanoma, respectively [21, 22]. The human MC-1R binds ·-MSH with the highest affinity albeit ACTH has an almost equal affinity, followed by ßMSH and Á-MSH [22, 23]. In addition to pigment cells,

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normal human keratinocytes [24], microvascular endothelial cells [25] and human dermal fibroblasts [26; Böhm et al., submitted] express MC-1R in vitro. Recently, human mast cells were also found to express MC-Rs; MC1R expression was detectable on the RNA level in both the human mast cell line HMC-1 and skin mast cells. However, less than 10% of HMC-1 cells expressed MC1R on the protein level and in skin mast cells MC-1R protein expression was absent [27]. In normal adult human skin MC-1R immunoreactivity was mainly detected the hair follicle epithelia, sebaceous glands, secretory and ductal epithelia of sweat glands, and some periadnexal mesenchymal cells whereas interfollicular epidermis was largely non-reactive [28]. The in situ MC-1R expression in sebaceous glands is in accordance with novel findings that also demonstrate MC-1R expression in the human sebocyte cell line SZ95 [29; Böhm et al., submitted]. The second MSH receptor subtype, MC-2R, was cloned shortly after the molecular characterisation of MC1R and represents the ‘bona fide’ receptor for ACTH [21, 30]. In contrast to the rest of the MC-Rs, it specifically binds ACTH but no other MC. Although its main physiological localization is the adrenal gland, where ACTH induces glucocorticoid production in the zona fasciculata and mineralocorticoid production in the zona glomerolosa, adipocytes from several mammal species have also been shown to express the MC-2R and MC-5R [31]. If MC-2R is expressed by human pre- or adipocytes is not known. Both the MC-3R and the MC-4R are mainly expressed within the central nervous system. In addition, MC-3R expression was reported in mouse peritoneal macrophages and implicated as a modulator of inflammation [32]. Recently, MC-4R gained increasing attention in the regulation of food intake and MC-4R since mutations were found to be associated with severe obesity [33, 34]. The MC-5R gene was the last of the MC-Rs to be cloned. The mRNA sequence published for MC-5R is a co-linear but truncated form of the sequence published for MC-2R [35]. MC-5R has been mapped to a number of exocrine glands including the preputial gland, lacrimal glands and Harderian gland of mice [36, 37]. Expression of MC-5R in the human sebaceous gland was recently detected in lysates of dissected human sebaceous glands by RT-PCR and Western blotting as well as by immunohistochemistry of normal facial skin employing an antibody directed against a peptide corresponding to 13 amino acids of the N-terminal human MC-5R. This antibody stained human epidermis, hair follicles, eccrine glands and endothelial cells in situ [38]. In addition, immuno-

There is a huge body of data that MCs are important regulators of the coat colour in many vertebrate species including man. Lerner and co-workers described skin darkening upon systemic administration of ·- and ß-MSH already in the early 60s [39]. Subcutaneous administration of synthetic melanocortins also induces skin tanning [40] and excessive endogenous levels of ·-MSH are associated with skin darkening such as in generalised melanosis due to metastatic melanoma [41]. In addition, it is well established that increased systemic levels of ACTH induce skin darkening, e.g., in Nelson syndrome or Addison disease. These pigmentary effects of ·-MSH and ACTH are mediated by the MC-1R expressed on pigment cells whose gene is encoded by the extension locus in the mouse. Mutations of extension uncoupling the receptor from its adenylate cyclase activity lead to defective eumelanin synthesis resulting in a coat colour variant with yellow fur [42]. Interestingly, in human individuals with red hair and fair skin, mutations of the MC-1R have also been reported [43]. Moreover, Arg151Cys, Arg160Trp and Aps294His MC-1R alleles were reported to have a more than 2-fold risk for melanoma [44]. Much progress has been made regarding the biochemical and molecular mechanism underlying the melanotropic effects of MCs; in culture, ·-MSH and ACTH are equipotent in their melanotropic effects on normal human melanocytes whereas Á-MSH is ineffective [23]. The inductive effects of ·-MSH or ACTH on eumelanin synthesis are associated with upregulation of tyrosinase, tyrosinase-related protein-1 (TRP-1) and TRP-2 [45] and appear to be mediated by Microphthalmia protein, a transcriptional activator of tyrosinase and TRP-1 [46]. In addition, ·-MSH also enhances the proliferation of normal human melanocytes in vitro [45]. The in situ presence of ACTH and ·-MSH within the epidermis and the upregulating effects of UV light on their expression strongly supports a role of MCs in the sun tanning response. Moreover, the upregulating effects of distinct pro-inflammatory cytokines on MC expression may be pathogenetically involved in postinflammatory hyperpigmentation. Interestingly, prolonged therapy with minocycline is associated with cutaneous hyperpigmentation which may be related to upregulation of ·-MSH [47].

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staining for MC-5R was detected in vitro by FACS analysis of HMC-1 cells and human skin mast cells [27].

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In the epidermis, MCs may have other functions besides regulation of pigmentation. It was reported that ·-MSH augements the proliferation of HaCaT keratinocytes and suppresses their differentiation-driven heat shock protein 70 expression [48]. Recently, it was reported that ·-MSH is capable of suppressing UV-induced H2O2 generation in HaCaT cells and melanoma cells [49]. ·-MSH may therefore assist in coping of the skin with oxidative stress following UV exposure. Another cutaneous structure being recognised as a target organ for the action of MCs is the pilosebaceous unit [1, 50–52]. However, most of the experimental studies on MCs and their effects on the hair follicle and sebaceous glands are restricted to rodents. For example, ACTH directly induces hair growth in the mink [53]. In the C57BL6 mice, intracutaneous injection of ACTH into telogen skin induces anagen development whereas in anagen skin it induces the premature onset of catagen [51]. ·-MSH also stimulates sebum secretion and lipogenesis in the preputial gland of the rat [52]. MC-5R-deficient mice have been shown to have impaired water repulsion and disturbed thermoregulation due to reduced lipid synthesis by sebaceous glands [37]. In man, however, there is only circumstantial evidence that MCs can regulate hair growth and sebaceous gland activity. Systemic administration of ACTH and increased peripheral blood levels of ACTH can lead to hypertrichosis [1]. Pregnancy and Parkinson’s disease are associated with incrased peripheral blood levels for ·-MSH and seborrhea, whereas in hypopituitarism sebum production is diminished [1, 50]. Recently, we detected an inhibitory effect of ·-MSH on the production of IL-8 by the human SZ95 sebocytes which express receptors for ·-MSH [29; Böhm et al., submitted]. By modulating the expression of this important chemokine, ·-MSH may regulate inflammatory responses in the pilosebaceous unit. The presence of the MC-1R in human dermal fibroblasts adds another dimension to the multifacetted biological activity of MCs. Human dermal fibroblasts in vitro respond to ·-MSH with increased expression of interstitial collagenase/matrix metalloproteinase-1 (MMP-1) [54]. This effect could link UV-induced MC expression to dermal photoageing in which repetitive induction of MMP-1 is one of the key events. Moreover, in a mouse model in which cutaneous fibrosis is elicited by repetitive injection with TGF-ß, ·-MSH suppressed this reaction [26; Böhm et al., submitted]. The mitigating effect of ·MSH on collagen synthesis by fibroblasts may be related to an inductive effect on the expression of IL-8 [55], a down-regulator of collagen type I and III.

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With regard to the skin immune system, it is well established that ·-MSH has potent modulatory activities on a variety of inflammatory and immune responses [56]. Interestingly, the carboxyterminal tripeptide of ·-MSH is sufficient for the immunomodulating activities [7, 57]. ·-MSH supresses the sensitisation and elicitation phase of the cutaneous immune response to contact allergens and induces hapten specific tolerance [58]. Preliminary observations on man show that topically applied ·-MSH also prevents nickel-induced contact dermatitis [1]. The immunomodulating activity of ·-MSH can be explained by effects on cytokine production and adhesion molecule expression by monocytes and antigen presenting cells. Accordingly, ·-MSH upregulates expression and release of IL-10 [59] while IL-1, IL-6 and TNF-· are down-regulated [60]. ·-MSH also down-regulates the expression of MHC class I molecules and significantly suppresses the expression of CD86 and CD40 on monocytes and dendritic cells [61, 62]. In vitro ·-MSH via modulating the function of antigen presenting cells also was found to regulate IgE synthesis of CD40 and IL-4 stimulated B-lymphocytes [63]. On the molecular level, ·-MSH was found to inhibit the activation of NF-ÎB, a transcription factor induced by various inflammatory agents including TNF·, IL-1, LPS and ceramide [64]. This effect of ·-MSH is not cell type-specific and includes keratinocytes and endothelial cells [64–66]. In another mouse model, ·-MSH was capable of suppressing endothelial damage induced by LPS [67]. This effect was associated with reduced expression of intercellular adhesion molecule-1, vascular adhesion molecule-1 and E-selection. Recently, mast cells were also found to be targets for MCs; ·-MSH in vitro inhibited antigen-stimulated histamine release, enhanced IL-3-dependent proliferation and down-modulated mRNA expression of IL1-ß, TNF-· and lymphotactin in murine mast cells [68]. On the other hand, ·-MSH increased histamine release in unstimulated human skin mast cells [27]. Taken together, these data show that the immunomodulatory activities of ·-MSH not only include the classical immunocompetent cells such as monocytes or antigen-presenting cells but also accessory cells such as mast cells or endothelial cells.

Melanocortin and Melanocortin Receptor Expression in Diseased Skin

MCs have been found to be overexpressed in a variety of neoplastic and inflammatory skin disorders including melanocytic naevi, melanoma, basal cell carcinoma, squa-

Böhm/Luger

mous cell carcinoma, keloids, psoriasis, scarring alopecia and acute UV dermatitis [9, 12, 69, 70]. Regarding pigment lesions immunoreactivity of ·-MSH, ACTH and ß-ED was more intense and diffuse in samples of nodular melanoma, vertically growing acral lentiginous melanoma, superficial spreading melanoma and metastatic melanomas compared to nevi. The amount of ·-MSH as determined by RIA was found to be markedly elevated in samples of cutaneous melanomas and lymph node metastases as compared to normal human skin [71]. Increased production of ·-MSH by melanomas may explain elevated ·-MSH serum levels of ·-MSH that correlate with the clinical stage of the disease and the tumor depth [72]. The potential role of MCs has also been investigated in tissue sections from patients with vitiligo. However, immunoreactivity for ·, ß, and Á3-MSH in lesional skin was not reduced as compared to control skin [11]. In contrast to several studies addressing expression of MCs in normal and diseased skin, little is known on the expression of the various MC-Rs in the skin under pathologic conditions. Xia et al. [73] were the first who reported increased MC-1R immunostaining in a human melanoma, which is in accordance with our findings [28]. Interestingly, MC-1R immunoreactivity in the melanoma sections was also detectable in the normal epidermis adjacent to the tumor, suggesting an up-regulating effect by tumor-derived factors. Recently, a novel splice variant

with similar binding affinity as compared to the originally identified MC-1R has been reported in human melanoma [74]. This splicoform has additional 65 amino acids at the C-terminal end of the human MC-1R. It will be interesting to check whether this splice variant has any diagnostic or prognostic value for melanoma, or whether other cutaneous tumors exhibit additional MC-1R splicoforms.

Outlook

The last decade has witnessed a tremendous progress in our knowledge of the MCs and their receptors. In addition to identifying novel biological activities of MCs we are at the beginning to decipher the relevance of the genetic polymorphism for MC-Rs. The presence of MC-Rs on various skin cell types and the pleiotropic effects of MCs have also opened strategies for pharmacological intervention. Some future challenges will be to design selective and powerful agonists and antagonists to the different MC-Rs and to develop strategies for directing these peptides to the desired cell type.

Acknowledgement This work was supported by a grant from the Volkswagenstiftung I/74582 to T.A. Luger.

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52 Thody AJ, Shuster S: Control and function of sebaceous glands. Physiol Rev 1989;69:383– 416. 53 Rose J: Adrenocorticotropic hormone (ACTH) but not alpha-melanocyte stimulating hormone (alpha-MSH) as a mediator of adrenalectomy induced hair growth in mink. J Invest Dermatol 1998;110:456–457. 54 Kiss M, Wlaschek M, Brenneisen P, Michel G, Hommel C, Lange TS, Peus D, Kemeny L, Dobozy A, Scharffetter Kochanek K, Ruzicka T: Alpha-melanocyte stimulating hormone induces collagenase/matrix metalloproteinase-1 in human dermal fibroblasts. Biol Chem Hoppe Seyler 1995;376:425–430. 55 Böhm M, Schulte U, Kalden DH, Luger TA: Alpha-melanocyte-stimulating hormone modulates activation of NF-ÎB and AP-1 and secretion of IL-8 in human dermal fibroblasts. Ann NY Acad Sci 1999;885:277–286. 56 Luger TA, Kalden D, Scholzen TE, Brzoska T: Alpha-melanocyte-stimulating hormone as a mediator of tolerance induction. Pathobiology 1999;67:318–321. 57 Lipton JM, Catania A: Antiinflammatory actions of the neuroimmunomodulator ·-MSH. Immunol Today 1997;18:140–145. 58 Grabbe S, Bhardwaj RS, Steinert M, Mahnke K, Simon MM, Schwarz T, Luger TA: Alphamelanocyte stimulating hormone induces hapten-specific tolerance in mice. J Immunol 1996;156:473–478. 59 Bhardwaj RS, Schwarz A, Becher E, Mahnke K, Riemann H, Aragane Y, Schwarz T, Luger TA: Pro-opiomelanocortin-derived peptides induce IL-10 production in human monocytes. J Immunol 1996;156:2517–2521.

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60 Lipton JM, Catania A: Mechanisms of antiinflammatory action of the neuroimmunomodulatory peptide alpha-MSH. Ann NY Acad Sci 1998;840:373–380. 61 Bhardwaj RS, Becher E, Mahnke K, Hartmeyer M, Schwarz T, Scholzen T, Luger TA: Evidence for the differential expression of the functional alpha melanocyte stimulating hormone receptor MC-1 on human monocytes. J Immunol 1997;158:3378–3384. 62 Becher E, Mahnke K, Brzoska T, Kalden DH, Grabbe S, Luger TA: Human peripheral bloodderived dendritic cells express functional melanocortin receptor MC-1R. Ann NY Acad Sci 1999;885:188–195. 63 Aebischer I, Stämpfli MR, Zürcher A, Miescher S, Urwyler A, Frey B, Luger TA, White RR, Stadler BM: Neuropeptides are potent modulators of human in vitro immunoglobulin E synthesis. Eur J Immunol 1994;24:1908– 1913. 64 Manna SK, Aggarwal BB: Alpha-melanocytestimulating hormone inhibits the nuclear transcription factor NF-kappa B activation induced by various inflammatory agents. J Immunol 1998;161:2873–2880. 65 Brzoska T, Kalden DH, Scholzen T, Luger TA: Molecular basis of ·-MSH/IL-1 antagonism. Ann NY Acad Sci 1999;885:230–238. 66 Kalden DH, Scholzen T, Brzoska T, Luger TA: Mechanisms of the antiinflammatory effects of ·-MSH: Role of transcription factor NF-ÎB and adhesion molecule expression. Ann NY Acad Sci 1999;885:254–261. 67 Sunderkötter C, Kalden DH, Brzoska T, Sorg C, Luger TA: ·-MSH reduces vasculitis in the local Shwartzman reaction. Ann NY Acad Sci 1999;885:414–418. 68 Adachi S, Nakano T, Vliagoftis H, Metcalfe DD: Receptor-mediated modulation of murine mast cell function by alpha-melanocyte stimulating hormone. J Immunol 1999;163:3363– 3368.

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Prevalence of Endocrine Dysfunction in HIV-Infected Men Norbert H. Brockmeyer Alexander Kreuter Arnim Bader Ute Seemann Georg Reimann Department of Dermatology, Ruhr-University, Bochum, Germany

Key Words Endocrine dysfunction W HIV-1 W Gonadal function W Adrenocortical function W Thyroid function W Tubular insufficiency W Hypergonadotropic hypogonadism

mary or tertiary origin, a substitution of hormones should be taken into consideration. Copyright © 2001 S. Karger AG, Basel

Introduction Abstract Objective: Endocrine dysfunction is a common problem in patients with human immunodeficiency virus infection (HIV). We therefore evaluated the endocrine function in 31 male homosexual HIV-1-infected men: mean age 37 B 7.2 years (range 24–52). Methods and Materials: Blood was obtained for baseline T3, T4, TSH, LH, FSH, prolactin, testosterone, ACTH and cortisol values. Endocrine function tests were performed as TRH, CRH, ACTH, LH-RH and HCG tests. Results: Thyroid function: There was a temporarily increased TSH in 3 of 17 patients but normal levels for T3, T4 and fT4 (without thyroid antibodies). One patient showed signs of latent hyperthyroidism (no response in TRH test). Adrenocortical function: Two patients had adrenal insufficiency. They showed a normal baseline cortisol level, an elevated ACTH level and no increase in cortisol levels after stimulation with CRH. All other patients revealed normal responses on the CRH/ACTH tests. Gonadal function: 9 patients had elevated FSH levels (tubular insufficiency), 4 patients additionally had increased LH levels (hypergonadotropic hypogonadism). 5 patients showed signs of tertiary hypogonadism (low LH and testosterone, increase of LH after stimulation with LH-RH). Conclusion: In disorders of thyroid and adrenocortical function of pri-

ABC

© 2001 S. Karger AG, Basel 0301–0163/00/0546–0294$17.50/0

Fax + 41 61 306 12 34 E-Mail [email protected] www.karger.com

Accessible online at: www.karger.com/journals/hre

Aquired immunodeficiency syndrome (AIDS) is a severe multivisceral and life-threatening affection. Also in the age of highly active antiretroviral therapy (HAART) human immunodeficiency virus (HIV) and resulting consequences remain a major challenge. Lung, brain and digestive complaints that result in opportunistic infections and neoplasm’s are the most documented. Alterations in endocrine function are also observed in HIVinfection. Direct destruction of endocrine organs by the virus itself or by invasive infection with opportunistic organisms (e.g. CMV, toxoplasmosis) as well as side effects caused by antiretroviral therapy have been reported. There exists no detailed data about the exactly prevalence of endocrine dysfunction in HIV. However, the number of patients in the present studies are to small to draw final conclusions.

Material and Methods We initiated this clinical trial to evaluate endocrine function in 31 male homosexual HIV-1 positive patients. Patients were grouped according to the CDC criteria (4 CDC A2, 5 CDC B2, 2 CDC B3, 7 CDC C2, 13 CDC C3). Mean age was 37 B 7.2 (range 24–52 years). All patients were naive to antiretroviral therapy. Examinations were

Prof. Dr. N.H. Brockmeyer Department of Dermatology, Ruhr-University Bochum Gudrunstrasse 56, D–44791 Bochum, (Germany) Tel. +49 234 509 3470, Fax +49 234 509 3475 E-Mail [email protected]

performed before initiating HAART. Blood was obtained for baseline T3, T4, TSH, LH, FSH, prolactin, testosterone, ACTH, and cortisol. Endocrine function tests were performed as TRH-, CRH-, ACTH-, LH-RH-, and HCG-tests.

Results

Thyroid function revealed no major abnormalities. TSH was temporarily increased in 3/17 patients but levels for T3, T4, fT4 (without thyroid antibodies) were within the normal range. Only one patient showed signs of latent hyperthyroidism (no response in TRH-test). Concerning adrenocortical function, two patients had adrenal insufficiency. They showed a normal baseline cortisol level, an elevated ACTH level and no increase of cortisol levels after stimulation with CRH. All other patients revealed normal responses on CRH-/ACTH-test. Only gonadal function revealed pathologic parameters, nine patients had elevated FSH levels consistent with tubular insufficiency. Four patients additionally had increased LH levels showing a hypergonadotropic hypogonadism. Five patients showed signs of a tertiary hypogonadism (low LH and testosterone, increase of LH after stimulation with LH-RH).

Discussion

Under HAART, several adverse drug reactions have been observed. Lipodystrophy syndrome, consisting of peripheral lipodystrophy, central adiposity, dyslipidaemia and insulin resistance has been described with a prevalence up to 80% [1]. Drug-induced mitochondrial dysfunction as well as cytokines and hormones have been suggested as an important pathophysiological origin. Morphologic and metabolic characteristics of lipodystrophy are similar to those of the mitochondrial disorder multiple symmetric lipomatosis, suggesting that mitochondrial toxicity may be the main mechanism [2].

A French group showed, that serum cholesterol, very low density lipoproteins (VLDL), high density lipoproteins (HDL), low density lipoproteins (LDL), triglycerides, and ApoB:apolipoprotein A1 (ApoB) were significantly increased in lipodystrophy-positive compared with lipodystrophy-negative patients. Serum dehydroepiandrosterone (DHEA) was significantly lower, cortisol: DHEA ratio was increased. The association between cortisol:DHEA ratio, lipid alterations and lipodystrophy leads to the speculation of an imbalance between peripheral lipolysis and lipogenesis [3]. Another group compared accelerated bone mineral loss in HIV-infected patients on HAART including a protease inhibitor (PI), HIV-infected patients without PI and healthy seronegatives [4]. They showed, that osteopenia and osteoporosis are unique complications in the treatment with PI, appearing to be independent of adipose tissue maldistribution. In conclusion, our data on a large serial investigation indicate, that thyroid and adrenocortical dysfunction is not increased in HIV-infection. Thus, hypogonadism occurs frequently in HIV-infected men. A decreased plasma concentration of total and free testosterone in 35 and 26% of patients has been described [5]. The non-specific response to the stress led by the disease does not give an explanation to the observed abnormality. The aetiology and complete clinical significance of these changes continues to be investigated. A substitution of testosterone should be discussed. An other study compared endocrine function in patients with HIV wasting syndrome with other HIV-positive patients without wasting. Total and free testosterone levels were significantly lower in patients with wasting [6]. The sometimes non-specificity of the clinical manifestations of endocrine dysfunction masked by the classic signs of AIDS and HAART, should not make forget the systematic search of glandular abnormalities as soon as a symptomatology is conspicuous. The recognition of endocrine disorders is essential for optimal therapy because, unknown, their evolution is dramatic.

References 1

2

Behrens GM, Stoll M, Schmidt RE: Lipodystrophy syndrome in HIV infection: What is it, what causes it and how can it be managed? Drug Saf 2000;23:57–76. Kakuda TN: Pharmacology of nucleoside and nucleotide reverse transcriptase inhibitor-induced mitochondrial toxicity. Clin Ther 2000; 22:685–708.

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Christeff N, Melchior JC, de Truchis P, Perronne C, Nunez EA, Gougeon ML: Lipodystrophy defined by a clinical score in HIV-infected men on highly active antiretroviral therapy. 1999;13:2251–2260. Tebas P, Powderly WG, Claxton S, Marin D, Tantisiriwat W, Teitelbaum SL, Yarasheski KE: Accelerated bone mineral liss in HIVinfected patients receiving potent antiretroviral therapy. AIDS 2000;14:63–67.

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Targeted Somatic Mutagenesis in Mouse Epidermis Arup Kumar Indra 1 Mei Li 1 Jacques Brocard Xavier Warot Jean-Marc Bornert Christelle Gérard Nadia Messaddeq Pierre Chambon Daniel Metzger Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Collège de France, Illkirch, France

Key Words Somatic mutagenesis W Cre/Lox W Tamoxifen W Keratinocytes W Epidermis W Hair follicle W Mouse

Abstract Gene targeting in the mouse is a powerful tool to study mammalian gene function. The possibility to efficiently introduce somatic mutations in a given gene, at a chosen time and/or in a given cell type will further improve such studies, and will facilitate the generation of animal models for human diseases. To create targeted somatic mutations in the epidermis, we established transgenic mice expressing the bacteriophage P1 Cre recombinase or the tamoxifen-dependent Cre-ERT2 recombinase under the control of the human keratin 14 (K14) promoter. We show that LoxP flanked (floxed) DNA segments were efficiently excised in epidermal keratinocytes of K14-Cre transgenic mice. Furthermore, Tamoxifen administration to adult K14-Cre-ERT2 mice efficiently induced recombination in the basal keratinocytes, whereas no background recombination was detected in the absence of ligand treatment. These two transgenic lines should be very useful to analyse the functional role of a number of genes expressed in keratinocytes. Copyright © 2001 S. Karger AG, Basel

1

These authors contributed equally to this work.

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Introduction

The skin is a complex organ composed of the epidermis and its appendages (hair follicles) which are separated from the dermis by a basement membrane. The epidermis is a highly dynamic stratified epithelium made principally of keratinocytes [1]. The inner most basal cells that are attached to the basement membrane form a proliferative layer, from which keratinocytes periodically withdraw from the cell cycle and commit to terminally differentiate, while migrating into the next layers known as the spinous and granular layers, which together represent the suprabasal layers. Terminally differentiated keratinocytes or squames form the cornified layer or corneum. Squamous keratinocytes are lost daily from the surface of the skin, and are continuously replaced by newly differentiating cells. Hair follicles that develop through a series of mesenchymal-epithelial interactions during embryogenesis are also dynamic structures. They are also principally composed of keratinocytes, and their outer root sheath (ORS) is contiguous with the epidermis. Once formed, hair follicles periodically undergo cycles of regression, rest and growth, through which old hairs are eventually replaced by new ones [2, 3]. Homologous recombination in embryonic stem cells and transgenesis have made it possible to specifically target and mutate any genetic element in the germ line of mice [4,5]. Using these techniques, it is now possible to generate more or less at will mutations which result either

Pierre Chambon IGBMC, BP 163 F–67404 Illkirch Cedex (France) Tel. +33 3 88 65 32 13/15/10, Fax +33 3 88 65 32 03 E-Mail [email protected]

K14-Cre-ERT2 transgenic mice were sufficient to induce the site-specific genetic modification in all keratinocytes of the epidermis and outer root sheath of the hair follicles for several months, thus showing that recombination occured in most if not all the epidermal stem cells. These tools should be very useful to study epidermal gene function.

in gene inactivation (knock out), overexpression or more discrete predetermined gene changes, which are all useful to assess physiological and pathological gene functions. For instance, mutations aimed at mimicking those observed in inherited or acquired diseases have been engineered in the germ line of mice to generate models of human diseases. There are, however, some serious limitations to this strategy, which are essentially due to the introduction of the gene modification into the germ line. For instance, a germ line mutation can be lethal, cause complex pleiotropic effects or, on the contrary, its effects could be fully compensated during development. In many instances this will prevent the determination of the role of a given gene product in a defined subset of cells at a given time of the animal life, and also the establishment of mouse model systems for human diseases generated by somatic mutations [6, 7]. To overcome these limitations, strategies for conditional gene targeting in mice, based on cell-type specific or inducible expression of the bacteriophage P1 Cre recombinase, have recently been developed [8–10 and refs therein]. Indeed, the Cre recombinase can efficiently excise a DNA segment flanked by two loxP sites (floxed DNA) in animal cells. Placing the Cre gene under the control of either a cell-specific or an inducible promoter can lead, through the excision of the floxed DNA segment, to either spatially or temporally controlled somatic mutations, respectively. To generate somatic mutations in a defined gene, at a given time of the life of the animal and in a specific cell type, we have recently generated conditional tamoxifen-inducible Cre recombinases by fusing Cre with mutated ligand binding domains of the human estrogen receptor (Cre-ERT and Cre-ERT2) which bind tamoxifen (Tam), but not estrogens [11, 12]. We have shown that floxed DNA segments are efficiently excised in vivo, after administration of Tam to transgenic mice expressing Cre-ERT or Cre-ERT2 [11, 13–17]. To create targeted somatic mutations in the epidermis, with and without temporal control, we established transgenic mice expressing Cre-ERT2 and Cre, respectively, under the control of the human keratin 14 (K14) promoter, which is selectively active in the epidermal layer during embryogenesis and is essentially restricted to keratinocytes of the basal layer of the epidermis and outer root sheath of the hair follicle in adult animals [18, 19]. We show that floxed DNA sequences are efficiently excised in keratinocytes during skin formation of K14-Cre fetuses, and that floxed DNA sequences were excised in all keratinocytes of the epidermis and outer root sheath of adult animals. Furthermore, five days of Tam treatment of

To introduce somatic mutations in the forming epidermis, we engineered transgenic mice expressing the Cre recombinase under the control of the human keratin 14 (K14) promoter (fig. 1A) [20], that is active at the body surface of the mouse embryo as early as 9.5 dpc and is strongly upregulated by 14.5 dpc [18, 19, 21]. In postnatal mice, its activity is essentially restricted to the dividing basal layer keratinocytes of the epidermis, the outer root sheath (ORS) of hair follicles, and some other stratified squamous epithelia, e.g. oral and tongue epithelia [22]. Three K14-Cre transgenic founder animals were identified by PCR and Southern blot analysis of tail DNA. To analyse the recombinase activity, these lines were bred with RXR·+/af2(I) Cre reporter mice, which contain one WT RXR· allele and one RXR· allele carrying a floxed marker gene in the intron located between exon 9 and 10 (RXR·+/af2(I), fig. 1B) [23]. The WT RXR· allele (+) and the recombined RXR· af2(I) allele [RXR· af2(II)] can be simultaneously detected by polymerase chain reaction (PCR), using one set of primers (fig. 1B). PCR analysis of DNA extracted from various organs isolated from 6week-old RXR·+/af2(I) mice harboring a K14-Cre transgene [K14-Cre(tg/0)/RXR·+/af2(I)] revealed the presence of high levels of the RXR· af2(II) allele in epidermis, as well as in tongue and salivary gland, and of lower levels in the

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Materials and Methods Transgenic Lines The K14-Cre, K14-Cre-ERT2, RXR·+/af2(I) and ROSAfl/+ Cre reporter line (R26R) were as described [17, 20, 23, 24]. Mice were genotyped as described [11, 20]. Tamoxifen (Tam) was prepared and administred to mice according to Metzger and Chambon [10]. Histochemistry ß-Galactosidase histochemistry was performed on 10-Ìm-thick frozen sections, stained with X-Gal (5-bromo-4-chloro-3-indolyl ß-Dgalactoside), or on 2 Ìm semi-thin sections as described [13]. Cre immunohistochemistry was performed according to Brocard et al. [13].

Results and Discussion Efficient Cre-Mediated Recombination in Epidermal Keratinocytes of K14-Cre Transgenic Mice

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staining was already seen in 15.5 dpc fetal epidermis (data not shown), and remained homogenous in epidermis and hair follicles of adult double transgenic mice (fig. 2B, compare c and d), confirming the efficiency of Cremediated recombination. Thus, floxed DNA segments are efficiently and selectively deleted in interfollicular epidermis and hair follicles of K14-Cre transgenic animals.

Fig. 1. Characterization of Cre recombinase activity in K14-Cre transgenic lines. A Structure of the K14-Cre transgene [20]. The human K14 promoter, the Cre coding sequence and the simian virus 40 polyadenylation signal (polyA) are represented by black, grey and open boxes. The rabbit ß-globin intron is depicted by a line. B Genomic structure of the RXR· WT, the RXR· af2(I) target allele and the recombined RXR· af2(II) allele [23], and PCR strategy (primers 3 and 4; [11]) to analyse Cre-mediated excision of the floxed marker. C PCR detection of Cre-mediated DNA excision in mice. PCR was performed on DNA isolated from the indicated organs of 6 week-old K14-Cre(tg/0)/RXR·+/af2(I) mice. The position of the PCR products amplified from the WT RXR· allele (+) and the RXR· af2(II) allele are indicated.

eye, whereas no recombination could be detected in other tissues of all three lines (fig. 1C; and data not shown). To further characterize these lines, K14-Cre transgenic mice were bred to ‘floxed’ ROSA26 Cre reporter transgenic mice (R26R in ref. 24; called thereafter ROSAfl/+) to yield K14-Cre(tg/0)/ROSAfl/+ double transgenic mice, in which translation of the ß-galactosidase of the broadly active Cre reporter transgene occurs only upon Cremediated DNA excision (fig. 2A). X-Gal staining performed on newborns of all three double transgenic lines revealed strong ß-galactosidase activity in epidermis and hair follicles (fig. 2B, compare a and b; and data not shown). X-Gal staining could also be detected in tongue and salivary gland epithelial cells, but not in other tissues (data not shown). Semi-thin section analysis showed that it was uniformly distributed in the epidermis of one of the double transgenic line, whereas it was patchy in the newborn and adult epidermis of the other transgenic lines (data not shown). In the best line, an homogenous X-Gal

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Efficient Tam-Induced Cre-Mediated Recombination in Epidermal Keratinocytes of Adult K14-Cre-ERT2 Transgenic Mice To perform temporally-controlled somatic mutagenesis in epidermis, we established K14-Cre-ERT2 transgenic mice expressing the Cre-ERT2 recombinase (induced upon treatment with low levels of Tam [14]) under the control of the human keratin K14 promoter (fig. 3A). Three transgenic lines were identified by PCR and Southern blot analysis of tail DNA. To characterize the expression pattern of the chimeric recombinase in the skin, immunohistochemistry analyses were performed on frozen tail skin sections from 8 week-old Tam-treated Cre-ERT2 animals, using confocal microscopy and an anti-Cre antibody and DAPI to stain cell nuclei. Basal cells were specifically Cre-positive in the epidermis of all three transgenic lines (fig. 3B, and data not shown). One of these lines exhibiting Cre-ERT2 expression in essentially all the basal keratinocytes was selected for further studies. To characterize its recombinase activity, hemizygous Cre-ERT2 mice were crossed with ROSAfl/+ mice to yield K14-Cre-ERT2(tg/0)/ROSAfl/+ double transgenic mice. Eight week-old animals were injected for 5 days with 0.1 mg Tam, and X-Gal staining was performed on tail skin sections at various times. Whereas no X-Gal staining could be detected in the skin of untreated or vehicle-treated K14Cre-ERT2(tg/0)/ROSAfl/+ mice, some keratinocytes of the epidermis and outer root sheath of hair follicle were X-Gal stained 5 days after the beginning of Tam injection. Furthermore, 30 and 60 days after Tam treatment robust XGal staining was observed in all keratinocytes of the epidermis and outer root sheath of hair follicle (fig. 3C, and data not shown). As suprabasal cells are renewed in 7–10 days in the mouse tail epidermis, these results show that recombination had occurred in most if not all epidermal stem cells. Importantly, the lack of X-Gal staining in the absence of ligand treatment clearly indicates that the recombinase activity of Cre-ERT2 is fully dependent on Tam binding. To verify that Cre-mediated floxed DNA excision also occured in other epithelia in which K14 promoter is active, K14-Cre-ERT2(tg/0) mice were bred with RXR·+/af2(I) reporter mice. As expected, conversion of the

Indra/Li/Brocard/Warot/Bornert/Gérard/ Messaddeq/Chambon/Metzger

Fig. 2. Characterization of Cre recombinase activity in skin of a K14-Cre transgenic line. A Structure of the fl allele

before and after Cre-mediated recombination [24]. SA, splice acceptor site; neo 4xpA, neomycin resistance cassette with 4 polyadenylation sites. B X-Gal staining of 10-Ìm skin sections taken from the back region of newborn (a, b) and 12-week-old (c, d) mice. a, c Transgenic ROSAfl/+; b, d double transgenic K14-Cre(tg/0)/ROSAfl/+. Sections were counterstained with safranin. Arrows point to the dermal-epidermal junction. hf, hair follicle. Scale bar, 16 Ìm.

Fig. 3. Characterization of Tam-induced Cre recombinase activity in the skin of an K14-Cre-ERT2 transgenic line. A Structure of the K14Cre-ERT2 transgene [17]. B Pattern of Cre-ERT2 expression in the tail

epidermis of K14-Cre-ERT2 transgenic mice. Immunohistochemistry with anti-Cre antibody was performed on 10-Ìm-thick sections of tail biopsies taken from 8 week-old K14-Cre-ERT2 transgenic mice one day after the last Tam injection (1 mg for 5 consecutive days). The red color corresponds to the staining of Cre-ERT2, and the blue color corresponds to the DAPI staining of the nuclei. The white color of the basal keratinocyte nuclei results from the superimposition of the red color of the anti-Cre signal and the blue color of the DAPI staining. B and S, basal and suprabasal layers, respectively. Scale bar, 25 Ìm. C Kinetics of ß-galactosidase expression in tail epidermis of K14-Cre-ERT2(tg/0)/ROSAfl/+ bigenic mice. Eight-week-old K14Cre-ERT2(tg/0)/ROSAfl/+ bigenic mice were injected daily with Tam (0.1 mg) from day 0 to 4. Tail biopsies were collected just before the first Tam injection (a) and 5 (b), 30 (c) and 60 (d) days after Tam treatment, and 10-Ìm frozen sections were X-Gal stained. D and E, dermis and epidermis, respectively. hf, hair follicle. Scale bar, 16 Ìm. D PCR detection of Cre-ERT2-mediated DNA excision in mice. Eight-week-old K14-Cre(tg/0)/RXR·+/af2(I) mice were injected daily for 5 days with 0.1 mg Tam. PCR was performed on DNA isolated from the indicated organs one day after the last Tam injection. The position of the PCR products amplified from the WT RXR· allele (+) and the RXR· af2(II) allele is indicated.

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RXR· af2(I) allele into RXR· af2(II) allele was observed not only in the skin and tail, but also in the tongue, salivary gland, eye, stomach and oesophagus of 8-week-old Tamtreated K14-Cre-ERT2(tg/0)/RXR·+/af2(I) mice, whereas no excision was detected in vehicle-treated control animals (fig. 3D, and data not shown).

Conclusion

of the epidermis and hair follicle ORS. The possibility to efficiently induce Cre-ERT2 recombinase activity at 10fold lower Tam doses than those required to efficiently induce the chimeric recombinase in the previously described K14-CreERtam mice [19] should facilitate the establishment of animal models for skin diseases [17].

Acknowledgements

The K14-Cre transgenic line described in this study allows efficient Cre-mediated recombination in epidermal keratinocytes. Recombination occured before 15.5 dpc, resulting in floxed DNA excision in all keratinocytes of the epidermis and outer root sheet (ORS) of the hair follicle in new born and adult mice, in agreement with results obtained with a similar line described by Vasioukhin et al. [19]. These lines will be valuable to study gene function during skin morphogenesis. Furthermore, efficient Cre-mediated recombination was induced in the keratinocytes of the basal layer and ORS of hair follicle by treatment of K14-Cre-ERT2 adult mice with low levels of Tam, thus allowing to produce mice in which a specific gene is selectively ablated in the adult in all keratinocytes

We are grateful to P. Soriano and B. Mascrez for Rosa R26R and RXR·+/af2(I) mice, respectively, and to Y. Lutz and M. Oulad-Abdelghani for biotin-conjugated anti-Cre antibody. We thank S. Bronner, N. Chartoire and R. Lorentz for technical assistance, as well as the staff of the animal facility. This work was supported by funds from the Centre National de la Recherche Scientifique, the Institut National de la Santé et de la Recherche Médicale, the Collège de France, the Hôpital Universitaire de Strasbourg, the Association pour la Recherche sur le Cancer, the Fondation pour la Recherche Médicale, the Human Frontier Science Program, the Ministère de l’Éducation Nationale de la Recherche et de la Technologie and the European Economic Community. M.L. was supported by fellowships from the Association pour la Recherche sur le Cancer and the Fondation pour la Recherche Médicale, A.K.I. by a fellowship from the Université Louis Pasteur (Strasbourg), and J.B. and X.W. by fellowships from the Ministère de l’Education Nationale, de la Recherche et de la Technologie and from the Fondation pour la Recherche Médicale.

References 1 Fuchs E: Of mice and men: Genetic disorders of the cytoskeleton. Mol Biol Cell 1997;8:189– 203. 2 Hardy MH: The secret life of the hair follicle. Trends Genet 1992;8:55–61. 3 Paus R, Cotsarelis G: The biology of hair follicles. New Eng J Med 1999;341:491–497. 4 Capecchi MR: The new mouse genetics: Altering the genome by gene targeting. Trends Genet 1989;5:70–76. 5 Jaenisch R: Transgenic animals. Science 1988; 240:1468–1474. 6 Rajewsky K, Gu H, Kühn R, Betz UAK, Müller W, Roes J, Schwenk F: Conditional gene targeting. J Clin Invest 1996;98:600–603. 7 Olson EN, Arnold HH, Rigby PWJ, Wold BJ: Know your neighbors: Three phenotypes in null mutants of the myogenic bHLH gene MRF4. Cell 1996;85:1–4. 8 Nagy A: Cre recombinase: The universal reagent for genome tailoring. Genesis 2000;26: 99–109. 9 Metzger D, Feil R: Engineering the mouse genome by site-specific recombination. Curr Opin Biotechnol 1999;10:470–476. 10 Metzger D, Chambon P: Site- and time-specific gene targeting in the mouse. Methods 2001;24: 71–80. 11 Feil R, Brocard J, Mascrez B, LeMeur M, Metzger D, Chambon P: Ligand-activated sitespecific recombination in mice. Proc Natl Acad Sci USA 1996;93:10887–10890.

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12 Feil R, Wagner J, Metzger D, Chambon P: Regulation of Cre recombinase activity by mutated oestrogen receptor ligand-binding domains. Biochem Biophys Res Comm 1997;237:752– 757. 13 Brocard J, Warot X, Wendling O, Messaddeq N, Vonesch JL, Chambon P, Metzger D: Spatio-temporally controlled site-specific somatic mutagenesis in the mouse. Proc Natl Acad Sci USA 1997;94:14559–14563. 14 Indra AK, Warot X, Brocard J, Bornert JM, Xiao JH, Chambon P, Metzger D: Temporallycontrolled site-specific mutagenesis in the basal layer of the epidermis: Comparison of the recombinase activity of the tamoxifen-inducible Cre-ERT and Cre-ERT2 recombinases. Nucl Acid Res 1999;27:4324–4327. 15 Imai T, Chambon P, Metzger D: Inducible sitespecific somatic mutagenesis in mouse hepatocytes. Genesis 2000;26:147–148. 16 Imai T, Jiang M, Chambon P, Metzger D: Impaired adipogenesis and lipolysis in the mouse upon Cre-ERT2-mediated selective ablation of RXR· in adipocytes. Proc Natl Acad Sci USA 2001;98:224–228. 17 Li M, Indra AK, Warot X, Brocard J, Messaddeq N, Kato S, Metzger D, Chambon P: Skin abnormalities generated by temporally controlled RXR· mutations in mouse epidermis. Nature 2000;407:633–636.

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18 Byrne C, Tainsky M, Fuchs E: Programming gene expression in developing epidermis. Development 1994;120:2369–2383. 19 Vasioukhin V, Degenstein L, Wise B, Fuchs E: The magical touch: Genome targeting in epidermal stem cells induced by tamoxifen application to mouse skin. Proc Natl Acad Sci USA 1999;96:8551–8556. 20 Li M, Chiba H, Warot X, Messaddeq N, Gérard C, Chambon P, Metzger D: RXR· ablation in skin keratinocytes results in alopecia and epidermal alterations, Development 2001; 128:675–688. 21 Vassar R, Rosenberg M, Ross S, Tyner A, Fuchs E: Tissue-specific and differentiationspecific expression of a human K14 keratin gene in transgenic mice. Proc Natl Acad Sci USA 1989;86:1563–1567. 22 Wang X, Zinkel S, Polonsky K, Fuchs E: Transgenic studies with a keratin promoter-driven growth hormone transgene: Prospects for gene therapy. Proc Natl Acad Sci USA 1997;94: 219–226. 23 Mascrez B, Mark M, Dierich A, Ghyselinck N B, Kastner P, Chambon P: The RXR· liganddependent activation function 2 (AF-2) is important for mouse development. Development 1998;125:4691–4707. 24 Soriano P: Generalized lacZ expression with the ROSA26 Cre reporter strain. Nature Genet 1999;21:70–71.

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Interaction of Vitamin D and Retinoid Receptors on Regulation of Gene Expression Ana Maria Jimenez-Lara 1 Ana Aranda Instituto de Investigaciones Biomédicas ‘Alberto Sols’, CSIC-UAM, Madrid, Spain

Key Words Vitamin D W Retinoic acid W Hormone response elements W Gene expression

Abstract Vitamin D and retinoic acid (RA) receptors (VDRs and RARs, respectively), bind as heterodimers with the retinoid X receptor (RXR) to hormone response elements (HREs) in target genes. In some cases RA and vitamin D can cooperate to stimulate transcription through the same HRE. However, VDR/RXR heterodimers bind in a transcriptionally unproductive manner and without a defined polarity on certain RA response elements, and under these circumstances vitamin D inhibits the response to RA. Although competition for binding to DNA may contribute to this inhibitory response, titration of common coactivators by VDR also appears to be involved in transrepression. Therefore, the transcriptional response to RA and vitamin D depends on a complex combinatory pattern of interaction among different receptors wih DNA and coactivators. Copyright © 2001 S. Karger AG, Basel

1 Present address: Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Illkirch, France.

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The nuclear receptors for vitamin D (VDR) and retinoic acid (RAR) are ligand-inducible transcription factors which bind, preferentially as heterodimers with the retinoid receptor RXR [1], to DNA sequences known as hormone response elements (HREs); VDREs in the case of VDR and RAREs in the case of RAR. Transactivation by nuclear receptors depends on the presence of a highly conserved ligand-dependent transcriptional activation function (AF2) located at the C-terminus which is responsible for ligand-dependent recruitment of a multisubunit coactivator complex which allows chromatin remodeling and creates a permissive state for promoter activation [2, 3]. VDREs and RAREs typically consist of two PuGG/ TTCA motifs configured as palindromes or direct repeats (DRs). VDR preferentially mediates ligand-dependent transactivation via a DR separated by 3 nucleotides (DR3), whereas RAR transactivates via DR2 or DR5 elements [4, 5]. Although the orientation and spacing of the half-sites can determine selective transcriptional responses, specificity is not total and some elements can bind different heterodimers. Furthermore, although binding to the HRE is a prerequisite for transcriptional stimulation, only a subset of DNA sequences which act as high affinity receptor binding elements function as response elements [6–8]. Since DR elements are inherently asymmetric, heterodimers may bind to them with two distinct

Ana Aranda Instituto de Investigaciones Biomédicas Arturo Duperier 4 E–29029 Madrid (Spain) Tel. +34 91 5854642, Fax +34 91 5854587, E-Mail [email protected]

RA-dependent transactivation of the RAR-ß2 or growth hormone promoters due to transcriptional interference of the VDR on the DNA elements responsible for transactivation by RAR. Competition of inactive RXR/VDR heterodimers with active RXR/RAR heterodimers for binding to DNA, as well as titration by VDR of limiting amounts of coactivators required for transactivation by RA, appear to be involved in transrepression by vitamin D.

Materials and Methods Plasmids and Transfections DR5-tk-CAT consists of a copy of the RARE present in the human RARß2 promoter [9] between positions –53/–37 (5)-GTCGAGGGTAGGGTTCACCGAAAGTTCACTCGTCGAC-3)) ligated in front of the thymidine kinase promoter of pBL-CAT8+. In DR4tk-CAT and DR3-tk-CAT, one or two oligonucleotides, respectively, between both hemi-sites of the element have been deleted [8]. Reporter CAT plasmids containing the rat growth hormone gene promoter (from nucleotide –530 to +7) or the human RARß2 promoter (from nucleotide –124 to +14), as well as expression vectors for human VDR, VDR mutants, RXR· and RAR· cloned in pSG5 have been described previously [6–8, 10, 11]. GH4C1 cells were transfected by electroporation and Cos-7 cells were transfected with calcium phosphate as reported [9]. CAT activity was determined in untreated cells and in cells incubated with 1 ÌM RA and/or 100 nM vitamin D for 48 h. The results are expressed as the mean B standard deviation of the CAT values obtained.

Fig. 1. Transcriptional activity of vitamin D and RA on various response elements. a Pituitary GH4C1 cells were transfected with

10 Ìg of thymidine kinase-CAT reporter plasmids containing the DR5 element of the RARß2 gene (ßRARE), or the same element separated by 4 or 3 nucleotides (DR4 and DR3, respectively). b The cells were transfected with a plasmid containing the human RARß2 promoter ligated to CAT. The position of the ßRARE is indicated. In c the cells were transfected with the rat growth hormone promoter that contains a palindromic RARE. After transfection the cells were treated with RA and/or vitamin D (Vit.D, black bars) as indicated, and the level of CAT activity was determined.

polarities. Indeed, it has been established that on DR3 and DR5, RXR occupies the 5) half-site and the heterodimeric partner (e.g. VDR and RAR, respectively) occupies the 3) half-site [5]. We have analyzed transcriptional responses to vitamin D and RA in cells which coexpress VDR, RAR and RXR. RA and vitamin D cooperate to stimulate transcription through DR3 elements. However, vitamin D represses

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Mobility Shift Assays and Crosslinking Gel retardation assays were performed with in vitro translated VDR, RAR and RXR and the oligonucleotide: 5)-GGGTAGGGTTCACCGAAAGTTCACTCG-3) as described [9]. Gapped oligonucleotides containing BrdU residues were used for crosslinking experiments [8]. The DR5 probe consisted in a 5) half-site: 5)-TCGAGGGTAGGG(BrdU)(BrdU)CACCG-3) and a 3) half-site: 5)-AAAG(BrdU)(BrdU)CACTCGCACTCG-3). The DR3 probe used contained the 5) hemi-site: 5)-CAGACCAACAAGG(BrdU)(BrdU)CAC3), and the 3) hemi-site: 5)GAGG(BrdU)(BrdU)CACGTCTCTAAAGG-3). These probes were annealed to the corresponding contiguous complementary strand, and either the 5) or the 3) half-sites were labeled with [Á-32P]ATP. Full length GST-VDR or GST-RAR (80 ng) and/or the same amount of GST-¢ABRXR (in which the Nterminal 109 aminoacids containing region A/B of RXR· have been deleted) were incubated with the gapped probes and crosslinked with UV light [8]. The DNA-protein complexes were separated in SDSpolyacrylamide gels and identified by autoradiography.

Results and Discussion

Previous studies have suggested that the sequence, spacing and relative orientation of core recognition motifs determine the affinity and specificity of interactions be-

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Fig. 2. a VDR/RXR heterodimers bind to the DR5 (ßRARE). In

vitro translated RAR, VDR and/or RXR (1 Ìl) were used as indicated (lanes 3–7) in gel retardation assays with an oligonucleotide conforming the ßRARE. Lane 1 shows the mobility of the unretarded oligonucleotide and lane 2 shows the non-specific band formed by the unprogrammed reticulocyte lysate (r.l.). The positions of RAR/ RXR and VDR/RXR heterodimers are shown by arrows at the left. b The RXR/VDR heterodimer binds without a defined polarity on a DR5 element. The 5) or the 3) motif in gapped DR5 (lanes 1–10) and DR3 (lanes 11–13) elements containing BrdU residues were labeled with 32P. Purified recombinant RAR, VDR and the truncated ¢ABRXR, as fusion proteins with GST, were incubated separatedly

or mixed with the oligonucleotides as indicated at the top of each panel. The receptors were crosslinked to the DR5 or DR3 with UV light, and the protein-DNA adducts were electrophoresed under denaturing conditions. The more rapidly migrating band corresponds to ¢ABRXR-DNA adducts, and the more slowing migrating ones to RAR-DNA or VDR-DNA which have a similar mobility. The oligonucleotides and the 32P-labeled motif used are indicated below each lane. c VDR displaces RAR from the DR5. Gel retardation assays with the ßRARE oligonucleotide and 1 Ìl of RXR and RAR in the presence of increasing concentrations (between 0 and 6 Ìl) of VDR. The amount of in vitro translated receptors or reticulocyte lysate (r.l.) used is indicated at the top.

tween RXR/VDR and RXR/RAR heterodimers and their respective response elements. To analyze the influence of the spacing between the half-sites of DR elements, pituitary GH4C1 cells which express these receptors endogenously were transfected with the DR5, DR4 and DR3tk-CAT reporters. The DRs consist of two half-sites of the sequence -PuGTTCA- spaced by 5, 4 or 3 nucleotides, respectively (DRT elements). Figure 1a shows that an

increase in the spacing changed the specificity of the transcriptional response. Incubation with RA resulted in a strong activation of the DR5 reporter, whereas vitamin D was unable to stimulate this construct. Deletion of one nucleotide resulted in a substantial loss of response to RA, but did not increase the response to vitamin D. In contrast, deletion of 2 nucleotides converted the RARE into a VDRE, although a weak response of DR3-tk-CAT to RA

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was still observed. Interestingly, whereas RA acted additively with vitamin D, increasing the activity of the DR3 reporter plasmid, the vitamin markedly inhibited RAmediated stimulation of the DR5 construct. Similar results were obtained with reporter plasmids containing two copies of the -PuGGTCA- motif spaced by 3, 4 or 5 nucleotides (DRG elements) [7]. The influence of RA and vitamin D in a construct which contains the DR3 element present in the osteopontin promoter was also analyzed. Vitamin D transactivated this promoter, and incubation with RA potentiated the effect of vitamin D (not illustrated). Therefore vitamin D can either interfere the response to RA on a DR5 element, or it can have an additive effect with the retinoid on a DR3 element. Vitamin D was also a potent inhibitor of the transcriptional response of natural promoters to RA. As shown in figure 1b, vitamin D strongly reduced RA-mediated transactivation of the RARß2 promoter which contains the DR5T motif (ßRARE). Furthermore, vitamin D repressed the response of the rat growth hormone (GH) promoter to RA (fig. 1c). In this case repression is mediated through transcriptional interference with a palindromic element that confers responsiveness to RA as well as to the thyroid hormone [12]. These findings demonstrate an important repressive role of VDR on some transcriptional responses mediated by RA receptors in pituitary cells. However, the interaction of vitamin D and retinoid signaling is complex, because depending on the response element these ligands can also cooperate to stimulate transcription. A balance between stimulatory and inhibitory effects of vitamin D must create a sensitive and complex transcriptional network. Transrepression of RA-dependent responses by vitamin D can be also found in other cell types, since RA-dependent activation of the RARß2 promoter is also repressed by vitamin D in Cos-7 and HeLa cells after expression of VDR [10, 13]. To study whether the repressive action of VDR could involve competition for DNA binding, in vitro translated VDR, RAR and RXR were used for gel retardation assays with the DR5T element (fig. 2a). In the absence of RXR, neither RAR nor VDR bound strongly to this element. However, in the presence of the heterodimeric partner, not only RAR (lane 5) but also VDR (lane 7) bound with high affinity to the DR5. Therefore, there is a marked discrepancy between the strong in vitro binding of RXR/ VDR to this element and the lack of transcriptional activation in vivo, indicating that the RXR/VDR heterodimer binds in a transcriptionally inactive manner to the RA-responsive element. This lack of response can be

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attributed, at least in part, to the fact that this heterodimer does not show a preferred orientation on the DR5 element, whereas it binds with a normal polarity on a DR3 (fig. 2b). When ¢ABRXR/RAR heterodimers were crosslinked to the DR5, ¢ABRXR bound covalently to the 5) motif (lane 9), while RAR always bound to the 3) motif (lane 10). However, no such binding polarity of oABRXR/VDR heterodimers on the DR5 was observed. As illustrated in lanes 7 and 8, VDR bound to both the 5) and the 3) hemi-sites with a similar intensity, and the same was true for ¢ABRXR. In contrast, as shown in lanes 12 and 13, the polarity of binding to the DR3, which acts as a VDRE, was 5)-RXR-VDR-3). These data suggest that the conformation of RXR/VDR bound to DR5 and DR3 elements may be different, which may facilitate or exclude interaction with other regulatory factors. It is likely that direct or indirect interactions between the bound receptor heterodimers and other coregulators, neighboring transcription factors or components of the basal transcriptional machinery might be influenced by the conformation of the heterodimers in the differently spaced DRs. A competitive DNA binding mechanism between active RAR/RXR heterodimers and inactive VDR/RXR heterodimers could contribute to transrepression of the RA-dependent transactivation of the DR5-containing promoters by vitamin D. In fact, in gel retardation assays VDR displaced RAR/RXR binding to the DR5. As illustrated in figure 2c, VDR reduced RAR/RXR binding to DNA in a dose-dependent manner, and this reduction was accompanied by an increase in binding of the inactive VDR/RXR heterodimer (lanes 4–8). Although DNA binding could be important for full potency dominant inhibitory activity of VDR, other mechanisms must contribute to this inhibition. This is based in the finding that a N-terminally truncated VDR (¢1–111 VDR) which is unable to bind DNA also displays repressor activity in Cos-7 cells (fig. 3b) and GH4C1 cells (not illustrated), although the repression is somewhat weaker than that mediated by the native receptor. This truncated VDR contains the AF-2 domain responsible for ligand-dependent transactivation. The ability of a receptor lacking the last 12 aminoacids to repress RA-dependent activation of the RARß2 promoter was also analyzed. This receptor, in which the C-terminal amphipathic helix which contains the AF-2 domain has been deleted, heterodimerizes and binds normally to DNA, and is expressed at the same levels as the native VDR after transfection in cells [10]. However, this deletion abrogates ligand-dependent interaction of VDR with coactivators

Jimenez-Lara/Aranda

[11]. As illustrated in figure 3b, the AF-2 mutant showed a markedly reduced ability to mediate a ligand-dependent inhibition of the RA response. Furthermore, mutants L417S and E420Q in the core AF2 domain, as well as a mutant in a highly conserved lysine residue (K262A) which is required for coactivator recruitment were also unable to mediate transrepression [13]. The requirement of the residues responsible for AF-2 activity for transrepression strongly suggests that titration of common coactivators or associated proteins for VDR and RAR could be involved in the inhibitory effect of vitamin D. In agreement with this hypothesis, expression of E1A, which can act as a RARß2 promoter-specific coactivator [14], significantly reversed repression by vitamin D [10]. In summary, our results indicate that a combination of the relative affinities of different receptor heterodimers for the various response elements, as well as the concentrations of the different receptors and coactivators determine the final transcriptional response to vitamin D and RA. Binding of RXR/VDR heterodimers to DRs with different transcriptional outcomes may generate selectivity and expand the interactions between these receptors and other factors to create a sensitive and specific transcriptional complex.

Acknowledgments This work has been supported by grants PB94-0094 from the DGICYT and PM97-0135 from the DGES.

Fig. 3. The AF-2 domain of VDR participates in transrepression of the RARß2 promoter by vitamin D. a Schematic representation of

the VDR and VDR mutants. The N-terminal A/B domain is followed by the C region which contains the DBD; the flexible hinge D region connects the DNA binding domain with region E/F which contains the ligand binding domain, the dimerization domain, and a C-terminal AF2. b Cos-7 cells were transfected with the reporter plasmid and a noncoding vector, or with expression vectors for VDR, for ¢1–111 VDR, or for a receptor in which the last 12 C-terminal residues have been deleted (¢AF-2 VDR). The cells also received 0.2 Ìg of RAR. CAT activity was determined in cells treated with RA in the presence and absence of vitamin D.

References 1 Mangelsdorf DJ, Evans RM: The RXR heterodimers and orphan receptors. Cell 1995;83: 841–850. 2 Lemon BD, Freedman LP: Increasing the complexity of coactivation in nuclear receptor signaling. Cell 1999;97:5–8. 3 Glass C, Rosenfeld MG: The coregulator exchange in transcriptional functions of nuclear receptors. Genes Dev 2000;14:121–141. 4 Umesono K, Murakami KK, Thompson CC, Evans RM: Direct repeats as selective response elements for the thyroid hormone, retinoic acid and vitamin D receptors. Cell 1991;65:1255– 1266. 5 Glass CK: Differential recognition of target genes by nuclear receptor monomers, dimers and heterodimers. Endocrinol Rev 1994;15: 391–407. 6 Kurokawa R, DiRenzo J, Boehm M, Sugarman J, Gloss B, Rosenfeld MG, Heyman RA, Glass CK: Regulation of retinoid signalling by receptor polarity and allosteric control of ligand binding. Nature 1994;371:528–5317.

Regulation of Gene Expression by Vitamin D and Retinoid Receptors

7 Garcia-Villalba P, Jimenez-Lara AM, Aranda A: Vitamin D interferes with transactivation of the growth hormone gene by thyroid hormone and retinoic acid. Mol Cell Biol 1996;16:318– 327. 8 Jimenez-Lara AM, Aranda A: The vitamin D receptor binds in a transcriptionally inactive form and without a defined polarity on a retinoic acid response element. FASEB J 1999;13: 1073–1081. 9 De Thé H, Vivanco Ruiz MdM., Tiollais P, Stunnenberg HG, Dejean A: Identification of a retinoic acid responsive element in the retinoic acid receptor ß gene. Nature 1990;343:177– 180. 10 Jiménez-Lara AM, Aranda A: Vitamin D represses retinoic acid-dependent transactivation of the retinoic acid receptor-ß2 promoter: The AF-2 domain of the vitamin D receptor is required for transrepression. Endocrinology 1999;140:2898–2907.

11 Castillo AI, Jimenez-Lara AM, Tolon RM, Aranda A: Synergistic activation of the prolactin promoter by vitamin D receptor and GHF1: Role of the coactivators CREB-binding protein and steroid hormone receptor coactivator1 (SRC-1). Mol Endocrinol 1999;13:1141– 1154. 12 Bedo G, Santisteban P, Aranda A: Retinoic acid regulates growth hormone gene expression. Nature 1989;339:251–254. 13 Jimenez-Lara AM, Aranda A: Lysine 246 of the vitamin D receptor is crucial for ligand-dependent interaction with coactivators and transcriptional activity. J Biol Chem 1999;274: 13503–13510. 14 Folkers GE, van der Saag PT: Adenovirus E1A functions as a cofactor for retinoic acid receptor ß (RARß) through direct interaction with RARß. Mol Cell Biol 1995;15:5868–5878.

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Development of Efficient Transient Transfection Systems for Introducing Antisense Oligonucleotides into Human Epithelial Skin Cells Sabine Fimmel Antje Saborowski Constantin E. Orfanos Christos C. Zouboulis Department of Dermatology, University Medical Center Benjamin Franklin, The Free University of Berlin, Berlin, Germany

Key Words Antisense W Transfection W Sebaceous gland W Cell line W Keratinocytes W Androgen receptor

Abstract Systemic treatment with antisense oligonucleotides is confounded by the dual problems of potential cytotoxicity of antisense oligonucleotides and carrier molecules such as cationic lipids. Treatment of pathologic conditions affecting the skin may avoid these problems to a large degree due to local application. The success of antisense strategies has been limited by the poor uptake of the transfection reagent and inadequate intracellular compartmentalization. Human skin epithelial cells, therefore, are attractive experimental tools for testing both in vitro and in vivo antisense therapies. In the present study, we determined commercially available liposomes which reproducibly induced a nontoxic increase of oligonucleotide uptake in cultured SZ95 sebocytes and keratinocytes. The final protocol for SZ95 sebocytes was a 4hour incubation with DOTAP in a 2:1 (w/w) lipid/oligonucleotide ratio in serum-free medium. The fluoresceinlabeled (ATCG)5 random oligonucleotide molecules were detected within the nucleus. The optimum transfection system for primary keratinocytes was poly-L-ornithine (12 Ìg/ml) in a medium without bovine pituitary

ABC

© 2001 S. Karger AG, Basel 0301–0163/00/0546–0306$17.50/0

Fax + 41 61 306 12 34 E-Mail [email protected] www.karger.com

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extract over 4 hours. The uptake of the oligonucleotide increased in the presence of the polycation and oligonucleotide molecules were localized in the cytoplasm of keratinocytes. Oligonucleotide transfection with the help of cationic lipids did not affect the expression of androgen receptor and of the house-keeping gene ß-actin. Thus, cationic lipids are useful for delivery of antisense oligonucleotides into skin cells in vitro and may be used for topical application on animal and human skin. Copyright © 2001 S. Karger AG, Basel

Introduction

Antisense oligonucleotides have the ability to enter living cells and block the expression of specific genes. Systemic treatment with antisense oligonucleotides is confounded by the dual problems of lack of oligonucleotide stability and potential toxicity of the oligonucleotides themselves and/or of the carrier molecule, such as cationic lipids. Treatement of pathologic conditions affecting the skin, however, may overcome these problems to a large degree by the local application of antisense oligonucleotides to an affected area. In this context, skin models are attractive experimental tools for testing both in vitro and in vivo antisense therapies [1–4].

Prof. Dr. Christos C. Zouboulis Department of Dermatology, University Medical Center Benjamin Franklin The Free University of Berlin, Fabeckstrasse 60–62 D–14195 Berlin (Germany) Tel. +49 30 84456910, Fax +49 30 84456908, E-Mail [email protected]

The success of antisense strategies has been limited, at least in part, by the poor uptake of these agents into most cell types and inadequate intracellular compartmentalization. In this study, we report the development of efficient and economically reasonable transient transfection systems for introducing antisense oligonucleotides in human epithelial skin cells, namely in sebocytes and epidermal keratinocytes.

Materials and Methods Cell Cultures Immortalized human facial sebocytes (SZ95 sebocytes) [5] were maintained in Sebomed® medium (Biochrom, Berlin, Germany) containing 10% (v/v) fetal calf serum (FCS) and 5 ng/ml recombinant human epidermal growth factor (Biochrom) at 5% CO2 and 37 ° C and used at passages 50–55. Primary human epidermal keratinocytes from neonatal foreskin were cultured in serum-free keratinocyte medium (Gibco BRL, Berlin, Germany) supplemented with 5 ng/ml recombinant human epidermal growth factor and 50 Ìg/ml bovine pituitary extract. The keratinocytes were transfected between passages 2 and 5. Transient Transfection of SZ95 Sebocytes and Keratinocytes SZ95 sebocytes were incubated with the following commercially available transfection reagents: Tfx-10, Tfx-20, Tfx-50 (Promega, Mannheim, Germany), DAC-30 (Eurogentec, Seraing, Belgium) and DOTAP (Roche, Mannheim, Germany) to test their cytotoxicity in the absence of antisense molecules. The Tfx reagents are a mixture of a synthetic cationic lipid molecule [N,N,N),N)-tetramethylN,N)-bis(2-hydroxyethyl)-2,3-di(oleoyloxy)-1,4-butanediammonium iodide] and L-dioleoyl phosphatidylethanolamine (DOPE). All Tfx reagents contain the same concentration of the cationic lipid component, but are formulated with different molar ratios of the fusogenic lipid DOPE. DAC-30 is a liposomal formulation of a monocationic biodegradable cholesterol derivative. DOTAP is a liposomal formulation of the cationic lipid N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl sulfate. Primary human epidermal keratinocytes from neonatal foreskin were transfected with poly-L-ornithine, Polybrene (both from Sigma, Deisenhofen, Germany) and with pFx-5 (PerFect Lipids, Invitrogen, Karlsruhe, Germany). The reagent pFx-5 is the composition of a 1:1 mixture of two cationic lipids. To test the panel of lipids, cells were seeded in 96-well plates to reach a 40–60% confluence by the next day. Twenty-four hours before treatment the culture medium was substituted by the transfection medium (serum- and antibiotic-free, only supplemented with recombinant human epidermal growth factor). The prediluted polycations were added at concentrations ranging from 2 to 51 Ìg/ml and the cells were incubated at 37 ° C for 1 to 24 h. After incubation, the transfection mixture was replaced with complete medium and the cells were left to grow for 24 h. Cytotoxicity Assay The cytotoxic effect of the cationic lipids was determined with a lactate dehydrogenase (LDH) assay (Roche) after different transfection times. This colorimetric assay for the quantification of cell

Transient Transfection of Epithelial Skin Cells

death and cell lysis is based on the measurement of LDH activity released from the cytosol of damaged cells into the supernatant. Fluorescence Microscopy The detection of the cellular uptake of random oligonucleotides was performed using conventional fluorescence microscopy. SZ95 sebocytes and keratinocytes were seeded on 18-mm coverslips in culture dishes at an approx. 40% confluence. SZ95 sebocytes were then transfected with a mixture of 0.4 ÌM fluorescein-labeled (ATCG)5 random oligonucleotide and DOTAP (5 Ìg/ml). The keratinocytes were transfected with poly-L-ornithine (12 Ìg/ml). After an incubation time between 1 and 24 h cells were viewed as live specimens. The distribution of the fluorescence was analyzed by an Olympus fluorescence microscope. Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) Total RNA was isolated from cells using a total RNA isolation system (Qiagen, Hilden, Germany). After denaturation (65 ° C, 5 min), 5 Ìg RNA was transcribed into cDNA using a ready-to-goT-primed first-strand kit (Amersham Pharmacia, Freiburg, Germany). cDNA fragments of androgen receptor (AR) and ß-actin were amplified using the following primers: ß-actin forward (5)-AGC CTC GCC TTT GCC GA-3)), reverse (5)-CTG GTG CCT GGG GCG-3)); AR forward (5)-GAA GAC CTG CCT GAT CTG TG-3)), reverse (5)-AAG CCT CTC CTT CCT CCT GT-3)). A total of 2 Ìl cDNA solution was amplified for 35 cycles according to the following program: 1 min at 94 ° C, 1 min at 60 ° C (AR) or 67 ° C (ß-actin) and 1 min at 72 ° C. The PCR products were visualized by ethidium bromide staining. ß-Actin served as an external standard.

Results

Optimum Transfection Conditions Experiments were initially carried out on 96-well plates, but the method has been adapted to 6-well plates and 100-mm plates without loss of efficiency. Each test was performed sixfold. The conditions for each experiment are noted in the figure captions. Cell number, serum content, charge ratio and transfection time have been varied to optimize the transfection efficiency for SZ95 sebocytes and keratinocytes. The optimum grade of confluence for SZ95 sebocytes was assessed to be ^80% from the start to the end of the transfection and the recovery phase. The cell density of keratinocytes had to be higher for transfection to exclude excessive cell death. The cytotoxic effect of the cationic lipid DOTAP in SZ95 sebocytes increased significantly with the serum concentration in the transfection medium (2% and 5% FCS). Serum-free conditions were required for optimal performance (fig. 1). The toxicity rate doubled with the transfection reagent DAC-30 (4 Ìg/ml) in presence of 5% FCS (data not shown). DOTAP-containing vesicles

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Fig. 1. Relationship between serum concentration and cytotoxicity

of the transfection agent DOTAP in correlation with the lipid concentration. SZ95 sebocytes were transfected with different concentrations of DOTAP (2, 5, 10 Ìg/ml) in presence of fetal calf serum (5%, 2%, 0%). The lactate dehydrogenase levels are the results of 3 separate experiments performed in 6-fold wells and are presented in correlation to the values of native cells.

Table 1. Charge ratio of different cationic

lipids for sebocyte transfection

random oligonucleotide (ATCG)5. SZ95 sebocytes were transfected for 24 h with DAC-30 with different oligonucleotide concentrations (0.5–1.0 ÌM ). The controls were untreated SZ95 sebocytes.

Charge ratio of reagent to random oligonucleotide

Transfection reagent, Ìg/ml Tfx-10

Tfx-20

Tfx-50

DAC-30

DOTAP

2:1 3:1 4:1

26 36 51

13 19 27

6 8 12

2 4 8

2 5 10

showed moderate cytotoxic effect compared with the other tested lipids, DAC-30 and the panel of Tfx reagents. Transfection efficiencies in serum-free medium were equivalent to transfection efficiencies obtained in serumcontaining medium but only exhibited a moderate toxicity. The bovine pituitary extract content of the medium had no influence on the transfection of the primary keratinocytes with pFx-5 or with poly-L-ornithine (data not shown). For better comparison of SZ95 sebocytes with the keratinocytes, the keratinocyte transfection was performed in medium without bovine pituitary extract content.

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Fig. 2. Cytotoxicity depended on charge ratio of the lipid DAC-30 to

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The charge ratio of transfection reagents to the antisense oligonucleotide was a critical parameter (table 1). The amount of a positive charge contributed by the cationic lipid component has to exceed the amount of negative charge contributed by the oligonucleotide (fig. 2). Low concentrations of DAC-30 (4 Ìg/ml) and DOTAP (2 Ìg/ml) in absence of serum in a charge ratio 2:1 and 3:1 exhibited cytotoxic effects. Lower amount of lipid/random oligonucleotide (ATCG)5 (0.5–1.0 ÌM ) while keeping the ratio constant was an optimizing parameter (fig. 2). High amounts of Tfx-10 and Tfx-50 compensated apparently the deficiency of the serum-free medium; but

Fimmel/Saborowski/Orfanos/Zouboulis

Fig. 3. Transfection with Tfx-10 as an example for non-successful transfection systems. SZ95 sebocytes were incubated with 26 Ìg/ml lipid over several time periods. After an incubation time longer than 4 h Tfx was strongly toxic.

Fig. 4. Efficient transfection of keratinocytes with minimal cytotoxic effect after 24 h with poly-L-ornithine (12 Ìg/ml). The transfection was followed by a 5-min 25% DMSO shock after 2, 4 or 24 h. The agent polybrene (30 Ìg/ml) induced high cell toxicity.

the transfection of SZ95 sebocytes with Tfx-reagents was unsuccessful. The success of keratinocyte transfection with pFx-5 was dependent on the oligonucleotide concentration rate and a constant lipid concentration. With decreasing amounts of oligonucleotides (1–0.5 ÌM) and pFx-5 ratio 1:1 the cytotoxic effect was diminished. Optimum transfection times varied depending on cell type and the transfection reagent. To determine the optimum transfection time SZ95 sebocytes were incubated with DOTAP from 1 to 36 h. The optimum transfection duration with DOTAP combined with random oligonucleotides was 4 h. The risk of cell death during the transfection interval was higher with DAC-30 and increased significantly after 4 h incubation time with Tfx-10 (fig. 3). The final protocol for SZ95 sebocyte transfection was a 4-hour incubation with DOTAP in a 2:1 (w/w) lipid/oligonucleotide ratio in serum-free medium. The optimum transfection system for primary keratinocytes was established with the polycation L-ornithine and dimethyl sulfoxide (DMSO) shock. The poly-L-ornithine concentration was 12 Ìg/ml transfection medium without bovine pituitary extract for 4 h, followed by a 4min 25% DMSO shock. The transfection time could be

prolonged until 24 h without increasing the cytotoxic effect. Polybrene (30 Ìg/ml), another reported transfection reagent [6], induced a higher rate of cell damage (fig. 4).

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Proof of Transfection Efficiency by Fluorescence Microscopy Fluorescein-labeled random oligonucleotides were used to assess the intracellular distribution of oligonucleotides in the presence of the transfection reagents DOTAP in SZ95 sebocytes and poly-L-ornithine in keratinocyte cultures. In both cell types, transfection efficacy increased proportionally to the tranfection time and was detected by an increase of cellular fluorescence. Cellular fluorescence was localized within the nucleus in SZ95 sebocytes and in cytoplasmic structures in keratinocytes (fig. 5a, b). Expression of AR and ß-Actin after Cell Transfection As demonstrated in fig. 6, incubation of SZ95 sebocytes and keratinocytes with the corresponding transfection reagent had no influence on the levels of expression of AR and ß-actin.

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Fig. 6. Expression of ß-actin and androgen receptor mRNA in native keratinocytes (a) and SZ95 sebocytes (d). Lane b represents polyL-ornithine-treated keratinocytes and lane e SZ95 sebocytes incubated with DOTAP. Lane c is a negative control.

Fig. 5. Subcellular localization of fluorescein-labeled random oligonucleotide (ATCG)5. SZ95 sebocytes (a) and keratinocytes (b) were grown on glass slides. Cells were incubated with 0.4 ÌM fluorescein(ATCG)5 plus 5 Ìg/ml DOTAP (SZ95 sebocytes) or 12 Ìg/ml polyL-ornithine (keratinocytes) for 4 h at 37 ° C. In SZ95 sebocytes the oligonucleotides could be detected within the nucleus, whereas in keratinocytes the fluorescence was observed into the cytoplasm.

Discussion

Cationic lipids have been shown to be effective aids for increasing internalization of antisense oligonucleotides. Questions still remain, however, as to the efficacy of a given liposome type in a variety of cell systems and the suitability of these agents considering their reported toxicity. Cationic lipids enhance cellular uptake and activity of antisense oligonucleotides by increasing the amount of oligonucleotide into the cells and altering its intracellular distribution [2]. The aim of this study was to determine commercially available liposomes which reproducibly induce a non-

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toxic increase of oligonucleotide uptake in cultured SZ95 sebocytes and epidermal keratinocytes. The experiments described the parameters involved in the optimum transient transfection of these cells in vitro. From the tested panel of lipids the polycation DOTAP was the most successful agent in transfecting SZ95 sebocytes and the polycation L-ornithine was most effective in transfecting keratinocytes. Using these lipids, we could establish effective transfection systems with only moderate cytotoxicity. It is likely that variations in culture conditions (including confluence [7]), oligonucleotide concentrations and charge ratios used in the different investigations result in the varying cell toxicity observed. The lipid to oligonucleotide ratio is also very important. Sufficient lipid amounts are required to create complexes with the oligonucleotide giving a net positive charge [8]. It is considered that the positive charge of the lipid-oligonucleotide complex facilitates the interaction with the negatively charged cell surface. Excess lipids, however, are toxic to the cells, resulting in changes of cell morphology, slow growth and eventually cell death. For SZ95 sebocytes, a charge ratio 2:1 of DOTAP, and for keratinocytes a charge ratio of approximately 1:1 was found to be optimum. This was also observed by White et al. [9] who suggested that charge neutralization was only required for optimum interaction of the oligonucleotide-liposome complex with the cell surface. A sucessful transfection results in fluorescein-labeled random oligonucleotide distribution within the cells that can be easily detected by fluorescence microscopy. As previously reported in a number of cell types including keratinocytes, uptake characteristics for molecules of 15–20 nucleotides are not dependent on the oligonucleotide

Fimmel/Saborowski/Orfanos/Zouboulis

sequence [10, 11]. Therefore, we tested the efficiency of the transfection with random oligonucleotides (ATCG)5. DOTAP-containing lipid vesicles not only enhanced the rate of oligonucleotide uptake into SZ95 sebocytes but also markedly changed the subcellular distribution of the oligonucleotides. This finding is in agreement with reports examining the cellular distribution of oligonucleotides [12, 13]. The major difference in the presence of cationic lipids was the localization of oligonucleotides in the cell nucleus in SZ95 sebocytes and in the cytoplasm in keratinocytes. Accumulation within the nucleus appeared to be mediated by diffusion through nuclear pores, because it was not affected by ATP depletion [13]. The advantage of DOTAP-containing vesicles is that oligonucleotides can be introduced into large numbers of cells, the main disadvantage is moderate toxicity in the presence of serum. Using DOTAP-containing vesicles, we were able to demonstrate successful uptake of random oligonucleotides in the SZ95 sebocytes as well as using poly-L-ornithine in keratinocytes without affecting the expression of AR and the house-keeping gene ß-actin. Thus, cationic lipids are useful for delivering oligonucleotides into sebocytes and keratinocytes in culture and potentially could be used for the delivery of antisense oligonucleotides by topical application in animal and human skin.

Acne and seborrhoea, most common diseases encountered by dermatologists, are mostly androgen-dependent disorders [14]. The effect of androgens on the skin is influenced among others by the number of nuclear androgen receptor molecules available in skin cells. Androgens are the best-known stimulators of sebaceous gland activity, enhancing lipogenesis, proliferation and terminal differentiation of human sebocytes in vivo and in vitro. Androgen activity on the skin can be inhibited by systemic administration of compounds without androgen activity which have strong affinity for the AR and antagonize binding of androgens [14–16]. A new technology to realize the same purpose could be the antisense strategy used to block specifically the AR on the mRNA level. Our successful delivery systems are useful tools towards this direction in ongoing studies.

Acknowledgements This work was supported by a research grant of the Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie (BMBF 0311957A – Project 3.06) in cooperation with LVMH Recherche & Développement, Sain-Jean de Braye, France, and in part by the Research Committee of the Medical Faculty, The Free University of Berlin and the Sonnenfeld Stiftung, Berlin.

References 1 Stein CA, Cheng YC: Antisense oligonucleotides as therapeutic agents – is the bullet really magical? Science 1993;261:1004–1012. 2 Bennett RM: As nature intended? The uptake of DNA and oligonucleotides by eukaryotic cells. Antisense Res Dev 1993;3:235–241. 3 Lebleu B: Delivering information-rich drugs – prospects and challenges. Trends Biotechnol 1996;14:109–110. 4 Lebleu B, Robbins I, Bastide L, Vives E, Gee JE: Pharmacokinetics of oligonucleotides in cell culture. Ciba Found Symp 1997;209:47– 54. 5 Zouboulis CC, Seltmann H, Neitzel H, Orfanos CE: Establishment and characterization of an immortalized human sebaceous gland cell line (SZ95). J Invest Dermatol 1999;113:1011– 1020. 6 Neid M, McCance DJ: Poly-L-ornithine-mediated transfection of human keratinocytes. J Invest Dermatol 1995;105:668–671.

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7 Noonberg S, Garavoy MR, Hunt CA: Characteristics of oligonucleotide uptake in human keratinocyte cultures. J Invest Dermatol 1993; 101:727–731. 8 Lewis JG, Lin K, Kothvale A: A serum-resistent cytofectin for cellular delivery of antisense oligodeoxynucleotides and plasmid. Proc Natl Acad Sci USA 1996;93:3176–3181. 9 White PJ, Fogarty R, McKean S, Venables D, Werther G, Wraight C: Oligonucleotide uptake in cultured keratinocytes: Influence of confluence, cationic liposomes, and keratinocyte cell type. J Invest Dermatol 1999;112:699– 705. 10 Zhao Q, Song Z, Waldschmidt EF, Krieg AM: Oligonucleotide uptake in human hematopoetic cells is increased in leukemia and related to cellular activation. Blood 1996;88:1788–1795. 11 Loke SL, Stein C, Zhang H, Cohen JS, Neckers M: Characterization of oligonucleotide transport into living cells. Proc Natl Acad Sci USA 1989;86:3474–3478.

12 Yakubov L, Deeva V, Zarytova E, Yurschenko L, Vlassow V: Mechanism of oligonucleotide uptake by cells: Involvment of specific receptors. Proc Natl Acad Sci USA 1989;86:6454– 6458. 13 Leonetti J, Mechti G, Degols G, Lebleu B: Intracellular distribution of microinjected antisense oligonucleotides. Proc Natl Acad Sci USA 1991;88:2702–2706. 14 Zouboulis CC: Acne: Current aspects on pathology and treatment. Dermatol Experiences 1999;1:6–37. 15 Diamanti-Kandarakis E: Current aspects of antiandrogen therapy in women. Curr Pharm Des 1999;5:707–723. 16 Singh SM, Gauthier S, Labrie F: Androgen receptor antagonists (antiandrogens): structure-activity relation. Curr Med Chem 2000;7: 211–247.

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A Novel Pathway for Hormonally Active Calcitriol Bodo Lehmann Peter Knuschke Michael Meurer Department of Dermatology, Carl Gustav Carus Medical School, Dresden University of Technology, Dresden, Germany

Key Words Calcitriol W Keratinocyte W Microdialysis W UVB W Vitamin D3

Abstract Calcitriol [1·,25(OH)2D3], the hormonally active form of vitamin D3 (D3) is produced in both renal and extrarenal tissues. Our findings demonstrate that physiological doses of UVB radiation at 300 nm induce the conversion of 7-dehydrocholesterol (7-DHC) via preD3 and D3 into calcitriol in the pmol range in epidermal keratinocytes. The hydroxylation of photosynthesized D3 to calcitriol is strongly suppressed by ketoconazole, a known inhibitor of cytochrome P450 mixed function oxidases. The UVBinduced formation of calcitriol in human skin is demonstrable in vivo by the microdialysis technique. These results suggest that human skin is an autonomous source of hormonally active calcitriol.

further metabolized in the kidney [4, 5] to calcitriol [1·,25(OH)2D3], the hormonal active form of D3. Except from its calcitropic action, 1·,25(OH)2D3 has antiproliferative and prodifferentiative effects on epidermal keratinocytes [6–8]. However, the calcitriol concentration required to suppress growth of keratinocytes in vitro are substantially higher than present in blood [6]. On the other hand, it has been shown that cultured keratinocytes can convert exogeneous calcidiol (25OHD3) to calcitriol [9]. The physiological importance of this pathway however is unclear because only trace amounts of calcitriol were synthesized from calcidiol by skin ex vivo [10]. Previously, we were able to demonstrate that in cultures of human keratinocytes biologically inactive D3 can be converted to 1·,25(OH)2D3 [11, 12]. Since the skin is both the site of D3 synthesis and a target organ for 1·,25(OH)2D3 it is important to know whether UVB-induced photolysis of 7-DHC with consecutive synthesis of D3 can influence the epidermal production 1·,25(OH)2D3.

Copyright © 2001 S. Karger AG, Basel

Methods Introduction

Cutaneous vitamin D3 (D3) is generated by UVBinduced photolysis of 7-dehydrocholesterol (7-DHC) [1]. Once formed, D3 is transported to the liver [2, 3] where it is metabolized to calcidiol (25OHD3) which is

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Cell Culture HaCaT cells (used with the permission of Prof. N.E. Fusenig of the German Cancer Research Center, Heidelberg) were seeded at a density of 5 ! 104 cells/cm2 in culture dishes (n30 mm) and grown in DMEM supplemented with 5% (v/v) FCS. Cultures were maintained at 37 ° C and 5% CO2 in air. After synchronization the

Bodo Lehmann, Department of Dermatology Carl Gustav Carus Medical School, Dresden University of Technology Fetscherstrasse 74, D–01307 Dresden (Germany) Tel. +49 351 458 2692, Fax +49 351 458 4338 E-Mail [email protected]

medium was replaced by serum-free DMEM supplemented with 1.0% (w/v) of highly purified BSA (Sigma). At this time cells were preconfluent (equal to " 0.7 ! 106 cells/dish). Cell numbers and their viability were assessed using a CASY® 1 cell counter (Schärfe System GmbH, Reutlingen, Germany). The viability was always 693%. Incubation Conditions 7-DHC (final concentration: 25 ÌM, 30 nmol/dish) dissolved in ethanol (final concentration: 0.83% (v/v)), was added to the cultures. Control experiments were carried out: (1) with cultures in presence of 7-DHC without irradiation; (2) solvent alone with and without irradiation; (3) medium containing 7-DHC in absence of cells with irradiation. Irradiation was followed by 16 h incubation in dark. The medium and detached cells were extracted with methanol:chloroform (1:1). In the chloroform phase pre-D3, D3, 7-DHC and 1·,25(OH)2D3 were determined. UV Irradiation Monochromatic light (bandwidth 5 nm) was generated by a Dermolum HI monochromator (1-kVA Xe lamp, Müller, Germany). The distance between light source and the bottom of open culture dish (n 30 mm) was 14 mm. The beam had a diameter of 15 mm. The culture dish was continously turned during irradiation and the beam was positioned at the radius of the rotating dish. The UVB doses used were adapted to these experimental conditions (effective dose = Deff = calculated dose ! 0.25). Irradiances were measured with a calibrated thermopile. HPLC Analysis NP-HPLC. Merck/Hitachi; column: LiChroCART 250-4, Superspher Si 60, 5 Ìm; eluent 1 (n-hexane:2-propanol = 95:5 (v/v); flow rate: 0.5 ml/min) for the determination of pre-D3, D3 and 7-DHC; eluent 2 (n-hexane:2-propanol:methanol = 87:10:3 (v/v/v); flow rate: 1.0 ml/min) for fractionation of calcitriol (retention time: 21 min). The peaks of pre-D3, D3 and 7-DHC (retention times: 8.20 min, 10.56 min and 11.58 min respectively) were quantified by UV detection at 265 nm. HPLC fractions containing calcitriol were collected and analyzed for calcitriol using a radioreceptor assay (Nichols Institute). The calcitriol generated was identified by co-chromatography of the 3H-labeled standard. RP-HPLC. Rechromatography of calcitriol on a Hibar© column, 250-4, LiChrospher 100RP-18, 5 Ìm (Merck, Darmstadt, Germany); eluent: methanol:water = 85:15 (v/v), flow rate: 1 ml/min; retention time of synthetic [3H]1·,25(OH)2D3: 10.0 min. GC-MS Analysis Dried fractions from HPLC were derivatized to TMS ether derivatives and analyzed by GC-MS. The derivatized sample (1 Ìl) was directly injected manually into a model 5890/II gas chromatograph equipped with a 25 m ! 0.2 mm HP-1 capillary column (cross-linked methylsiloxane, 0.33 Ìm) and interfaced with a model 5989A MS Engine (Hewlett-Packard, Palo Alto, CA, USA).

Fig. 1. Relationship between wavelengths of UVB light and the gen-

eration of D3 and 1·,25(OH)2D3 in HaCaT cells. Cultures preincubated with 25 ÌM 7-DHC were irradiated at several wavelengths between 285 nm and 320 nm (dose: 30 mJ ! cm –2) followed by 16 h incubation. Each point represents the mean of two independent experiments.

20 kD. The abdomen was selected as site for insertion of two independent microdialysis probes (UVB irradiation and control, respectively). When local anesthesia at the point of insertion was done, an apheresis needle (1.60 ! 32 mm) was inserted as a guide, as superficially as possible for 15 mm. The probe was inserted through the guide, which was then withdrawn and taped in position. The site of insertion and the area above the tip of the probe were marked. The probe was first flushed with Ringer’s solution using a CMA 107 microdialysis pump for 5 min at a flow rate of 15 Ìl/min. Thereafter the flow was changed to 0.3 Ìl/min, and the dialysate was fractionated into microvials for 3 h each (control). The second probe was uncovered after 3 h and irradiated wih monochromatic UVB at 300 nm (doses: 14 or 28 mJ ! cm –2, equal to 0.7 or 1.4 MED, respectively). The microvials were changed every 3 h until 24 h after UVB exposure. The content of 1·,25(OH)2D3 in 50 Ìl dialysate of each fraction was determined by radioreceptor assay (see above).

Results and Discussion

Microdialysis Investigations were carried out during winter. The microdialysis probe used (CMA 70, CMA/Microdialysis Research AB, Sweden) has a shaft length of 60 mm and a membrane length of 10 mm. The steel shaft of the probe has an outer diameter of 0.64 mm. The diameter at the tip of the probe where the dialysis membrane is located is 0.5 mm. The molecular weight cutoff point for the membrane is

In vitro experiments using the HaCaT cell line demonstrated the UVB-induced conversion of exogeneous 7-DHC via previtamin D3 (pre-D3) and D3 to 1·,25(OH)2D3 in these cells. The rate of formation of 1·,25(OH)2D3 in HaCaT cells depends on the UVB wavelength used for irradiation and is very similar to that of D3, showing maxima at around 303 nm (fig. 1). This finding points to a close relation between UVB-induced D3 synthesis and consecutive formation of 1·,25(OH)2D3.

Synthesis of Calcitriol in the Skin

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313

Fig. 2. Inhibitory effect of ketoconazole and 1,2-dianilinoethane on hydroxylation of D3 created after irradiation with UVB to 1·,25(OH)2D3 in HaCaT cells. Cultures containing 25 ÌM 7-DHC were irradiated at 300 nm (dose: 30 mJ ! cm –2). Immediately after irradiation, ethanol (control), ketoconazole (10 ÌM ) or 1,2-dianilinoethane (10 ÌM ) were added to the cultures, and further incubation for 16 h at 37 ° C was done in the dark. Concentrations obtained are depicted as relative percent B SD of control (100 B 8% equal to 1990 B 159 fmol calcitriol) of three independent experiments. *** p ! 0.001 compared to control.

3. Microdialysis probe for cutaneous monitoring of 1·,25(OH)2D3 after UVB irradiation of human skin. Skin was irradiated at 300 nm at 14 mJ ! cm –2 or 28 mJ ! cm –2 (equal to 0.7 and 1.4 MED, respectively) and not irradiated (control). Flow rate of dialysis fluid: 0.3 Ìl/min, volume of fraction: 54 Ìl/3 h, total duration of microdialysis: 27 h. Calcitriol was determined in 50 Ìl dialysate. Data are depicted as mean values of two independent experiments.

Calcitriol obtained after NP-HPLC (eluent 2) of extracts of irradiated HaCaT cells (300 nm; dose: 30 mJ ! cm –2; irradiance: 0.28 mW ! cm –2) was identical with synthetic standard calcitriol as shown by co-migration with 3H1·,25(OH)2D3 in NP and RP-HPLC systems. No calcitriol was detectable in extracts of unirradiated HaCaT cells. Studies of the TMS derivatives of synthetic and generated calcitriol by GC-MS demonstrated identical retention times as well as full-scan EI mass spectra. Ketoconazole [13] dose-dependently inhibited the UVB-induced generation of calcitriol in HaCaT cells when added to the culture immediately after irradiation (fig. 2). In contrast, the radical scavenger and antioxidant 1,2-dianilinoethane [14] showed only marginal inhibitory effects on the generation of 1·,25(OH)2D3 (fig. 2). This finding points to an enzymatically catalyzed pathway of D3 to 1·,25(OH)2D3 as previously found after exogenous addition of D3 to cultured keratinocytes [11, 12]. Calculations indicate that only 0.007% of the UVBirradiated 7-DHC (30 nmol/dish) is converted to 1·,25(OH)2D3 (equal to 2.093 fmol/106 cells). For comparison, the normal range of the concentration of 1·,25(OH)2D3 in human serum is 68 B 27 fmol/ml [15].

Finally, we have been able to show in preliminary experiments that the UVB-induced formation of calcitriol at 300 nm in human skin can be demonstrated using the microdialysis technique (fig. 3). Calcitriol concentrations in the dialysate were at a maximum 12–18 h after UVB irradiation and increased with rising UVB doses up to 28 mJ ! cm –2 (equal to 1.4 MED). It can be concluded from our findings that the metabolism of photosynthesized D3 to 1·,25(OH)2D3 in keratinocytes is obviously catalyzed by P450 mixed function oxidases. Our findings demonstrate the UVB-triggered conversion of 7-DHC to substantial amounts of 1·,25(OH)2D3 in HaCaT keratinocytes. In vivo experiments using the microdialysis technique demonstrated cutaneous generation of 1·,25(OH)2D3 12–18 h after the skin was UVB irradiated at 300 nm in therapeutical doses. These findings are of potential importance for the proliferation, differentiation and apoptosis of keratinocytes as well as immunsuppression in skin under influence of UVB. Also, one could speculate whether the therapeutical effect of UVB in hyperproliferative skin diseases such as psoriasis can be partially attributed to the action of epidermally synthesized calcitriol.

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Fig.

Lehmann/Knuschke/Meurer

References 1 Holick MF, MacLaughlin JA, Clark MB, Holick SA, Potts, JT: Photosynthesis of of previtamin D3 in human skin and the physiologic consequences. Science 1980;210:203–205. 2 Ponchon G, Kennan AL, DeLuca HF: Activation of vitamin D by the liver. Clin Invest 1969;48:2032–2037. 3 Horsting M, DeLuca HF: In vitro production of 25-hydroxycholecalciferol. Biochem Biophys Res Comm 1969;36:251–256. 4 Holick MF, Schnoes HK, DeLuca HF: Identification of 1,25-dihydroxycholecalciferol, a form of vitamin D3 metabolically active in the intestine. Proc Natl Acad Sci USA 1971;68: 177–181. 5 Lawson DE, Fraser DR, Kodicek E, Morris HR, Williams DH: Identification of 1,25-dihydroxycholecalciferol, a new kidney hormone controlling calcium metabolism. Nature 1971; 230:228–230.

Synthesis of Calcitriol in the Skin

6 Matsumoto K, Hashimoto K, Nishida Y, Hashiro M, Yoshikawa K: Growth-inhibitory effects of 1,25-dihydroxyvitamin D3 on normal human keratinocytes cultured in serum-free medium. Biochem Biophys Res Commun 1990;166:916–923. 7 Hosomi J, Hosoi J, Abe E, Suda T, Kuroki T: Regulation of terminal differentiation of cultured mouse epidermal cells by 1·,25-dihydroxyvitamin D3. Endocrinology 1983;113: 1950–1957. 8 Smith EL, Walworth NC, Holick MF: Effect of 1·,25-dihydroxyvitamin D3 on the morphologic and biochemical differentiation of cultured human epidermal keratinocytes grown in serum-free conditions. J Invest Dermatol 1986; 86:709–714. 9 Bikle DD, Nemanic MK, Elias P: 1,25-Dihydroxyvitamin D3 production by human keratinocytes. J Clin Invest 1986;78:557–566. 10 Bikle DD, Halloran BP, Riviere JE: Production of 1,25-dihydroxyvitamin D3 by perfused pig skin. J Invest Dermatol 1986;102:796–798.

11 Lehmann B, Pietzsch J, Kämpf A, Meurer M: Human keratinocyte line HaCaT metabolizes vitamin D3 and 1·-hydroxyvitamin D3 to 1·,25-dihydroxyvitamin D3. J Dermatol Sci 1998;18:118–127. 12 Lehmann B, Rudolph T, Pietzsch J, Meurer M: Conversion of vitamin D3 to 1·,25-dihydroxyvitamin D3 in human skin equivalents. Exp Dermatol 2000;9:97–103. 13 Wilkinson CF, Hetnarski K, Cantwell GP, DiCarlo FJ: Structure-activity relationships in the effects of 1-alkylimidazoles on microsomal oxidation in vitro and in vivo. Biochem Pharmacol 1974;23:2377–2386. 14 Sietsema WK, DeLuca HF: Retinoic acid 5,6epoxidase: properties and biological significance. J Biol Chem 1982;257:4265. 15 Hollis BW: Assay of circulating 1,25-dihydroxyvitamin D involving a novel single-cartridge extraction and purification procedure. Clin Chem 1986;32:2060–2063.

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Successful Treatment of Non-Segmental Vitiligo: Systemic Therapy with Sex Hormone-Thyroid Powder Mixture Ko Nagai Makoto Ichimiya Kazuo Yokoyama Yoshiaki Hamamoto Masahiko Muto Department of Dermatology and Biomolecular Recognition, Yamaguchi University School of Medicine, Ube, Japan

Key Words Vitiligo W ·-Melanocyte-stimulating hormone (·-MSH)

Abstract We previously reported a patient with generalized vitiligo improved by oral administration of the drug for menopausal syndrome (sex hormone-thyroid powder mixture). In this study, we reevaluated the efficiency of this drug for vitiligo, and examined its pharmacological action in melanogenesis. Copyright © 2001 S. Karger AG, Basel

Patients and Treatment Fifteen non-segmental vitiligenous patients (10 females, 5 males) were treated with oral administration of two tablets of Methalmon-F™ (NIHONZOKI Pharmaceutical Co., Ltd., Tokyo, Japan) daily. The age of the patients ranged from 13 to 68 years. The average age was 56.4 years. Antinuclear factor and autoantibodies against thyroid gland were positive in 4 and 3 cases respectively. Immunohistochemical Study Biopsy specimens were obtained both from depigmented skin before treatment and from repigmented skin after treatment. The expressions of ACTH and ·-MSH were analysed immunohistochemically with the use of antibodies against ACTH (purchased from YLEM, Italy) and ·-MSH (ICN, USA). Staining was performed with the alkaliphosphatase-labelled streptavidin method.

Introduction

Non-segmental vitiligo is thought to be an autoimmune disorder [1]. However, the pathomechanism of vitiligo is still unknown, and traditional therapeutic methods are incomplete to cure the disease. We have used Metharmon-F™ for the treatment of non-segmental vitiligo with better outcome [2]. Metharmon-F™ contains estrogen which has been reported to increase tyrosinase activity of melanocytes [3]. On the other hand, estrogen inhibits proliferation of melanocyte growth in some cell lines [4]. Here we examine pharmacological action of the drug for vitiligenous melanocytes.

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Results

As shown in table 1, after two or three months of treatment, 7 patients showed repigmentation (repigmented ratio: 7/15). Metharmon-F™ therapy was more effective in menopausal female patients (repigmented ratio: 6/10). Male patients were generally resistant to the therapy. There was no relation between the effectiveness of the therapy and the presence of autoantibodies. A side effect was observed in one case (vertigo). Immunohistochemical analyses revealed the expression of ·-MSH in lesional melanocytes. Expression of ACTH showed no alteration during the therapy (table 2). Ko Nagai Minami-Kogushi 1-1-1 Ube 755-8505 (Japan) Tel./Fax +81 836 22 2271

Table 1. Case profiles of Metharmon-FTM trial group

Table 2. Expression of ACTH and ·-MSH in melanocytes in vitiligo lesional skin

Case

Age

Sex

Repigmentation Combined therapy Autoantibodies

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

49 57 68 51 53 54 67 53 60 52 24 63 27 13 19

female female female female female female female female female female male male male male male

+ + + + + + – – – – + – – – –

topical steroid topical steroid topical steroid topical steroid topical steroid topical steroid topical steroid topical steroid

ANF (!100, diffuse) ANF (!20, speckled)

Depigmented skin Repigmented skin

ACTH

·-MSH

– –

– ++

TGAb, TPOAb TGAb TGAb ANF (!20, speckled)

topical steroid

topical steroid

ANF (!100, diffuse)

TGAb = Anti-thyroglobulin antibody; TPOAb = anti-thyroxin peroxidase antibody.

Discussion

Up to now it is very difficult to cure vitiligo, although many therapeutic methods have been reported. We used Metharmon-F™, a sex hormone-thyroid powder mixture for the treatment of vitiligo with high repigmented ratio. Each tablet of Metharmon-F™ is composed of pregnenolone (1.0 mg), androstenedione (1.0 mg), androstenediol (0.5 mg), testosterone (0.1 mg), estorone (5 Ìg) and thyroid powder (7.5 mg). Ranson et al. reported that estradiol increased thyrosinase activity of human melanocytes [3]. However estrogen is also an apoptosis inducer, and re-

References

1 2

3

Treatment of Non-Segmental Vitiligo

ported to inhibit melanocyte growth. Then, we examined another possibility to explain the effectiveness of this drug for vitiligo. ·-MSH is a proopiomelanocortin-derived peptide which is produced mainly in pituitary gland. However it has been reported that the peptide is produced in keratinocytes and melanocytes [5, 6]. In this study, we revealed the increase of ·-MSH expression in melanocytes treated with Metharmon-F™. Therefore the possibility that this sex hormone-thyroid powder mixture might induce melanogenesis via ·-MSH expression in melanocytes.

Bystryn JC: Immune mechanisms in vitiligo. Immunol Ser 1989;46:447–473. Muto M, Furumoto H, Ohmura A: Successful treatment of vitiligo with a sex steroid-thyroid hormone mixture. J Dermatol 1995;22:770– 772. Ranson N, Posen S, Mason R: Human melanocyte as a target tissue hormone: In vitro studies with 1·-25 dihydroxyvitamine D3, melanocyte stimulationg hormone and estradiol. J Invest Dermatol 1998;91:593–598.

4

5

6

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Lama G, Angelucci C, Bruzzese N: Sensitivity of human melanoma cells to oestrogens, tamoxifen and quercetin: Is there any relationship with type I and type II oestrogen binding site expression? Melanoma Res 1998;8:313– 322. Slominski A, Paus R, Wortsman J: On the potential role of proopiomelanocortin in skin physiology and pathology. Mol Cell Endocrinol 1993;93:C1–C6. Lunce J, Pieron C, Sherbet GV: Alpha-melanocyte-stimulating hormone immunoreactivity in melanoma cells. Pathobiology 1990;58:193– 197.

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Liposomal Ursolic Acid (Merotaine) Increases Ceramides and Collagen in Human Skin Daniel B. Yarosh Dawn Both David Brown AGI Dermatics, Freeport, N.Y., USA

Key Words Merotaine W Hydroxy-ceramides W Procollagen W Keratinocytes W Fibroblasts W Barrier function W Glucocorticoid receptors

Abstract Skin wrinkling and xerosis associated with aging result from decreases of dermal collagen and stratum corneum ceramide content. This study demonstrates that ursolic acid incorporated into liposomes (MerotaineTM) increases both the ceramide content of cultured normal human epidermal keratinocytes and the collagen content of cultured normal human dermal fibroblasts. In clinical tests, Merotaine increased the ceramide content in human skin over an 11-day period. Merotaine has effects on keratinocyte differentiation and dermal fibroblast collagen synthesis similar to retinoids. However, unlike retinoids, Merotaine increases ceramide content of human keratinocytes. Ursolic acid may bind to members of the glucocorticoid receptor family to initiate changes in keratinocyte gene transcription. Copyright © 2001 S. Karger AG, Basel

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Introduction

During the aging process, skin wrinkling occurs as a result of reductions of collagen in the dermis and epidermal atrophy [1, 2]. In addition, reduced barrier function and xerosis occur as a result of reduced stratum corneum thickness and ceramide content [3, 4]. Studies of the aging process show that ceramides are the lipid class most decreased with age [4]. Sun exposure accentuates the skin wrinkling process by suppression of collagen I synthesis and stimulation of collagenase release from inflammatory cells, fibroblasts and keratinocytes [1, 5]. Ursolic acid (URA) is a non-toxic naturally occurring triterpenoid that is found in a variety of plants with medicinal properties, notably rosemary [6]. Previous studies have shown that URA has both anti-inflammatory and anti-tumor properties, including inhibition of tumor promotion in mouse skin [7, 8]. Unfortunately, URA is extremely insoluble and is often lost from commercial extracts of rosemary (unpubl. results). Integration of URA in the lipid bilayers of liposomes overcomes this problem. Our preparation of liposomal URA is called Merotaine, after the waxy part of the apple peel, where URA is also found. We have examined the effect of URA on skin physiology.

Daniel B. Yarosh AGI Dermatics, 205 Buffalo Avenue Freeport, NY 11520 (USA) Tel. +1 516 868 9026, Fax +1 516 868 9143 E-Mail [email protected]

Fig. 1. Induction of ceramides in human keratinocytes following treatment with 1% Merotaine. Cultured normal human epidermal keratinocytes were treated on days 0 and 3, and harvested on day 6. (Top) HPTLC’s showing results from 3 different experiments (1, 2, 3). (Bottom) Phase contrast micrograph of keratinocytes on day 6. Note the lipid-containing vacuoles in the cells treated with Merotaine.

Fig. 2. Induction of ceramides in human skin following treatment with 0.3% or 1% Merotaine for 3 and 11 days. Values are relative to those obtained for adjacent areas treated with Empty Liposome Lotion (X B SD; n = 3). * p ! 0.05 (ANOVA).

Materials and Methods URA (Sigma, France) was encapsulated in phosphatidyl choline:cholesterol (2.5:1) liposomes at 3 mM URA, as determined by HPTLC. Liposomes and 1% Carbopol 981 lotions (pH 8.0) were prepared containing no test article (empty liposomes), 1% Merotaine or with all-trans retinoic acid 0.83 mM. Normal human epidermal keratinocytes were cultured until 60–70% confluence and then treated with empty liposomes (vehicle control) or Merotaine. Following treatments, cells were harvested and counted, and the lipids extracted [9]. The human subjects (3 Caucasian males, 45–55) applied lotions twice per day for 11 days to their forearms before lipids were extracted [10]. Both the cell culture lipids and human skin lipids were separated by silica gel HPTLC, developed by heat and the ceramides quantified by computerized image analysis.

Liposomal Ursolic Acid Induces Ceramides

Results

Treatment of keratinocytes for 6 days with 1% Merotaine increased total ceramides by approximately 3-fold (fig. 1, top). The accumulation of lipids in vacuoles is apparent in the keratinocytes (fig. 1, bottom). The induction of ceramides by Merotaine was not detectable until 3 days after treatment, then rose 2.5-fold after 5 days of treatment, 3-fold after 7 days of treatment and 4.5-fold after 9 days of treatment (data not shown). Treatment of human subjects with lotion containing 0.3% or 1% Merotaine resulted in induction of ceramides, with increases of hydroxy-ceramides generally greater than non-hydroxy-ceramides (fig. 2). In fact, following 3

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Fig. 3. Chemical structures of ligands of nuclear receptors and URA.

days of treatment, non-hydroxy ceramides were decreased approximately 12% by 0.3% or 1% Merotaine. In contrast, hydroxy ceramides were increased by approximately 18% following 3 days of treatment with either 0.3% or 1% Merotaine. Following 11 days of treatment, both non-hydroxy- and hydroxy-ceramides were increased approximately 30% by 0.3% Merotaine. However, at this same time period, non-hydroxy- and hydroxy-ceramides were increased only 7% and 18% respectively, by 1% Merotaine. Collagen production within cultured normal human dermal fibroblasts was measured using antibodies against procollagen, and was near the limit of detection in untreated cells. However, it was increased approximately 70-fold by 0.3% Merotaine (data not shown). Procollagen was 2.9-fold higher in cells treated with 1% Merotaine than in those treated with 0.3% Merotaine. During differentiation of cultured keratinocytes, expression of keratin 1, keratin 10, and involucrin increases

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[11]. We examined these markers of differentiation to determine if induction of ceramides by Merotaine occurs as a simple byproduct of keratinocyte differentiation, or independent of keratinocyte differentiation. We found that both retinoic acid in liposomes, and 30 ÌM URA in Merotaine reduced differentiation (data not shown). Thus, retinoids reduce ceramides because they differentiate keratinocytes, while Merotaine increases ceramides in a way apparently unrelated to keratinocyte differentiation.

Discussion

This study demonstrates that liposome-encapsulated URA (Merotaine) increases ceramides and collagen in human skin. Reductions of both ceramides and collagen occur as a result of the aging process [1, 4] and are associated with reduced barrier function, and dry flaky skin [3,

Yarosh/Both/Brown

4]. During aging, stratum corneum ceramides decrease by approximately 10–15% per decade after the age of 20 [3, 4]. This study has shown that treatment with lotion containing 0.3% Merotaine for 11 days increases both nonhydroxy- and hydroxy-ceramides by approximately 30%, which is equivalent to the losses that would be incurred as a result of two to three decades of aging. Both retinoic acid and Merotaine reduced markers of differentiation, and increased procollagen synthesis. Whereas induction of ceramide synthesis is usually associated with keratinocyte differentiation, we found that induction of ceramides by Merotaine occurs along with the hallmarks of dedifferentiation. Some insight into the mechanism of action of URA comes from comparing its chemical structure to other related molecules. URA shares some structural similarities to the family of estradiols, which are ligands for nuclear receptors and tran-

scription activators (fig. 3). In fact, URA competes with RU486 for glucocorticoid receptors in the regulation of matrix metalloproteinase-9 [12]. One notable characteristic of estrogen binding to glucocorticoid receptors is an increase in collagen [13], as is seen with URA. In addition, activated glucocorticoid receptors suppress keratin gene expression [14], one of the signs of dedifferentiation, and this is also found after URA treatment. However, URA does not share structural similarities with the retinoic acid family (fig. 3), which binds another class of nuclear receptors and increases collagen while decreasing ceramides and keratins. Therefore, it is likely that URA and retinoic acid do not share the same mechanism even if their effects on collagen and keratin are similar. The difference is that URA, not retinoids, stimulates the production of ceramides in keratinocytes and human skin.

References 1 Griffiths, CEM, Voorhees JJ: Topical retinoic acid for photoaging: Clinical response and underlying mechanisms. Skin Pharmacol 1993; 6(suppl 1):70–77. 2 Uitto J, Bernstein EF: Molecular mechanisms of cutaneous aging: Connective tissue alterations in the dermis. J Invest Dermatol 1998;3: 41–44. 3 Imokawa G, Abe A, Jin K, Higaki Y, Kawashima M, Hidano A: Decreased level of ceramides in stratum corneum of atopic dermatitis: An etiologic factor in atopic dry skin. J Invest Dermatol 1991;96:523–526. 4 Rogers J, Harding C, Mayo A, Banks J, Rawlings A: Stratum corneum lipids: the effect of ageing and the seasons. Arch Dermatol Res 1996;288:765–770. 5 Griffiths CEM, Russman AN, Majmudar G, Singer RS, Hamilton TA, Voorhess JJ: Restoration of collagen formation in photodamaged human skin by Tretinoin (retinoic acid). N Engl J Med 1993;329:530–535.

Liposomal Ursolic Acid Induces Ceramides

6 Liu J: Pharmacology of oleanolic acid and ursolic acid. J Ethnopharmacol 1995;49:57–68. 7 Huang MT, Ho CT, Wang ZY, Ferraro T, Lou YR, Stauber K, Ma W, Georgiadis C, Laskin JD, Conney AH: Inhibition of skin tumorigenesis by rosemary and its constituents carnosol and ursolic acid. Cancer Res 1994;54:701– 708. 8 Suh N, Honda T, Finlay HJ, Barchowsky A, Williams C, Benoit NE, Xie QW, Nathan C, Gribble GW, Sporn MB: Novel triterpenoids suppress inducible nitric oxide synthase (iNOS) and inducible cyclooxygenase (COX-2) in mouse macrophages. Cancer Res 1998;58: 717–723. 9 Ponec M, Weerheim A: Retinoids and lipid changes in keratinocytes. Methods Enzymol 1990;190:30–41.

10 Bontè F, Saunois A, Pinguet P, Meybeck A: Existence of a lipid gradient in the upper stratum corneum and its possible biological significance. Arch Dermatol Res 1997;289:78–82. 11 Gendimenico GJ, Mezick JA: Pharmacological effects of retinoids on skin cells. Skin Pharmacol 1993;6(suppl 1):24–34. 12 Cha HJ, Park MT, Chung HY, Kim ND, Sato H, Seiki M, Kim KW: Ursolic acid-induced down-regulation of MMP-9 gene is mediated through the nuclear translocation of glucocorticoid receptor in HT1080 human fibrosarcoma cells. Oncogene 1998;16:771–778. 13 Affinito P, Palomba S, Sorrentino C, DiCarlo C, Bifulco G, Arienzo MP, Nappi C: Effects of postmenopausal hypoestrogenism on skin collagen. Maturitas 1999;15:239–247. 14 Radoja N, Komine M, Jho SH, Blumenburg M, Tomic-Canic M: Novel mechanism of steroid action in skin through glucocorticoid receptor monomers. Mol Cell Biol 2000;20:4328–4339.

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Association of Insulin Resistance with Hyperandrogenia in Women Michel Pugeat Pierre Henri Ducluzeau Marika Mallion-Donadieu Fédération d’Endocrinologie de l’Hôpital de l’Antiquaille, Hospices Civils de Lyon, and INSERM U329, Lyon, France

Key Words Androgens W Insulin resistance W Hyperandrogenia W Acanthosis nigricans W Hirsutism W Alopecia

Abstract In humans, the skin is a target tissue for androgen action; hair growth and sebum secretion are under active androgen control. An increased production or metabolism of testosterone, the main active androgen, shows up clinically in dermatological symptoms such as hirsutism, hyperseborrheic acne and alopecia. Polycystic ovary syndrome (PCOS) is the most frequent androgen disorder of ovarian function. PCOS patients have amenorrhea or severe oligomenorrhea, increased testosterone levels and most often enlarged polycystic ovaries on ultrasound examination. In addition, many PCOS patients have a tendency to accumulate abdominal fat and/or to develop obesity. Some also display a particular metabolic pattern including an atherogenic lipid profile, glucose intolerance and an increased fasting insulin level, which is known to be closely linked with an insulin resistant state. Several studies have now reported that PCOS patients show increased incidence of type 2 diabetes and cardiovascular disease. In addition to being a target for androgens the skin has abundant insulin receptors on the keratinocyte surface membrane and acanthosis nigricans is a common symptom of severe insulin resistance among patients with insulin receptor disorders. How-

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ever, acanthosis nigricans could also be present in PCOS women given evidence of the intensity of their insulin resistance. This presentation will review the mutual relationship between hyperandrogenia and insulin resistance, with particular attention paid to: (1) insulin secretion and insulin sensitivity in PCOS; (2) the complexity of the molecular mechanisms involved in insulin resistance; (3) the paradoxical relationship between insulin resistance and hyperandrogenia; (4) the current genetic studies; and (5) new avenues for long-term treatment of PCOS women. Copyright © 2001 S. Karger AG, Basel

General Background of Hyperandrogenia in Women

In women, increased production, delivery or bioactivity of testosterone, the main active androgen, can achieve hyperandrogenia. Dermatological symptoms such as hirsutism (excessive hair growth with a male pattern), recurrent acne with increased sebum production and/or alopecia are generally the clinical evidence of hyperandrogenia. In rare situations, laboratory investigations will suggest an androgen secreting tumour or a non-classical form of 21hydroxylase deficiency because serum levels of testosterone or its precursors, ¢4-androstenedione or 17-hydroxyprogesterone, are massively increased [1]. However, in most patients with a complaint of hirsutism and/or acne,

Prof. Michel Pugeat Fédération d’Endocrinologie de l’Hôpital de l’Antiquaille Hospices Civils de Lyon F–69321 Lyon Cedex 05 (France) Tel. +33 4 72 38 50 88, Fax +33 4 72 38 65 86, E-Mail [email protected]

serum testosterone will be slightly increased and difficult to distinguish from normal non-hirsute women. Indeed, patients with clinical hyperandrogenia may have normal androgen production but increased skin delivery of free testosterone because sex hormone-binding globulin (SHBG), the specific transport system of sex-steroid hormones, is decreased [2–3]. It is interesting to note that many studies have shown that low SHBG levels are inversely correlated to increased body mass index (BMI); a finding generally explained by the potent inhibitory activity of insulin on SHBG liver production [4]. The biological activity of testosterone can also be amplified by increased 5·-reductase activity that converts testosterone to a more active androgen, dihydrotestosterone (DHT), which has a higher affinity for the androgen receptor [5]. Finally, increased transactivation activity of the androgen receptor could also be associated to skin hypersensitivity to androgens in patients with the so-called idiopathic hirsutism [6]. The ovaries are the main source of androgens and their secretion is achieved under the control of the pituitary luteinizing hormone (LH). Adrenals also contribute to androgen production by secreting dehydroepiandrosterone (DHEA) and its ester sulphate, DHEAS. DHEA is a weak androgen, but can be transformed in more active androgens, androstenedione and testosterone, in peripheral tissues such as liver, muscle, and skin [7]. The adrenal secretion of DHEA is under the control of the pituitary adrenal corticotrophin hormone (ACTH). DHEA production is as large as cortisol but follows a different pattern during life with a progressive increase from prepuberty to adulthood and a progressive but profound decline during ageing. Although increased DHEA secretion can be associated to pituitary adenoma, as described in Cushing disease, many hirsute patients have a functional oversecretion of adrenal androgens that is dissociated from normal cortisol secretion and whose mechanisms are still poorly understood.

Insulin and Androgen Secretion

Some evidence that hyperandrogenia could be associated to insulin disorder was suggested by the description of ‘diabète des femmes à barbe’ by Achard and Thiers (1921) who reported that severe facial hirsutism in women could be associated to obesity and early development of type 2 (non-insulin-dependent) diabetes [8]. The polycystic ovary syndrome (PCOS) first described by Stein and Leventhal (1935) is now recognised as the most fre-

Association of Insulin Resistance with Hyperandrogenia in Women

quent androgen disorder of ovarian function in premenopausal women [9]. PCOS patients have clinical hyperandrogenia with amenorrhea or severe oligomenorrhea. They have increased testosterone or androstenedione levels and typically show enlarged ovaries with dozens of small peripheral cysts when ultrasound is performed. Many PCOS patients have a tendency to accumulate abdominal fat and/or to develop obesity. They also display a particular metabolic profile including an atherogenic lipid profile (an increase in triglyceridemia and a decrease in HDL-cholesterol), glucose intolerance and an increased fasting insulin level, which is known to be closely linked with an insulin resistance state. Acanthosis nigricans is a common symptom of severe insulin resistance in patients with insulin receptor disorders. Despite the absence of any abnormality of the insulin receptor, several patients have PCOS associated with acanthosis nigricans. This association was first described as the HAIR-AN syndrome for hyperandrogenism insulin resistance and acanthosis nigricans [10].

Reciprocal Relationship of Hyperinsulinemia and Hyperandrogenia

There is some evidence that androgen administration can induce insulin resistance in both males and females, however most studies have shown that insulin resistance is poorly improved in PCOS patients receiving antiandrogens or drugs to suppress ovarian androgen secretion such as birth pill control or GnRH agonist [see ref. 11]. On the other hand, insulin receptors as well as insulin-like growth factor 1 (IGF-1) receptors are abundant on the ovaries and the synergistic effect of insulin on the LHdependent androgen secretion has been shown on ovarian thecal and stroma cells in vitro [see ref. 11]. By decreasing insulin secretion, insulin-sensitizing agents such as metformin and troglitazone have been shown to also decrease ovarian androgen secretion in PCOS patients and finally to improve their rate of ovulation [review in ref. 12]. These findings suggested that ovarian androgen excess is linked to insulin secretion in most patients with PCOS.

Insulin Secretion and Insulin Sensitivity in PCOS

Several studies have clearly shown that PCOS patients have increased fasting insulin levels and increased response to an oral glucose challenge test. This is not

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restricted to obese patients but can also be found in nonobese patients [13–16]. However, it is important to note that defective insulin secretion seems to be more pronounced in patients with a first degree relative with type 2 diabetes [15]. Hyperinsulinemia observed in more than half of the PCOS patients is certainly a result of a decrease in the peripheral actions of insulin, and one explanation for hyperinsulinemia in PCOS patients is that they are insulin resistant. The concept of insulin resistance emerges from the normal dose-response relationship between insulin and specific metabolic processes in insulin sensitive tissues. Insulin resistance occurs when normal circulating concentrations of insulin are insufficient to regulate normal physiological response. To assess insulin sensitivity in vivo, the ‘gold standard’ technique is the euglycaemic hyperinsulinaemic clamp procedure that provides the most precise characterisation of insulin action on carbohydrate metabolism in vivo. It simply requires an insulin infusion and, to maintain euglycemia, a glucose infusion. After an appropriate period of steady state to suppress endogenous glucose production, mainly from the liver, the rate of glucose infusion is equal to the rate of peripheral glucose disposal. The calculated steady state glucose disposal is an indicator of insulin action on the whole body. Using euglycaemic hyperinsulinemic clamp, insulin resistance has been documented by several studies. Dunaif et al. [13] reported that women with PCOS have decreased glucose disposal and that insulin resistance was not restricted to obese women with PCOS. In a recent study we found that 50% of non-obese patients with PCOS were insulin resistant, insulin resistance being more pronounced in PCOS patients with a familial history of diabetes [Ducluzeau et al., in preparation].

Molecular Mechanisms Involved in Insulin Resistance of PCOS

The cellular mechanisms of insulin-resistance have been studied by several groups and are still a matter of extensive investigation. In vitro data obtained in adipocytes from PCOS women have shown normal insulin receptor binding capacity and affinity, normal total phosphorylation of the insulin receptor ß-subunit, but an abnormal dose-dependent effect of insulin on glucose transport, suggesting a post-receptor defect [17]. To go further into the molecular mechanism of insulin resistance, phosphorylation studies of the insulin receptor ßsubunit in fibroblasts from PCOS patients have shown

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that around half of the PCOS patients have an increased basal phosphorylation of serine residue, which contrasts with the blunted insulin-stimulated autophosphorylation of the tyrosine residue. This reduced autophosphorylation may be responsible for insulin resistance in these particular PCOS patients [18]. In addition, it was demonstrated that a factor located in the fibroblast membrane was responsible for the excessive serine-phosphorylation of the insulin receptor ß-subunit. Thus insulin resistance may have resulted from a cell membrane-associated factor, presumably a serine/threonine kinase, that phosphorylates serine residues of the insulin receptor ß-subunit, thereby inhibiting insulin signaling. These findings create a unified hypothesis defended by our colleagues in the US [19]. The hypothesis proposes that increased serine kinase activity may have two consequences. First, it could induce an increase in phosphorylation of serine residues in the insulin receptor ß-subunit and consequently a decrease in tyrosine kinase activity of the insulin receptor, explaining insulin resistance. Second, it could also increase the serine phosphorylation of P450-17·, the key enzyme of androgen synthesis. This enzyme ensures 17·hydroxylase activity for the synthesis of 17-hydroxyprogesterone and 17,20-lyase activity for the synthesis of androstenedione, the precursor of testosterone. Increased serine phosphorylation of P450-17· may increase 17,20lyase activity and is directly associated to functional ovarian as well as adrenal hyperandrogenia in PCOS patients [20]. The genetic basis of such abnormalities remains to be defined. However, 50% of the PCOS patients studied by Andrea Dunaif had normal receptor tyrosine kinase activity [18], suggesting a defect downstream of insulin receptor phosphorylation. The insulin receptor can phosphorylate intracellular substrates such as IRS-1 and IRS-2 and initiate signal transduction and pleiotropic actions of insulin [21–22]. The activation of phosphatidylinositol 3-kinase (PI 3-kinase) by tyrosine-phosphorylated IRS-1 appears to be the pathway for insulin-mediated glucose transport [23]. Insulin-stimulated glucose uptake is largely mediated by the GLUT-4 muscle and adipose glucose transporter, which undergoes a translocation from an intracellular pool to the plasma membrane in response to insulin stimulation. In adipocyte cell membranes from PCOS patients, a decreased content of GLUT-4 glucose transporters has been shown [24]. As expected, after three days of a high carbohydrate diet to improve insulin secretion, a 40% decrease in GLUT-4 content in adipocyte membranes was found in obese normal women compared with lean women. However, independent of obesity, patients

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with PCOS showed a 36% decrease in GLUT-4 content. There was a highly significant positive correlation between GLUT-4 content and insulin-stimulated glucose transport. This study suggested that insulin resistance in PCOS patients is associated with decreased GLUT-4 abundance on the adipocyte plasma membrane [24].

Genetic and PCOS

Familial aggregation of PCOS has been reported by several studies and is consistent with a genetic basis. Most studies have proposed a single dominant gene with high penetrance [25], but some have suggested epigenetic transmission with few causative genes [26]. A recent study from the US tested 37 candidate genes that are involved in steroid metabolism, synthesis and regulation, gonadotropin action, the insulin signalling pathway and obesity [27]. They used an affected-sib pair test for linkage analysis and a transmission/disequilibrium test for association between alleles of the candidate genes and the PCO phenotype as well as the hyperandrogenia phenotype. They report that the strongest evidence for linkage was found with follistatin. Follistatin is a specific binding protein for activin, a paracrine factor that inhibits androgen production by ovarian thecal cells, stimulates the pituitary secretion of FSH, and also stimulates insulin secretion [see ref. 27]. Therefore increasing expression of follistatin accounts all at once for the decreased FSH that explains chronic anovulation, the increased androgen secretion and abnormal insulin secretion that are the features of PCOS. However, when enough families were sampled [27], the evidence of linkage of follistatin with PCOS became less significant [28]. This approach opens a new paradigm that might be quite

effective for identifying dominant genes when a complete gene scan study has been achieved.

New Avenues for Long-Term Treatment of Insulin Resistance in PCOS Women

In PCOS patients, low calorie diets resulting in weight loss have been shown by several investigators to efficiently decrease fasting insulin levels and also to improve hyperandrogenism by increasing SHBG and decreasing androgens levels [29–31]. Interestingly, diet weight loss only achieved reduction in 17-hydroxyprogesterone in PCOS obese but not in ovulatory obese women [32]. These findings confirmed that ovarian overproduction of 17-hydroxyprogesterone is a specific feature of PCOS [20] and decreasing insulin secretion [32] can reduce that ovarian hyperactivity. The biguanide metformin is a drug commonly used for increasing insulin sensitivity without producing hypoglycemia. Several studies have investigated the potential benefit of metformin administration in obese patients with PCOS [12]. The results of metformin administration in lean PCOS patients [33] support the idea that metformin has a specific molecular effect on insulin secretion and/or on insulin signal transduction. Because metformin has not been demonstrated to have adverse teratogenic effect it is a new tool for restoring ovulation in many PCOS patients but requires further clinical investigation [34]. In addition, and more importantly, the benefit of metformin administration on metabolic and lipid disorder has opened a new field of insulin sensitizing agents in the primary prevention of type 2 diabetes and cardiovascular risk in PCOS patients [35–38].

References 1 Androgen secreting ovarian neoplasms; in Azziz R, Nestler and Devailly D (eds): Androgen Excess Disorders in Women. Philadelphia/ New York, Lippincott-Raven, 1997, pp 555– 568. 2 Rosner W: The functions of corticosteroidbinding globulin and sex hormone-binding globulin: Recent advances. Endocr Rev 1990; 11:80–91. 3 Hammond GL: Potential functions of plasma steroid-binding proteins. Trends Endocrinol Metab 1995;6:298–304.

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4 Pugeat M, Crave JC, Elmidani M, Nicolas MH, Garoscio-Cholet M, Lejeune H, Déchaud H, Tourniaire J: Pathophysiology of sex hormonebinding globulin (SHBG): Relation to insulin. J Steroid Biochem 1991;40:841–849. 5 Serafini P, Ablan F, Lobo RA: 5alpha-Reductase in the genital skin of hirsute women. J Clin Endocrinol Metab 1985;60:349–355. 6 Vottero A, Stratakis CA, Ghizzoni L, Longui CA, Karl M, Chrousos GP: Androgen receptormediated hypersensitivity to androgens in women with nonhyperandrogenic hirsutism: Skewing of X-chromosome inactivation. J Clin Endocrinol Metab 1999;84:1091–1095.

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7 Labrie F, Bélanger A, Simard J, Liuu-The V, Labrie C: DHEA and peripheral androgen and estrogen formation: Intracrinology; in Bellino FL, Daynes RA, Hornsby PJ, Lavrin DH, Nestler JE (eds): Dehydroepiandrosterone (DHEA) in aging. Ann NY Acad Sci 1995;74: 16–28. 8 Jeffcoate W, Kong MF: Diabète des femmes à barbe: a classic paper reread. Lancet 2000;356: 1183–1185.

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9 Franks S: Medical Progress: Polycystic ovary syndrome. N Engl J Med 1995;28:853–861. 10 Barbieri RL: Hyperandrogenism, insulin resistance and acanthosis nigricans: 10 years of progress. J Reprod Med 1994;39:327–336. 11 Poretsky L: Insulin resistance and hyperandrogenism: Update 1994. Endocr Rev 1994;2: 125–129. 12 Pugeat M, Ducluzeau PH: Insulin resistance, polycystic ovary syndrome and metformin. Drugs 1999;58(suppl 1):41–46. 13 Dunaif A: Insulin resistance and the polycystic ovary syndrome: Mechanism and implications for pathogenesis. Endocrine Rev 1997;6:774– 800. 14 Holte J, Bergh T, Berne C, Berglund L, Lithell H: Enhanced early insulin response to glucose in relation to insulin resistance in women with polycystic ovary syndrome and normal glucose tolerance. J Clin Endocrinol Metab 1994;78: 1052–1058. 15 O’Meara NM, Blackman JD, Ehrmann DA, Barnes RB, Rosenfield RL, Jaspan JB, Polonsky KS: Defect in beta cell function in functionnal ovarian hyperandrogenism. J Clin Endocrinol Meatb 1993;76:1241–1247. 16 Morin-Papunen LC, Vauhkonen I, Koivunen RM, Ruokonen A, Tapanainen JS: Insulin sensitivity, insulin secretion, and metabolic and hormonal parameters in healthy women and women with polycystic ovarian syndrome. Hum Reprod 2000;15:1266–1274. 17 Ciaraldi TP, El-Roeiy A, Madar Z, Reichart D, Olefsky JM, Yen SSC: Cellular mechanisms of insulin resistance in polycystic ovary syndrome. J Clin Endocrinol Metab 1992;75:577– 583. 18 Dunaif A, Xia J, Book C, Schenker E, Tang Z: Excessive insulin receptor serine phosphorylation in cultured fibroblasts and in skeletal muscle: A potential mechanism for insulin resistance in the polycystic ovary syndrome. J Clin Invest 1995;96:801–810. 19 Zhang LH, Rodriguez H, Ohno S, Miller WL: Serine phosphorylation of human P450c17 increases 17,20 lyase activity: Implications of adrenarche and the polycystic ovary syndrome. Proc Nat Acad Sci USA 1995;92:10619– 10623.

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20 Ehrman DA, Barnes RB, Rosenfield RL: Polycystic ovary syndrome as a form of functional ovarian hyperandrogenism due to dysregulation of androgen secretion. Endocr Rev 1995; 13:322–353. 21 Aguirre V, White MF: Dysregulation of IRSproteins causes insulin resistance and diabetes. Curr Opin Endocrinol Diabetes 2000;7:1–7. 22 Czech MP, Corvera S: Signaling mechanisms that regulate glucose transport. J Biol Chem 2000;274:1865–1868. 23 Pessin JE, Saltiel AR: Signalling pathways in insulin action: Molecular targets of insulin resistance. J Clin Invest 2000;106:165–169. 24 Rosenbaum D, Haber RS, Dunaif A: Insulin resistance in polycystic ovary syndrome: decreased expression of GLUT-4 glucose transporters in adipocytes. Am J Physiol 1993;264: E197–E202. 25 Legro RS, Driscoll D, Strauss JF, Fox J, Dunaif A: Evidence for a genetic basis for hyperandrogenemia in polycystic ovary syndrome. Proc Nat Acad Sci 1998;95:14956–14960. 26 Franks S, Gharani N, Waterworth DM, Batty S, White D, Williamson R, McCarthy M: The genetic basis of polycystic ovary syndrome. Hum Reprod 1997;12:2641–2648. 27 Urbanek M, Legro RS, Driscoll DA, Azziz R, Ehrmann DA, Norman RJ, Strauss JF, Spielman RS, Dunaif A: Thirthy-seven candidate genes for polycystic ovary syndrome: strongest evidence for linkage is with follistatin. Proc Nat Acad Sci USA 1999;96:8573–8578. 28 Azziz R, Saenger P: The second international symposium on the developmental aspects of androgen excess. Toronto, Canada, 20 June 2000. Trends in Endocrinol and Metab 2000; 11:8:338–340. 29 Pasquali R, Antenucci D, Casimirri F, et al: Clinical and hormonal characteristics of obese amenorrheic hyperandrogenic women before and after weight loss. J Clin Endocrinol Metab 1989;68:173–179.

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30 Kiddy DS, Hamilton-Fairley D, Seppälä M, et al: Diet-induced changes in sex hormone binding globulin and free testosterone in women with normal or polycystic ovaries; correlation with insulin and insulin-like growth factor-I. Clin Endocrinol 1989;31:757–763. 31 Crave J, Fimbel S, Lejeune H, et al: Effects of diet and metformin administration on sex hormone-binding globulin, androgens, and insulin in hirsute and obese women. J Clin Endocrinol Metab 1995;80:2057–2062. 32 Jakubowicz DJ, Nestler JE: 17·-hydroxyprogesterone responses to leuprolide and serum androgens in obese women with and without polycystic ovary syndrome after dietary weight loss. J Clin Endocrinol Metab 1997;82:556– 560. 33 Nestler JE, Jakubowitcz D: Lean women with polycystic ovary syndrome respond to insulin reduction with decreases in ovarian P450c17· activity and serum androgens. J Clin Endocrinol Metab 1997;82:4075–4079. 34 Nestler JE, Jakubowicz DJ, William SE, et al: Effects of metformin on spontaneous and clomiphene-induced ovulation in the polycystic ovary syndrome. N Engl J Med 1998;338: 1876–1880. 35 Dahlegren E, Janson PO, Johansson S, et al: Polycystic ovary syndrome and risk for myocardial infarction-evaluated from a risk factor model based on a prospective study of women. Acta Obstet Gynecol Scand 1992;71:599–604. 36 Conway GS, Agrawal R, Betteridge DJ, Jacobs HS: Risk factors for coronary artery disease in lean and obese women with the polycystic ovary syndrome. Clin Endocrinol 1992;37:119– 125. 37 Robinson S, Henderson AD, Gelding SV, et al: Dyslipidemia is associated with insulin resistance in women with polycystic ovaries. Clin Endocrinol 1996;44:277–284. 38 Franks S: Polycystic ovary syndrome: Approaching the millennium. Hum Reprod 1997; 12(suppl):43–45.

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The Molecular Basis of Androgen Insensitivity Esther M. Nitsche Olaf Hiort Department of Pediatrics, Medical University of Lübeck, Germany

Key Words Sex differentiation W Androgen insensitivity W Androgen receptor W Genetics

Abstract Androgen action is mediated in the peripheral target cell via the androgen receptor (AR). The AR is a nuclear transcription factor, combining a DNA-binding and a hormone-binding domain with a large transactivation unit. Androgen insensitivity syndrome (AIS) as the clinical entity of defective androgen action with variable phenotypes in 46,XY patients is caused by mutations of the Xchromosomal AR gene. Most variations in the AR gene are point mutations inhibiting either hormone or DNA binding. However, even within the same family, the phenotype for a given mutation can vary widely. Only few influential factors have been identified for the phenotypic diversity. For mutations affecting hormone binding, ligand concentration variability during fetal life may be an important influence on residual androgen action. A second factor is the occurrence of postzygotic de novo mutations, which are present at a high rate in single-case families. These somatic mutations lead to expression of both mutant and wild-type AR in a single patient and thus allow androgen action despite a deleterious mutation of the AR gene. Third, residual androgen response may be mediated by additional transcripts of the AR gene which are present in several cell types and can be affected in a different pattern by splice-site mutations. Whether differential expression of AR-interacting pro-

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teins has an influence on phenotype has not yet been proven. Moreover, little is known about the regulation of AR-dependent genes. Their identification is needed to understand post-AR action and, hence, androgenic control of sexual differentiation and maturation. Copyright © 2001 S. Karger AG, Basel

Androgens are responsible for a wide range of biological effects during embryonic, fetal and postnatal life. Their effects are mediated via the androgen receptor (AR), a nuclear transcription factor. The majority of effects is due to binding of two distinct ligands to the androgen receptor, testosterone (T) and its 5-·-reduced metabolite dihydrotestosterone (DHT). In contrast to many other steroid hormone receptors, to this date only one type AR has been identified, presumably mediating all the different known androgen-dependent effects. The single copy gene for the AR is encoded on the X chromosome and mutations in the receptor gene lead to a wide spectrum of impaired androgen action, clinically described as androgen insensitivity (AIS) ranging from complete testicular feminization (complete androgen insensitivity, CAIS) to partial androgen insensitivity (PAIS) presenting with ambiguous genitalia and finally mild androgen insensitivity with impairment of spermatogenesis and consequently sub- to infertility in later life [1]. Moreover, a large number of conditions besides AIS have been associated with AR dysfunction: prostate cancer [2], breast cancer [3], androgenic alopecia in men [4] and hirsutism in women [5], as well as conditions that are not

Olaf Hiort, MD Department of Pediatrics, Medical University of Lübeck Ratzeburger Allee 160, D–23538 Lübeck (Germany) Tel. +49 451 500 2191, Fax +49 451 500 2184 E-Mail [email protected]

Fig. 1. Grades of virilization disorder of the external genitalia. Below are clinical examples of phenotypes grade 2–4. From Hiort et al., Deutsches Ärzteblatt 1999;96:A688.

obviously androgen regulated such as spinal and bulbar muscular atrophy [6] and rheumatoid arthritis [7]. The predominant roles of androgen action, however, are male sexual differentiation and virilization [8]. As an X-linked condition, AIS due to point mutations of the AR gene primarily afflicts individuals with 46,XY karyotype . Clinical observation also shows that the dysfunctional AR can lead to a variety of clearly visible abnormalities, but also that androgens and AR function do not seem to be essential for life, as life expectancy is not altered in these patients. Due to advancing molecular biological techniques, a stunningly large number of more than 300 patients with mutations in the AR have been reported during the past 10 years [9]. Unlike other hormone receptors there is no classical ‘hot spot’ for mutations. The large number of mutations alone, however, does not explain the variability of the clinical picture of AIS. Extended studies by different groups including our own have shown that the pheno-

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type caused by the same point mutation even in one family can vary greatly, suggesting additional factors modifying the effect of the mutated AR.

The Clinical Spectrum of AIS

Androgen insensitivity due to a mutation of the AR gene is a relatively rare disorder and occurs in about 1: 20,000 male births. AR gene mutations result in a wide spectrum of phenotypes. Similar to the classification of virilization in patients with adrenal hyperplasia, efforts have been made to classify the degree of impaired virilization in such patients (fig. 1: Classification according to Sinnecker et al. [10]) according to the aspect of the external genitalia. Patients with CAIS present as normal females with normal external genitalia and will be missed at birth. At puberty they develop normal breasts but reduced or lack of pubic or axillary hair. The gonads

Nitsche/Hiort

Fig. 2. The human androgen receptor – from

chromosomal localization to genomic structure, cDNA and protein domains. Modified from Hiort et al., Deutsches Ärzteblatt 1999; 96:A688.

(testes) in these patients are located either intra-abdominal or within the labia maiora. During embryonic development the testes produce adequate amounts of anti-mullerian hormone, thus these individuals have a blind ending vagina and lack mullerian structures, i.e. no uterus and no fallopian tubes. In PAIS, the phenotype can range from a predominantly female phenotype with signs of slight virilisation over patients with ambiguous genitalia to a predominantly male phenotype with various degrees of urogenital abnormalities such as hypospadia, micropenis and cryptorchdism. Minimal AIS due to mutation of the AR gene has been found in a small number of patients as the cause of impaired spermatogenesis and infertility [11].

The androgen receptor is a ligand-activated nuclear transcription factor and belongs to the steroid receptor family, which includes amongst others the estrogen receptors · and ß, the mineralo- and glucocorticoid receptors, the thyroid hormone receptor and the vitamin D receptor. All mentioned hormones have in common that they consist of three main functional domains: The transactivation domain, the DNA-binding domain and the ligandbinding domain. The AR gene is located closely to the centromere of the X chromosome at Xq11-12 and consists of 75–90 kb of genomic DNA containing 8 exons.

The largest domain is the transactivation domain located at the N-terminus. Two polymorphic repeat regions are within this domain, including a variable CAG (glutamine) repeat that is expanded in spinal and bulbar neuromuscular atrophy (Kennedy’s disease). The DNA-binding domain, which is the smallest and shows the largest degree of conservation, is encoded by exon 2 and 3 and contains two zinc finger proteins essential for specific DNA binding. The ligand-binding domain is located in the C-terminal region of the molecule encoded by exon 4–8 (fig. 2: Gene to protein in the AR). The mature protein consists of 12 ·-helices and 2 ß-sheets forming a central hydrophobic ligand-binding pit [12]. Point mutations leading to AIS are mostly located in the DNA-binding or ligandbinding domain of the molecule [9]. Following ligand binding and receptor dimerization, a complex process for which the presence of HSP70, HSP90 (heat shock proteins 70 and 90) as well as HSP40 (heat shock protein 40, DnaJ) were found to be essential [13], the hormone-receptor complex is translocated from the cytoplasm into to nucleus. Together with co-activator proteins such as TIF2 (transcriptional factor 2) SRC1 (steroid receptor coactivator1) or ARA24 it forms pre-initiation complexes, that bind to specific DNA sequences, androgen response elements (ARE), in the promoter regions of androgen-regulated genes to specifically mediate gene expression (fig 3: molecular events involved in induction of androgenic effect). Mutations leading to impaired virilization are found mainly in the hormone- and DNA-binding domain. Mod-

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Androgen Receptor Structure and Functional Features

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Fig. 3. Molecular events in androgen action. Testosterone enters the target cells, is partially converted to dihydrotestosterone. Both androgens are able to bind to the androgen receptor, which translocates to the nucleus, dimerizes, forms transactivation complexes, and binds to androgen responsive elements (ARE) in the target DNA to regulate transcription and translation of response proteins.

ification of receptor-mediated androgen action due to alterations of the N-terminal transactivation domain, which at the same time is the least conserved domain of the androgen receptor, seem to play an important role in a different context. The transactivational domain caught the interest of science, when the extension of the polymorphic trinucleotide repeat segment (CAG)n in this region encoding a polyglutamine tract was demonstrated in patients with X-linked spinal bulbar muscular atrophy (SBMA) [6]. Expansion of trinucleotide repeats are found responsible for a number of other neurologic diseases such as fragile X syndrome, Huntington’s disease, spinocerebellar ataxia type I, dentatorubral-pallidoluysian atrophy, Machado-Joseph’s disease and Friedreich’s ataxia [14]. Although the described regions are variable in healthy subjects, in patients affected by such disease, they are expanded beyond the expected number. In case of the AR in patients with SBMA 40 or more CAG repeats are found as opposed to a maximum of 35 in healthy people. The mechanisms by which the extension of the CAG repeat lead to disease remain unknown, but it has been postulated that some derivative of the full-length protein is a toxic product [15]. Nevertheless, slight impairment of virilization is present in some patients [16]. However, CAG repeat number variations are not only associated with neurologic dysfunction. A shortened CAG repeat of 22 or less seems to be associated with an increased risk of prostate cancer [2]. Men with a repeat of 18 or less were find to have a 50% increased risk of prostate cancer as compared to men whose AR gene carries 26

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or more repeats [17] and the shorter the repeat sequence the more aggressive the cancer was found to be [18]. The hypothesis is that shorter CAG repeats lead to an increased androgenicity and, hence, stimulation of the prostatic tissue, resulting in early onset and fast expansion of prostatic tumors. Excessively long CAG repeats as in SBMA interfere with androgen action and short CAG repeats enhance androgen action. On this basis, patients with impaired spermatogenesis were investigated for AR CAG repeat length and found in some studied to have normal yet significantly longer CAG repeats as compared to fertile controls [19]. This has been challenged by our own extended study which showed no significant difference in CAG repeat length between fertile and infertile males [11, 20]. In women with BRCA1 mutations carrying a long CAG repeat in the AR at the same time, were found to develop breast cancer earlier than in women with short CAG repeats in the AR gene [3]. Short CAG repeats and hence increased androgen action are also associated with malepattern baldness and [4] and the younger onset of rheumatoid arthritis in males [7]. The underlying molecular basis of increased androgen action via AR with shorter CAG repeats is not entirely clear yet. However, a nuclear co-activator protein (ARA24) has been identified that binds to the poly-glutamine tract of the AR. Binding is stronger to AR molecules with shorter CAG repeats thus leading to an increased transactivation of the AR-hormome co-activator complex [21].

Nitsche/Hiort

Fig. 4. Point mutations found in the human

androgen receptor gene in the German collaborative intersex study. Note that most mutations are amino acid substitutions localized in exons coding for the hormone binding domain of the androgen receptor.

Identification and Characterization of AR Gene Mutations

First approaches to AR function assessment were made by ligand binding studies in cultured genital skin fibroblasts of patients with AIS in comparison to normal controls. This method offered an estimation of quantitative as well as to a certain degree qualitative ligand binding to the androgen receptor. These studies were highly valuable at the time, however they required a genital skin biopsy, were labor intensive and time consuming, and they offered little information as to the underlying molecular defect [1]. In 1988 the cloning of the androgen receptor [22, 23] opened the possibilities of sequence analysis, which later on [24] was facilitated by new and faster techniques such as single strand polymorphism analysis for screening prior to sequencing of the exons. It was of little surprise that the mutations in the AR gene leading to complete or partial loss of AR functionality were found mainly in the DNAand ligand-binding domains (fig. 4: AR gene with mutations). However the mutations found are scattered quite randomly on exon 2–8, a hot spot where frequent functionally relevant mutations occur could not be identified. The obtained results offered information on the type of mutation. A single mutation can result in a stop codon instead of coding for an amino acid and hence cause premature termination of protein synthesis. As the protein is synthesized from the N-terminus, i.e. the transactivational domain, towards the C-terminus, i.e. the hormone binding domain, a truncated protein would most likely lack either a functional hormone-binding domain or both

hormone- and DNA-binding domains. A point mutation can also lead to an exchange of an amino acid, leading to an altered protein structure. Deletions and insertions can result in open reading frame shifts, which again lead to an altered ‘nonsense’ protein. A splice site mutation affecting the mRNA splicing may have the same effect [25]. In addition to the qualitative impairment of the mutated AR protein, there is evidence that in many mutations the quantity of expressed AR protein is also reduced [26, 27]. Except for mutations resulting in truncated receptor proteins, the sequencing results give little information on the degree of functional impairment, that is to be expected. For further functional characterization of an AR mutant, co-transfection techniques have been applied. A cell line lacking the AR is transfected with an expression vector carrying the mutated AR as well as with a second plasmid containing a suitable reporter gene, that can be activated via an androgen response element (ARE) located in the promoter region of the gene. When AR is being expressed in the presence of ligand, the degree to which the reporter gene is turned on is dependent on AR functionality. This technique again is highly valuable, yet has its clear limitations. The AR in these systems is overexpressed and available in far higher concentrations than in vivo, moreover a number of steps required for AR function in vivo as translocation of the molecule into the nucleus binding to additional transcription factors for differential gene regulation are not being evaluated and the investigation takes place in a non-human cell environment. Another attempt is the transfection of genital skin fibroblasts with a plasmid carrying a reporter gene and an ARE in the promoter region. Again in patients with CAIS

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the reporter gene activation was found extremely low, whilst the variability of normal controls was remarkable. However, it has been published that AR functionality of patients with PAIS and AR mutations can be further evaluated employing this method [28].

Facts and Speculations on Phenotype Variability in Patients with Androgen Insensitivity Syndrome (AIS) due to a Mutation in the Androgen Receptor

Up to date only very few factors have been identified that help explain the remarkable variability in the clinical picture of one and the same point mutation – even within the same family. For mutations in the hormone binding domain varying androgen levels during fetal life have been postulated, influencing the residual AR function. In some patients with de novo mutations, the mutation has occurred postzygotically, leading to a mosaic phenotype, as could be shown for patients by our own group [29]. With some cells carrying the mutant AR gene and some the wild type, the degree of AIS will depend on the number of cells affected by the somatic mutation and allow partial androgen sensitivity even in presence of a mutation leading to complete loss of AR function. Furthermore, several transcripts of the AR have been shown to be present in different cell types. These transcripts could be affected differently for instance in splice site mutations [25]. Finally, it may be speculated that allelic differences

or levels of expression of proteins interacting with the AR or AR-hormone complex at different stages could modify the residual activity of androgens in patients with AIS due to a mutation of the AR gene as was shown for the nuclear co-activator ARA24 in patients with altered CAG repeat lengths in the transactivation domain of the AR gene [21]. Many attempts have been made to identify androgenregulated genes and a number of those genes were isolated from different tissues. Some gained clinical importance like SHBG (sexual hormome-binding globulin) expressed in the liver and used as an inducible clinical marker for androgen sensitivity in AIS [10]. Most of these genes, however, were isolated from mostly epithelial tumors or tumor cell lines like breast and prostate cancer, LNCaP or MCF7 cells. As was demonstrated in animal models, not the epithelial AR is important for male differentiation but the mesenchymal functional AR alone induces formation of the male urogenital tract. Hence, it has to be assumed that the morphogens responsible for androgen-dependent development are expressed in the mesenchyme. Up to date a number of candidate genes have been isolated, but so far none of them was proven to be a relevant androgendependent morphogen [31] Further research in this area strongly depends on the isolation and identification of morphogen- and functional androgen-dependent genes for a better understanding of post-AR action and consequently of androgen-dependent differentiation and maturation.

References 1 Quigley CA, Bellis A, Marschke KB, El Awadi MK, Wislon EM, French FS: Androgen receptor defects: Historical, clinical and molecular perspectives. Endocr Rev 1995;16:271–321. 2 Irvine RA, Yu MC, Ross RK, Coetzee GA: The CAG and GGC microsatellites of the androgen receptor gene are in linkage with disequilibrium in men with prostate cancer. Cancer Res 1994;54:2861–2864. 3 Rebbeck TR, Kantoff PW, Krithirvas K, Neuhausen S, Blackwood MA, Godwin AK Daly MB, Narod SA, Garber JE, Lynch HT, Weber BL, Brown M: Modification of BRCA1-associated breast cancer risk by the polymorphic androgen receptor CAG repeat. Am J Hum Genet 1999;64:1371–1377. 4 Sawaja ME, Shalita AR: Androgen receptor polymorphisms (CAG repeat lengths) in androgenic alopecia, hirsutism and acne. J Cutan Med Surg 1998;3:9–15.

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5 Vottero A, Stratakis CA, Ghizzoni L, Longui CA, Karl M, Chrousos GP: Androgen receptor mediated hypesensitivity to androgens in women with nonhyperandrogenic hirsutism: Skewing of X chromosome inactivation. J Clin Endocrinol Metab 1999;84:1091–1095. 6 La Spada AR, Wilson EM, Lubahn DB, Harding AE, Fischbeck KH: Androgen receptor gene mutations in X-linked spinal and bulbar muscular atrophy. Nature 1991;352:77–79. 7 Kawasaki T, Ushiyama T, Ueyama H, Inoue K, Mori K, Ohkubo I, Hukuda S: Polymorphic CAG repeat of the androgenreceptor gene and rheumatoid arthritis. Ann Rheum Dis 1999;58: 500–502. 8 Hiort O, Holterhus PM: The molecular basis of male sexual differentiation. Eur J Endocrinol 2000;142:101–110. 9 Gottlieb B: Androgen receptor gene mutation database. http://www.mcgill.ca/androgendb/

10 Sinnecker GHG, Hiort O, Nitsche EM, Holterhus PM, Kruse K: Functional assessment and clinical classification of androgen sensitivity in patients with mutations of the androgen receptor gene. German Collaborative Intersex Study Group. Eur J Pediatr 1997;156:7–14. 11 Hiort O, Holterhus PM, Horter T, Schulze W, Kremke B, Bals-Pratsch M, Sinnecker GH, Kruse K: Significance of mutations in the androgen receptor gene in males with idiopathic infertility. J Clin Endocrinol Metab 2000;85: 2810–2815. 12 Yong EL, Tut TG, Ghadessy FJ, Prin G, Ratnam SS: Partial androgen insensitivity and correlations with the predicted three dimensional structure of the androgen receptor ligend binding domain. Mol Cell Endocrinol 1998;137:41– 50.

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13 Fliss AE, Rao J, Melville MW, Cheetham ME, Caplan AJ: Domain requirements of DnaJ-loke (Hsp40) moleculare chaperones on the activation of a steroid hormone receptor. J Biol Chem 1999;274:34045–34052. 14 Mitas M: Trinucleotide repeats associated with human disease. Nucl Acids Res 1997;25:2245– 2254. 15 Paulsen HL: Toward an understanding of polyglutamine neurodegeneration. Brain Pathol 2000;10:293–299. 16 Amato AA, Prior TW, Barohn RJ, Snyder P, Papp A, Mendell JR: Kennedy’s disease: A clinicopathologic correlation with mutations in the androgen receptor gene. Neurology 1993; 43:791–794. 17 Giovanucci E, Stampfer MJ, Krithivas K, Brown M, Dahl D, Brufsky A: The CAG repeat within the androgen receptor gene and ist relationship to prostate cancer. Proc Natl Acad Sci USA 1997;94:3320–3323. 18 Stanford JL, Just JJ, Gibbs M, Wicklund KG, Neal CL, Blumenstein BA, Ostrander EA: Polymorphic repeats in the androgen receptor gene: Molecular markers of prostate cancer risk. Cancer Res 1997;57:1194–1198. 19 Dowsing AT, Yong EL, Clark M, McLachlan RI, de Kretser DM, Trounson AO: Linkage between male infertility and trinucleotide repeat expansion in the androgen-receptor gene. Lancet 1999;354:640–643.

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20 Hiort O, Horter T, Schulze W, Kremke B, Sinnecker GHG: Male infertility and increased risk of diseases in future generations. Lancet 1999;354:1907–1908. 21 Hsiao PW, Lin DL, Nakao R, Chang C: The linkage of Kennedy’s neuron disease to ARA24, the first indentified androgen receptor polyglutamine region-associated coactivator. J Biol Chem 1999;274:20229–20234. 22 Chang CS, Kokontis J, Liao ST: Structural analysis of complemetary DNA and amino acid sequences of human and rat androgen receptor. Proc Natl Acad Sci USA 1988;85: 7211–7215. 23 Lubahn DB, Joseph DR, Sar M, Tan J, Higgs HN, Larson RE, French FS, Wilson EM: The human androgen receptor: Complementary desoxyribonucleic acid cloninf, sequence analysis and gene expression in prostate. Mol Endocrinol 1988 2:1265–1275. 24 Hiort O, Huang Q, Sinnecker GH, SadeghiNejad A, Kruse K, Wolfe HJ, Yandell DW: Single strand conformation polymorphism analysis of androgen receptor gene mutations in patients with androgen insensitivity syndromes: Applications for diagnosis, genetic counceling and therapy. J Clin Endocrinol Metab 1993;68:262–266. 25 Hellwinkel OJ, Bull K, Holterhus PM, Homburg N, Struve D, Hiort O: Complete androgen insensitivity caused by a splice donor site mutation in intron 2 of the human androgen receptor gene resulting in an exon 2-lacking transcript with premature stop-codon and reduced expression. J Steroid Biochem Mol Biol 1999; 68:1–9.

26 McIntosh I, Hamosh A, Dietz HC: Nonsense mutations and diminished mRNA levels. Nat Genet 1993;4:219. 27 Ruiz-Echevarria MJ, Gonzales CI, Peltz SW: Identifying the right stop: Determination how the surveillance complex recognizes and degrades an aberrant mRNA. EMBO J 1998;17: 575–589. 28 McPhaul M: Molecular defects of the androgen receptor. J Steroid Biochem Mol Biol 1999:69: 315–322. 29 Holterhus PM, Wiebel J, Sinnecker GHG, Brüggenwirth HAT, Sippell WG, Brinkmann AO, Kruse K, Hiort O: Clinical and molecular spectrum of somatic mosaicism in androgen insensitivity syndrome. Pediatr Res 1999;64: 648–690. 30 Holterhus PM, Sinnecker GHG, Hiort O: Phenotypic diversity and testosterone-induced normalization of mutant L712F androgen receptor function in a kindred with androgen insensitivity. J Clin Endocrinol Metab 2000; 85:3245–3250. 31 Nitsche EM, Moquin A, Adams PS, Guenette RS, Lakins JN, Sinnecker GH,Kruse K, Tenniswood MP: Differential display RT PCR of total RNA from human foreskin fibroblasts for investigation of androgen-dependent gene expression. Am J Med Genet 1996;63:231–238.

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Genetics of Peutz-Jeghers Syndrome, Carney Complex and Other Familial Lentiginoses Constantine A. Stratakis Unit on Genetics & Endocrinology, Developmental Endocrinology Branch, National Institute of Child Health & Human Development, National Institutes of Health, Bethesda, Md., USA

Key Words Peutz-Jeghers syndrome W Carney complex W Lentigines W Multiple neoplasia syndromes W STK11 gene W Protein kinase A W PRKAR1A gene W Cowden disease W PTEN gene

dation of the lentiginoses and their related syndromes identifies new pathways of growth control and cellular regulation that are important for endocrine signaling, tumorigenesis, cutaneous function and embryonic development. Copyright © 2001 S. Karger AG, Basel

Abstract Peutz-Jeghers syndrome (PJS, #175200) and Carney complex (CNC, OMIM#160980) are the two most common multiple neoplasia syndromes associated with lentiginosis. Both disorders are inherited in an autosomal dominant manner and they have recently been elucidated at the molecular level. PJS and CNC share manifestations with Cowden syndrome (or Cowden disease) (CS, OMIM#158350) and Bannayan-Riley-Ruvalcaba syndrome (BRR, OMIM#153480). The endocrine tumors of CS and PJS, which could classify these disorders as variant types of multiple endocrine neoplasias (MENs), are not present in most CS and BRR patients, but lentigines are shared by PJS, CNC and BRR. The serine-threonine kinase STK11 (or LKB1), located on 19p13, is mutated in more than half of all PJS kindreds. The R1· subunit of c-AMP-dependent protein kinase A, located on 17q22– 24, is mutated in 40% of CNC kindreds. The protein phosphatase PTEN is mutated in most cases of CS and in almost 50% of BRR kindreds, despite significant clinical heterogeneity in these syndromes. The molecular eluci-

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Accessible online at: www.karger.com/journals/hre

Introduction

Pigmented spots in the skin and the mucosa that are called lentigines (from the Latin lentigo, ‘small lentil’) are the hallmark of the familial lentiginosis syndromes. These disorders [presented in table 1 with their on-line Mendelian Inheritance in Man (OMIM) [1] catalog numbers] share with the multiple endocrine neoplasias (MENs) and phacomatoses an increased incidence of endocrine, neural and mesenchymal tumors [2]. Lentigines are flat, poorly circumscribed, variegated, brown-to-black macules with a small size, usually not larger than 0.5 cm [2]. The appearance of the lesion may differ in the various skin types; in African-Americans, for example, lentigines may be slightly raised, dark papules, similar to nevi. On histologic examination, lentigines show prominent rete ridges and basal cell layer hyperpigmentation associated with an increased number of melanocytes [2], which distinguishes them from the common

Constantine A. Stratakis, MD Unit on Genetics & Endocrinology, Developmental Endocrinology Branch National Institute of Child Health & Human Development, National Institutes of Health Building 10, Room 10N262, 10 Center Dr., Bethesda, MD 20892-1862 (USA) Tel. +1 301 402 1998, Fax +1 301 402 0574, E-Mail [email protected]

freckles or ephelides (fig. 1). The latter contain a normal number of melanocytes and are pigmented because of melanin donation to adjacent keratinocytes. Lentigines, on the other hand, are associated with true melanocytic hyperplasia. There are several other differences between lentigines and freckles, although, in most cases, histologically and clinically the lesions can be indistinguishable: lentigines are present in early childhood and occur preferentially, but not exclusively, on sun-exposed skin, whereas freckles are smaller (2–4 mm) and usually lighter macules that are found almost exclusively on sun-exposed skin areas, particularly in lightly pigmented persons. Furthermore, lentigines can be deeply pigmented in certain anatomic sites, such as the mucosae of the vermilion border of the lips, the labia majora of the vulva, and the inner and outer canthi of the conjunctivae. This fact is of particular clinical significance, since the presence of lentigines in these areas may suggest an association with one of the

Fig. 1. Diagram of the basal layer of the skin and melanocytes in the normal state (a), freckles (b) and lentigines (c): On histologic examination, lentigines show prominent rete ridges and basal cell layer hyperpigmentation associated with an increased number of melanocytes, which distinguishes them from common freckles (ephelides). Freckles contain a normal number of melanocytes and are pigmented because of melanin donation to adjacent keratinocytes. Lentigines, on the other hand, are associated with true melanocytic hyperplasia.

Table 1. Familial lentiginoses: clinical manifestations and genetics

Disease

MIM

Main manifestations

Genetics

Genes

Carney complex

160980

lentigines, myxomas, endocrine overactivity, schwannomas

AD, chr. 2p16 and 17q22–24 loci

PRKARIA, other

Peutz-Jeghers

175200

lentigines, polyps, neoplasias

AD, chr. 19p and 19q loci

STK11/LKB1, other

LEOPARD

151100

lentigines, cardiac, endocrine, developmental defects, mental deficiency, deafness

AD

Arterial dissections and lentiginosis

600459

dissecting aneurysms of aorta, renal, and carotid arteries connective tissue defects

AR (?)

Laugier-Hunziker disease

–

lentigines, other pigmented lesions

unknown

Lentiginosis

151000 150900

familial, benign lentigines only, occasionally other defects

AD

lentigines, developmental defects

AD, chrom 10q locus

Bannayan-Ruvalcaba- 153480 Riley syndrome

PTEN, other

AD = Autosomal dominant inheritance; AR = autosomal recessive inheritance; LEOPARD = lentigines, electrocardiographic abnormalities, ocular hypertelorism, pulmonary stenosis, abnormalities of the genitalia, retardation, deafness.

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Fig. 2. Pigmentation of a patient with PJS.

familial lentiginosis syndromes. It should be noted, however, that isolated pigmented macules are relatively common in the general population [2, 3], and that perhaps the most frequent type of familial lentiginosis is ‘benign, patterned, centrofacial lentiginosis’, which is particularly common among the African-American population [2–5]. The lentiginoses include the Peutz-Jeghers syndrome (PJS), Carney complex (CNC), LEOPARD, arterial dissections and lentiginosis, Laugier-Hunziker syndromes, Cowden syndrome (CS) (or Cowden disease) and Bannayan-Riley-Ruvalcaba (BRR) or Ruvalcaba-MyhreSmith or Bannayan-Zonana syndrome and segmental lentiginoses, all of which can be associated with a variety of developmental defects. The inheritance of most of these syndromes (with the exception of the arterial dissections and lentiginosis syndrome) is autosomal dominant; genetic loci or genes have been identified for PJS, CNC and CS/BRR syndromes, but not for other lentiginoses. Elucidation of the molecular defects responsible for these disorders is expected to shed light on aspects of early neural crest differentiation, the regulation of pigmentation, the development of autonomous endocrine function, and endocrine and nonendocrine tumorigenesis.

Peutz-Jeghers Syndrome

Clinical Features The syndrome was first described by Peutz [6] and Jeghers et al. [7]. The hallmark of the syndrome is the presence of pigmented spots on the lips, which are first

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present in early childhood (fig. 2). These lesions are associated with gastrointestinal hamartomatous polyps [8]. Gastrointestinal cancers are frequent in PJS; they may arise from genetic changes known to occur in colorectal carcinoma and other tumors, because the PJS-responsible gene(s) function as tumor suppressor(s) [8, 9]. PJS patients are also at an increased risk for breast, ovarian, testicular, uterine and cervical cancers, as well as non-malignant lesions in these tissues [8, 10–13]. A 49-year followup of the ‘Harrisburg family’, the kindred originally described by Jeghers et al., revealed that PJS is a premalignant condition associated with significant morbidity and increased mortality [14]. Among the 12 affected family members, 10 underwent 75 polypectomies, and 2 developed gastric cancer and duodenal carcinoma, respectively. Other investigators found that cancer developed in 15 of 31 patients from 13 unrelated families; the overall incidence of carcinoma in patients with PJS varies from 20 to 50%, and it appears at a relatively early age [10–20]. Among the nongastrointestinal neoplasms associated with PJS, endocrine tumors, including thyroid nodules and cancer, are the most frequent [9–13, 17, 18]. Genital tract neoplasms in female patients with PJS include ovarian neoplasms from both the epithelium and stromal cells, and also adenoma malignum of the cervix and adenocarcinoma of the endometrium [19, 20]. Male patients with PJS often have Leydig cell tumors, or a Sertoli cell tumor that is uniquely found in the two lentiginoses discussed in this chapter, PJS and CNC: large-cell, calcifying Sertoli cell tumor (LCCSCT) [71]. LCCSCT in PJS, like in CNC, may be associated with increased aromatization of adrenal or testicular androgens, which produces estradiol and other estrogens (estrone, in particular) that may lead to precocious puberty, pre-pubertal or peri-pubertal gynecomastia [3, 21]. Molecular Genetics One of the first reports on the genetics of PJS was that of a patient with the syndrome and a pericentric inversion of chromosome 6 [22]. However, other patients with similar chromosomal changes do not manifest symptoms of the disease. More recently, three families with PJS were studied by linkage analysis and were mapped to the short arm of chromosome 1 (1p) [23]; however, the finding ended up being spurious (most likely due to the small number of patients investigated) and was not confirmed in a more recent investigation [24]. A study of comparative genomic hybridisation (CGH) and loss-of-heterozygosity (LOH) analysis defined a susceptibility locus for PJS on 19p, around the microsatellite repeat marker

Stratakis

D19S886 [9]. LOH of 19p13 markers suggested that the gene functions as a tumor suppressor gene. Subsequently, most PJS families were linked to 19p13.3 [25], although some mapped to a second locus at 19q13.4 [26]. The PJS gene at 19p13.3, STK11 (for serine threonine kinase 11) or LKB1 was then identified by two research groups [27, 28]. More than half of the families with PJS have mutations in this gene, although the percentage varies greatly from one study to the other [29–34]. STK11/ LKB1 is a novel serine-threonine kinase containing 9 exons; it shows high homology, almost 84%, with XEEK1 (named for Xenopus egg and embryo kinase 1), a Xenopus cytosolic serine-threonine protein kinase [35]. A mouse homolog was also recently identified [36]. The kinase domain of the STK11/LKB1 gene is highly conserved between mouse and human. Although it has been suggested that mouse Lkb1 is a nuclear protein, wild-type STK11/LKB1 shows both nuclear and cytoplasmic localisation. A number of recent studies have elucidated the effects of inherited STK11/LKB1 mutations in PJS kindreds, based on the functional domains of the protein [37]; in most cases, elimination of the kinase activity underlies the molecular cause of the phenotype. Genetic heterogeneity in PJS appears to be not accompanied by clinical heterogeneity, as there are no known differences between the families that map to 19p13.3 and have STK11/LKB1 mutations and those that map elsewhere or do not have mutations in that gene.

Clinical Features CNC was first reported in 1985, after earlier observations on patients with pituitary-independent Cushing syndrome and an unusual adrenal pathology characterized by multiple, small, pigmented, adrenocortical nodules and internodular cortical atrophy had shown as association with myxomas and lentigines [3, 38–40]. The adrenal condition, which proved to be primary and bilateral, is now commonly referred to as primary pigmented nodular adrenal disease (PPNAD). In the early 1980’s several sporadic cases of PPNAD and two families, one from Cuba and another from Switzerland, were reported [38–41]. More cases were identified later, with various combinations of myxomas affecting multiple organs (heart, skin, and breast), spotty skin pigmentation (lentigines and blue nevi), and tumors of three endocrine organs (adrenal, pituitary, and testis) [42]. Subsequently, it was realized that the syndrome was transmitted in a manner consistent

with dominant inheritance [42, 43]. The characteristic pathology of PPNAD had been described in children and young adults with Cushing syndrome as early as 1949 and in a number of case reports thereafter [17, 43]. Also, several familial cases of cutaneous and cardiac myxomas associated with lentigines and blue nevi of the skin and mucosae had been described under the acronyms NAME (for nevi, atrial myxoma, myxoid neurofibromata, and ephelides) and LAMB (for lentigines, atrial myxoma, mucocutaneous myxoma, blue nevi) syndromes [44, 45]. In CNC, myxomas are often multiple, affect any or all cardiac chambers, occur at a relatively young age, and are equally distributed between the sexes [46]. The cutaneous myxomas have a predilection for the eyelids and external ear canals, although they may affect any part of the skin [47, 48]. Mammary myxoid fibroadenoma is also relatively frequent in the breast of CNC women [49], often associated with other multiple foci of myxomatous changes [49, 50]. Centrofacial spotty pigmentation in patients with CNC involves the vermilion border of the lips and the conjunctiva. The pigmented spots may be either (a) tan, irregularly shaped and poorly outlined, several millimeters in diameter, and freckle-like or (b) small, sharply delineated, and dark brown to black. The conjunctival pigmentation typically affects the lacrimal caruncle and the conjunctival semilunar fold and may involve the sclera. Skin pigmentation in CNC is of multiple pathologic types. Most of the lesions are lentigines or represent other examples of hypermelanosis (increased melanin in basal cells and throughout the thickness of the epidermis). Blue nevi (the usual type, as well as the exceptionally rare epithelioid type), combined and common junctional, dermal, and compound nevi, and café-au-lait spots (CALS) also occur in the syndrome [17, 51, 52]. Cushing syndrome, which appears to be the most common endocrine manifestation of CNC and affects about one-third of the patients, is always caused by PPNAD. In PPNAD, the glands are most commonly normal-sized or small and peppered with black or brown nodules set in a cortex that is usually atrophic. Despite their small size (less than 6 mm), the nodules are visible with computed tomography or magnetic resonance imaging of the adrenal glands [53]. Patients with CNC often present with a variant Cushing syndrome called ‘atypical’ (ACS) [54], which is characterized by an asthenic, rather than obese, body habitus caused by severe osteoporosis, short stature, and muscle and skin wasting. ACS was recognized as early as 1956 and has since been described in several cases of patients with Cushing syndrome [55–57]. More recently,

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this condition was uniquely associated with CNC [58, 59] including the case of a patient who presented 27 years after unilateral adrenalectomy [58]. Patients with ACS tend to have normal or near-normal 24-hour cortisol production, but this is characterized by the absence of the normal circadian rhythmicity of this hormone [54–60]. Occasionally patients present with ‘periodic Cushing syndrome’ (PCS), a variant of Cushing syndrome that is frequently found in children with the complex [59, 60]. All patients with PPNAD and classic CS, and most patients with ACS or PCS respond to dexamethasone with a paradoxical rise of cortisol production [58, 60]. In a recent study of the largest series reported to date [60], all patients with PPNAD responded to the graded administration of dexamethasone during Liddle’s test with a rise in both urinary free cortisol and 17-hydroxy-corticosteroid production. The test may be used diagnostically for the identification of PPNAD, even in patients that have normal baseline cortisol levels and do not have clinical stigmata of CS. About 10% of patients with CNC have a growth hormone (GH)-secreting pituitary adenoma that results in acromegaly [43]. Although most of the known patients with this condition had macroadenomas, a number of recently investigated cases show that abnormal 24-hour GH and prolactin secretion can precede the development of a pituitary tumor in CNC [61, 62]. The disorder, therefore, provides the unusual opportunity for prospective screening of affected patients without clinical acromegaly. Hyperplasia appears to be present in the pituitary gland of all patients with the complex, acromegaly and a GH-producing microadenoma operated to date [63]. Endocrine involvement in CNC also includes three types of testicular tumors: LCCSCT, adrenocortical rests, and Leydig cell tumor [17, 43, 64]. More than ¾ of affected male patients have one or more of these masses. LCCSCT, as in PJS, may secrete estrogens and cause precocious puberty, gynecomastia, or both [64]. It is our clinical impression that LCCSCTs in CNC are more benign than those in PJS, in at least as far as aromatization is concerned. Also, unlike PJS, the ovaries in CNC may be affected by cystic formation and occasionally epithelial cancer, but not by germ cell tumors [65]. Since 1985, three new components of the syndrome have been identified: psammomatous melanotic schwannoma, epithelioid blue nevus, and ductal adenoma of the breast [66–68]. Because thyroid follicular neoplasms, both benign and malignant, have been found in a number of patients, thyroid involvement appears to be a component of the syndrome [69].

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Molecular Genetics of CNC Two genetic loci have been determined for CNC by linkage analysis of polymorphic markers from likely areas of the genome [70, 71]. Initially, positive lod scores were obtained for nine markers on the short arm of chromosome 2, identifying an approximately 4-cM-long area in the cytogenetic band 2p16 (CNC locus), which was likely to contain the gene(s) responsible for the complex [70, 72]. This region was where another genetic syndrome, hereditary nonpolyposis colorectal cancer (HNPCC), had been mapped [70, 72]. The gene for HNPCC (hMSH2) codes for a protein that plays a direct role in DNA mismatch repair, increasing microsatellite stability and enhancing mutation avoidance in human cells – this gene was excluded from being a candidate for the complex [72]. Another recently identified gene, the hMSH6, also involved in DNA stability, is adjacent to hMSH2, but outside the defined region on 2p16 [70, 72]. Genomic instability in the form of telomeric associations and dicentric chromosomes is a frequent feature of fibroblasts derived from the myxoid and PPNAD tumors excised from patients with CNC [73–76]. Earlier investigations had found chromosomal instability in cultured skin fibroblasts, peripheral blood lymphocytes and adenomatous polyp cells established from patients with familial polyposis coli and PJS [77–79]. In these studies, no specific chromosomal breaks or exchange points were revealed, although several sites were involved in three or more rearrangements. Microsatellite analysis of the tumors excised from patients with CNC confirmed the significant genomic instability that accompanies tumorigenesis in this syndrome [76]. One of the first genes to be screened for mutations in CNC was the gsp proto-oncogene (GNAS1); however, one study showed that gsp mutations were not present in CNC tumors [80]. The locations of several genes that code for components of the guanine nucleotide-binding proteins (G-proteins) were excluded by linkage analysis [70]. Nevertheless, it seemed likely that the gene or genes responsible for CNC participate in G-protein-controlled or -related signalling systems, due to the similarities of CNC with McCune-Albright syndrome [17]. In the last 2 years, several families with CNC were described that did not map to chromosome 2 [81, 82]. Genetic heterogeneity was confirmed in this syndrome when a second locus on 17q22–24 was identified [71]. This was followed by the recent identification of the PRKARIA gene coding for the type I· regulatory subunit (RI·) of protein kinase A (PKA) as the gene responsible for CNC in most families that mapped to chromosome 17

Stratakis

Fig. 3. Detection of a PRKAR1A exon 4B frameshift mutation in a sporadic case of CNC (CAR23.03). a Typical CNC facial pigmentation in individual CAR23.03, an 11-year old boy with a testicular and adrenal tumors. b Partial pedigree of this patient. c DHPLC analysis of PRKAR1A exon 4B in the individuals shown in panel b. The arrow indicates an alteration in peak shape in CAR23.03 indicative of heteroduplex formation. d Sequence analysis of the

father (top), mother (middle), and affected son (bottom) showing the de novo generation of a sequence alteration in CAR23.03; cloning and sequencing identified a 578delTG PRKAR1A mutation.

and some sporadic cases of the disease [83, 84] (fig. 3). So far, all CNC-responsible mutations lead to truncation of the RI· protein. The LOH observed at the 17q22–24 locus in CNC tumors suggests that the PRKAR1A gene may function as a tumor suppressor gene in affected tissues [83]. A defective cyclic nucleotide-dependent pathway has long been considered a candidate mechanism for the various manifestations of CNC [17] including tumors similar to those of MAS [62, 63] and paradoxical responses to hormonal stimuli [60]. However, measurements of base-

line and post-stimulation intracellular cAMP levels in cultured tumors from patients with CNC (Stratakis CA, unpublished) and mutation analysis of the GNAS1 gene [80] gave negative results. Thus the defect in CNC was placed downstream from cAMP activation; thus, the PKA complex, a critical step in cAMP-dependent signaling, was a likely candidate for the identification of mutations in patients with CNC. How does one combine the cAMP-signaling defect with the identified LOH and possible tumor suppression function of PRKAR1A? One possibility is that truncated

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or absent RI· may inhibit the normal formation of the multimeric PKA enzymatic complex leading to dysregulated PKA activity [85]. Alternatively, the truncated RI· may interfere with the function of the normal components of the PKA complex in a dominant-negative manner (assuming that normal RI· has primarily an inhibitory role in CNC tissues) or, perhaps, RI· deficiency leads to overexpression of the genes coding for the other regulatory subunits of the PKA complex, as has been demonstrated in other contexts. Other possible mechanisms include an uninhibited catalytic subunit of the PKA complex which by itself when mutated leads to unregulated PKA activity, or a reduced turn-over of the cAMP molecule because several mutations interrupt the cAMP-binding domains of the RI· [83, 84]. The identification of the gene causing CNC on chromosome 17 left a group of families that appear to map collectively to chromosome 2 (although none of them with LOD score over 3) for which the syndrome has not been molecularly elucidated [70, 84]. There are also families that seem to map neither to chromosome 2 nor to chromosome 17, allowing for a third possible locus harboring gene(s) responsible for the complex. In tumors from patients with CNC, genetic changes of the chromosome 2p16 locus, including both copy number gain and loss, have been identified [85]. Interestingly, these changes are shared by both chromosome 2- and chromosome-17 mapping families, indicating, perhaps, a common molecular mechanism [85]. Given the substantial clinical overlap between syndromes like BRR, PJS, and CNC it is not unlikely that some patients, especially sporadic cases of CNC, will end up being diagnosed with one of these overlapping conditions. It is characteristic of the potential diagnostic errors in patients with one of the lentiginoses that one of the first families identified with CNC and the first one that was found to harbor PRKAR1A mutations was at first misdiagnosed with PJS [86]. It remains to be seen whether functional characterization of the responsible genes for all these disorders will provide a molecular basis for the clinical overlap.

Cowden and LEOPARD Syndromes and Other Lentiginoses

CS is associated with hamartomas and tumors of ecto-, meso- and endodermal origin affecting multiple organs [87]. Thyroid and breast masses, hamartomatous polyps, and mucocutaneous lesions (oral papillomatosis, acral

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keratosis, and multiple fibromas) occur consistently in this syndrome, which is also associated with anomalies of the skeletal and nervous systems [87–90]. RuvalcabaMyhre-Smith, Bannayan-Zonana or Bannayan-Riley-Ruvalcaba syndrome is also associated with hamartomatous intestinal polyposis; in addition, these patients have characteristic lentiginosis of the genitalia and developmental defects (macrocephaly, eye and skeletal anomalies) and myopathy [91–95]. LEOPARD syndrome is also known as the ‘multiple lentigenes syndrome’. First suggested by Gorlin et al. in 1969, the acronym describes the association of lentigenes, (multiple, darkly pigmented and present on the lips, but absent from other mucosal sites) electrocardiographic abnormalities, ocular hypertelorism (with other dysmorphic features), pulmonary stenosis, abnormalities of the genitalia (hypogonadism), (mental) retardation, and deafness (sensorineural) [96, 97]. This condition was also referred to as ‘cardiocutaneous syndrome’ in older reports, which had not recognized the pleomorphy of the phenotype [98– 100]. Watson syndrome, characterized by lentigenes, CALS, pulmonic stenosis, and mental retardation, as well as the association of heart defects, pigmented lesions, and deafness in other patients could represent variant forms of the LEOPARD syndrome. LEOPARD syndrome and its variants are inherited in an autosomal dominant manner. Variable expression within the same family, a feature of all the lentigenosis syndromes, is frequently seen. This is best exemplified by a kindred where the propositus, an 11-year-old boy, had many lentigenes and severe heart problems. His father and five out of six siblings with generalized lentigenosis had no other abnormalities, and nine other relatives from three generations who had lentigenes were otherwise healthy [100]. Neoplasms, although uncommon, are also present in LEOPARD syndrome and include rhabdomyosarcoma and granular cell myoblastoma [101]. The arterial dissections and lentiginosis (ADL) syndrome was recently described in two sets of siblings from a series of 240 patients with arterial dissections seen at the Mayo Clinic [102]. Six additional sporadic cases were reported, all with lentigines of the trunk and the extremities, particularly the lower legs. There were no other pigmented lesions, and the mucosae were most likely not affected. This disease shares with Marfan syndrome and other connective tissue disorders a predisposition to dissections of the aorta, renal artery, and extracranial internal carotid artery. The inheritance of ADL syndrome is not clear, although it was suggested to be autosomal recessive [102].

Stratakis

The PTEN gene is found mutated in CS and BRR [103, 104], syndromes that have substantial clinical heterogeneity. There are no other genetic defects identified for LEOPARD and the other, less frequent lentiginosis syndromes.

Notice ‘Lentigines’ may also be seen as ‘lentigenes’ in other publications; the diseases may also be called ‘lentigenoses’ instead of ‘lentiginoses’. In this review, we use ‘lentigen’ for the singular form and ‘lentigines’/ ‘lentiginosis’, except if ‘lentigenes’ was used in the source.

Acknowledgement The author would like to thank Dr. C. Eng, Ohio, USA.

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81 Basson CT, MacRae CA, Korf B, Merliss A: Genetic heterogeneity of familial atrial myxoma syndromes (Carney complex). Am J Cardiol 1997;79:994–995. 82 Taymans SE, Macrae CA, Casey M, Merliss A, Lin J-P, Rocchi M, Kirschner LS, Basson CT, Stratakis CA: A refined genetic, radiation hybrid, and phyiscal map of the Carney complex (CNC) locus on chromosome 2p16; evidence for genetic heterogeneity in the syndrome. Am J Hum Genet 1997;61(suppl):A84. 83 Kirschner LS, Carney JA, Pack S, Taymans SE, Cho YS, Cho-Chung Y, Stratakis CA: Mutations in the gene encoding the type Ia regulatory subunit of the protein kinase A (PRKARIA) in patients with Carney complex. Nat Genet 2000;26:89–92. 84 Kirschner LS, Sandrini F, Monbo J, Lin JP, Carney JA, Stratakis CA: Genetic heterogeneity and spectrum of mutations of the PRKAR1A gene in patients with the Carney complex. Hum Mol Genet 2000;9:3037–3046. 85 Scott JD: Cyclic nucleotide-dependent protein kinases. Pharm Ther 1991;50:123–145. 86 Taymans SE, Kirschner LS, Pack S, Ping Z, Stratakis CA: YAC-BAC contig of the Carney complex (CNC) critical region on 2p16 and copy number gain of 2p16 in CNC tumors: evidence for a novel oncogene? Am J Hum Genet 1999;65(suppl):A326. 87 Stratakis CA, Kirschner LS, Taymans SE, Tomlinson IPM, Marsh DJ, Torpy DJ, Giatzakis C, Eccles DM, Theaker J, Houlston RS, Blouin J-L, Antonarakis SE, Basson CT, Eng C, Carney JA: Carney complex, Peutz-Jeghers syndrome, Cowden disease, and BannayanZonana syndrome share cutaneous and endocrine manifestations, but not genetic loci. J Clin Endo Metab 1998;83:2972–2976. 88 Lloyd KM, Dennis M: Cowden disease: a possible new symptom complex with multiple system involvement. Ann Intern Med 1963;58: 136–142. 89 Brownstein MH, Wolf M, Bilowski JB: Cowden’s disease. Cancer 1978;41:2393–2398. 90 Eng C: Genetics of Cowden syndrome: Through the looking glass of oncology (review). Int J Oncol 1998;12:701–710. 91 Bannayan GA: Lipomatosis, angiomatosis, and macrencephalia. A previously undescribed congenital syndrome. Arch Pathol 1971;92:1– 5. 92 Gorlin RJ, Cohen MM, Condon LM, Burke BA: Bannayan-Riley-Ruvalcaba syndrome. Am J Med Genet 1992;44:307–314. 93 Zonana J, Rimoin DL, Davis DC: Macrocephaly with multiple lipomas and hemangiomas. J Pediatr 1976;89:600–603.

94 Zigman AF, Lavine JE, Jones MC, Boland CR, Carethers JM: Localisation of the Bannayan-Riley-Ruvalcaba syndrome gene to chromosome 10q23. Gastroenterology 1997; 113:1433–1437. 95 Fargnoli MC, Orlow SJ, Semel-Concepcion J, Bolognia JL: Clinicopathologic findings in the Bannayan-Riley-Ruvalcaba syndrome. Arch Dermatol 1996;132:1214–1218. 96 Gorlin RJ, Anderson RC, Blaw M: Multiple lentigines syndrome. Am J Dis Child 1969; 117:652–662. 97 Polani PE, Moynahan EJ: Progressive cardiomyopathic lentiginosis. Quart J Med 1972; 41:205–225. 98 Senanez H, Mane-Garzon F, Kolski R: Cardio-cutaneous syndrome (the ‘LEOPARD’ syndrome). Review of the literature and a new family. Clin Genet 1976;9:266–276. 99 Watson GH: Pulmonary stenosis, cafe-au-lait spots, and dull intelligence. Arch Dis Child 1967;42:303–307. 100 Gorlin RJ, Anderson RC, Moller JH: The Leopard (multiple lentigines) syndrome revisited. Birth Defects 1971;7:110–115. 101 Heney D, Lockwood L, Allibone EB, Bailey CC: Nasopharyngeal rhabdomyosarcoma and multiple lentigines syndrome: a case report. Med Pediatr Oncol 1992;20:227–228. 102 Schievink WI, Michels VV, Mokri B, Piepgras DG, Perry HO: A familial syndrome of arterial dissections with lentiginosis. N Engl J Med 1995;332:576–579. 103 Nelen MR, Padberg GW, Peeters EAJ, Lin AY, van den Helm B, Frants RR, Coulon V, Goldstein AM, van Reen MMM, Easton DF, Eeles RA, Hodgson S, Mulvihill JJ, Murday VA, Tucker MA, Mariman ECM, Starink TM, Ponder BAJ, Ropers HH, Kremer H, Longy M, Eng C: Localization of the gene for Cowden disease to chromosome 10q22–23. Nat Genet 1996;13:114–116. 104 Marsh DJ, Kum JB, Lunetta KL, Bennett MJ, Gorlin RJ, Ahmed SF, Bodurtha J, Crowe C, Curtis MA, Dasouki M, Dunn T, Feit H, Geraghty MT, Graham JM, Hodgson SV, Hunter A, Korf BR, Manchester D, Miesfeldt S, Murday VA, Nathanson KL, Parisi M, Pober B, Romano C, Tolmie JL, Trembath R, Winter RM, Zackai EH, Zori RT, Weng L-P, Dahia PLM, Eng C: PTEN mutation spectrum and genotype-phenotype correlations in Bannayan-Riley-Ruvalcaba syndrome suggest a single entity with Cowden syndrome. Hum Mol Genet 1999;8:1461–1472.

Horm Res 2000;54:334–343

343

Author Index Vol. 54, No. 5–6, 2000

Adler, Y.D. 251 Al-Azzawi, F. 259 Alesci, S. 281 Aranda, A. 301

Hamada, K. 243 Hamamoto, Y. 316 Hibberts, N.A. 243 Hiort, O. 327

Nagai, K. 316 Nitsche, E.M. 327

Bader, A. 294 Basu-Modak, S. 263 Böhm, M. 287 Bornert, J.-M. 296 Bornstein, S.R. 281 Both, D. 318 Brocard, J. 296 Brockmeyer, N.H. 294 Brown, D. 318

Ichimiya, M. 316 Indra, A.K. 296

Pelletier, G. 218 Pugeat, M. 322

Jenner, T.J. 243 Jimenez-Lara, A.M. 301

Randall, V.A. 243 Reimann, G. 294 Ricote, M. 275 Rosenfield, R.L. 269

Chambon, P. 296

Labrie, C. 218 Labrie, F. 218 Lehmann, B. 312 Li, M. 296 Luger, T.A. 287 Luu-The, V. 218

Kato, S. 243 Knuschke, P. 312 Kreuter, A. 294

De Oliveira, I. 243 Deplewski, D. 269 Desvergne, B. 263 Ducluzeau, P.H. 322 El-Alfy, M. 218

Mallion-Donadieu, M. 322 Merrick, A.E. 243 Messaddeq, N. 296 Messenger, A.G. 243 Metzger, D. 296 Meurer, M. 312 Michalik, L. 263 Mulligan, K. 259 Muto, M. 316

Fimmel, S. 306 Gérard, C. 296 Glass, C.K. 275 Greene, M.E. 269

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Orfanos, C.E. 251, 306

Saborowski, A. 306 Seemann, U. 294 Stratakis, C.A. 334 Tan, N.S. 263 Taylor, A.H. 259 Thornton, M.J. 243, 259 Wahli, W. 263 Warot, X. 296 Welch, J.S. 275 Yarosh, D.B. 318 Yokoyama, K. 316 Zouboulis, C.C. 217, 230, 251, 306

Subject Index Vol. 54, No. 5–6, 2000

Acanthosis nigricans 322 Acne 251, 281 Adrenal androgens 281 Adrenocortical function 294 Alopecia 281, 322 Androgen(s) 218, 322 – insensitivity 327 – receptor 259, 306, 332 Androgenic alopecia 251, 281 Antisense oligonucleotides 306 Balding 243 Barrier function 318 Beard 243 Calcitriol 312 Carney complex 334 CD36 275 Cell line 306 Cowden disease 334 Cre/Lox 296

Gonadal function 294 Growth factors 243

Oestrogen receptor-ß 259 Hair follicle 243, 259, 296 – loss 281 Hirsutism 251, 281, 322 Hormone activity 230 – metabolism 230 – receptors 230 – response elements 301 – synthesis 230 Human hair growth 243 Hydroxy-ceramides 318 Hypergonadotropic hypogonadism 294 Hyperandrogenia 322 Hyperandrogenism 281 IL-4 275 iNOS 275 Insulin resistance 322 Intracrinology 218

Dehydroepiandrosterone 218 Dermal papilla cells 243 DHEA 281

Keratinocyte(s) 296, 306, 312, 318 – proliferation 263 Lentigines 334

Endocrine dysfunction 294 Endocrinology 230 Epidermal water barrier 269 Epidermis 296 Estrogens 218 Fibroblasts 318 Gene expression 301 Genetics 327 Glucocorticoid receptors 318

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Nuclear receptors 275

Macrophages 275 Melanocortin(s) 287 – receptors 287 ·-Melanocyte-stimulating hormone 287, 316 Merotaine 318 Microdialysis 312 Mouse 296 – epidermis 263 Multiple neoplasia syndromes 334

Peutz-Jeghers syndrome 334 PPAR gene expression 263 PPAR-Á 275 PRKAR1A gene 334 PTEN gene 334 Procollagen 318 Protein kinase A 334 Retinoic acid 301 SAHA syndrome 251 Sebaceous gland(s) 218, 259, 269, 306 Seborrhoea 251 Sex differentiation 327 Skin 218, 259 – wound healing 263 Somatic mutagenesis 296 STAT-6 275 Stem cell factor 243 STK11 gene 334 Tamoxifen 296 Thyroid function 294 Transfection 306 Tubular insufficiency 294 UVB 312 Vitamin D 301 Vitamin D3 312 Vitiligo 316

345

Author Index Vol. 54, 2000 B = Book Review

Adami, S. 164 Adler, Y.D. 251 Al-Azzawi, F. 259 Albers, N. 174 Albertsson-Wikland, K. 120 Alesci, S. 281 Alfonso, M. 78 l’Allemand, D. 14 Andersson, A.M. 84 Araki, O. 49 Aranda, A. 301 Arufe, M.C. 78 Asaba, K. 69, 198 Asada, N. 203 Ayuso, S. 20 Bader, A. 294 Bamberger, A.-M. 32 Banegas, F. 6 Barac-Nieto, M. 38 Basu-Modak, S. 263 Beck, J.D. 44 Bedecarrı´s, P. 20 Belgian Study Group for Pediatric Endocrinology 120 Bergadı´, C. 20 Bieth, E. 92 Bilbao, J.R. 181 Binder, G. 60, 149 Blum, W.F. 26, 174 Boguszewski, M.C.S. 120 Böhm, M. 287 Bonfleur, M.L. 186 Bonnel, F. 6 Bornert, J.-M. 296 Bornstein, S.R. 281 Both, D. 318 Braga, V. 164 Brocard, J. 296 Brockmeyer, N.H. 294 Brown, D. 318 Busturia, M.A. 181 Campo, S. 20 Canovas, F. 6 Carrascosa, A. 131 Castaño, L. 181 Cezar de Freitas Mathias, P. 186 Chambon, P. 296 Charveron, M. 92

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Conte, F. 92 Cyteval, C. 6 Delitala, G. 101 De Oliveira, I. 243 Deplewski, D. 269 Desvergne, B. 263 De Zegher, F. 192 DiMéglio, A. 6 Diridollou, S. 92 Dörr, H.G. 26, 44 Dötsch, J. 26 Du Caju, M.V.L. 126 Ducluzeau, P.H. 322 Dunger, D.B. 192 Dura´n, R. 78 El-Alfy, M. 218 Elmlinger, M.W. 60 Escobar, M.E. 20 Fahlbusch, R. 44 Fanciulli, G. 101 Feldt-Rasmussen, U. 53 Fimmel, S. 306 Futamura, A. 169 Gall, Y. 92 Gartenmann, M. 98 Gaston, V. 1 Gérard, C. 296 Gicquel, C. 1 Glass, C.K. 275 Grabenbauer, G.G. 44 Gravena, C. 186 Greene, M.E. 269 Gröschl, M. 26 Grüters, A. 14 Gryngarten, M. 20 Guski, H. 74 Gussinyé, M. 131 Hagendorens, M.M. 126 Hamada, K. 243 Hamamoto, Y. 316 Hara, M. 169 Hashimoto, K. 69, 198 Hashimoto, Y. 169 Hebebrand, J. 174 Herpertz, S. 174 Hertz, H. 53 Hibberts, N.A. 243

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Hiort, O. 327 Honjo, M. 136, 203 Huang, J.-K. 143 Iba´ñez, L. 192 Ichimiya, M. 316 Ikeda, M. 136 Iliev, D.I. 149 Indra, A.K. 296 Inoue, M. 198 Jacobi, C.A. 74 Jaeger, M. 6 Jan, C.-R. 143 Jenner, T.J. 243 Jimenez-Lara, A.M. 301 Jouret, B. 92 Kaneda, T. 69 Kashima, K. 49 Kato, S. 243 Kilian, M. 74 Kimura, S. 169 Knuschke, P. 312 Kohama, T. 49 Kojima, A. 49 Konrad, D. 98 Kreuter, A. 294 Kurisaka, M. 198 Labrie, C. 218 Labrie, F. 218 Lassen, S. 53 Le Bouc, Y. 1 Lehmann, B. 312 Leriche, C. 149 Li, M. 296 Livieri, C. 164 Llanos, M.N. 157 Löning, T. 32 Lucinei Balbo, S. 186 Luger, T.A. 287 Luu-The, V. 218 Makino, S. 69 Mallion-Donadieu, M. 322 Mann, K. 174 Martin, E. 98 Marx, M. 44 Matsumoto, K. 136 Mautsch, I. 74 Merrick, A.E. 243

Messaddeq, N. 296 Messenger, A.G. 243 Metzger, D. 296 Meurer, M. 312 Michalik, L. 263 Migliavacca, D. 164 Milde-Langosch, K. 32 Mimoto, T. 69 Mizuide, M. 49 Mizuma, H. 49 Mori, M. 49 Mori, T. 198 Müller, J. 53, 84 Müller, J.M. 74 Mulligan, K. 259 Mullis, P.E. 107 Murakami, M. 49 Muto, M. 316 Nagai, K. 316 Nagano, I. 198 Naito, N. 136 Nakahara, K. 169 Nakajo, T. 198 Nakarai, H. 169 Nanamiya, W. 69 Neu, A. 104 (B) Nishioka, T. 69, 198 Nishiyama, M. 69 Nitsche, E.M. 327 Nordic Study Group for Growth Hormone Treatment in SGA Children 120 Oberste-Berghaus, C. 174 Ogiwara, T. 49 Ong, K. 192 Op De Beeck, L. 126 Orfanos, C.E. 251, 306 Otsuka, F. 198 Paganini, C. 164 Pelletier, G. 218 Pelz, B. 174 Petruch, U.R. 149 Piga, S. 101 Potau, N. 192 Poulsen, H.S. 53 Pugeat, M. 322 Radetti, G. 164 Randall, V.A. 243

Ranke, M.B. 60, 149 Rascher, W. 26 Reimann, G. 294 Rica, I. 181 Ricote, M. 275 Rochiccioli, P. 92 Ronco, A.M. 157 Rooman, R.P.A. 126 Rosenfield, R.L. 269 Saborowski, A. 306 Satoh, H. 169 Saul, G.J. 74 Schimke, I. 74 Schmiegelow, K. 53 Schmiegelow, M. 53 Schoenle, E.J. 98 Schulte, H.M. 32 Schwarze, C.P. 60

Author Index

Schweizer, R. 60 Seemann, U. 294 Sehested, A. 84 Senf, W. 174 Silverstein, D.M. 38 Skakkebæk, N.E. 84 Soupre, V. 1 Spitzer, A. 38 Stichel, H. 14 Stratakis, C.A. 334 Strotbek, G. 149 Suemaru, S. 69 Sultan, C. 6 Sys, S.U. 126 Takahashi, Y. 203 Takao, T. 69, 198 Tan, N.S. 263 Tauber, M. 92

Taylor, A.H. 259 Tecleme, P. 101 Terrades, P. 131 Thornton, M.J. 243, 259 Togo, M. 169 Tomasi, P.A. 101 Tomioka, S.-i. 49 Tsukamoto, K. 169 Tsunekawa, B. 136 Turlier, V. 92

Wahli, W. 263 Warot, X. 296 Watanabe, T. 169 Weber, K. 60 Welch, J.S. 275 Wenger, F.A. 74 Wollmann, H.A. 60, 149

Uchida, H. 136

Yarosh, D.B. 318 Yeste, D. 131 Yokoyama, K. 316 Yu, C.-C. 143

Va´zquez, J.A. 181 Vazquez, M.P. 1 Vicens-Calvet, E. 131

Zegher, F. de 120 Zouboulis, C.C. 217, 230, 251, 306

Wada, M. 136 Wagner, R. 26, 174

Horm Res Vol. 54, 2000

347

Subject Index Vol. 54, 2000

Acanthosis nigricans 322 Acetylcholinesterase activity 186 Acne 251, 281 Adrenal androgens 281 Adrenalectomy 69 Adrenocortical function 294 Adrenocorticotropic hormone 198 Alcohol drinking 169 Alopecia 281, 322 Androgen(s) 20, 192, 218, 322 – insensitivity 327 – receptor 259, 306, 332 Androgenic alopecia 251, 281 Antisense oligonucleotides 306 AP-1 transcription factors 32 Arachidonic acid 157 Areal bone mineral density 131 Autoantibodies 181 Balding 243 Barrier function 318 Beard 243 Beckwith-Wiedemann syndrome 1 BFTC cells 143 Binding affinity 136 Birthweight 192 Bladder cell carcinoma 143 Body mass index 169 Bone age 6 – markers 164 – maturation 6 – mineral density 164, 192 Brain tumors 53 Breast cancer 32 Ca2+ signaling 143 Calcitriol 312 Carney complex 334 Carpal bone 6 CD36 275 Cell line 306 – proliferation 136 Central diabetes insipidus 98 – precocious puberty 84 Child 53, 101 Childhood obesity 14 Choriocarcinoma 44 Citrate 26 Clomiphene 143 CNS 44 Combined pituitary hormone deficiency 107

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Computed tomography 6 Congenital adrenal hyperplasia 164 Constitutional delay in growth and puberty 126 Corticosterone 69 Cortisol 69, 174 Cowden disease 334 Cranial irradiation adverse effects 53 Cre/Lox 296 Cushing disease 198 Deglycosylation 157 Dehydroepiandrosterone 218 Dermal papilla cells 243 DHEA 281 Diabetes mellitus 181 Diagnostics 60 Diurnal rhythm 69 Dosimetry 53 Elasticity 92 Endocrine dysfunction 294 Endocrinology 230 Epidermal water barrier 269 Epidermis 296 Estradiol 131 Estrogens 218 Excitatory amino acid 78 Exercise 169 Fibroblasts 318 Fluoride 26 Freely moving rats 78 Fura-2 143 GAD 181 Gene expression 301 – promoter activation 136 Genetics 327 Germ cell tumours 44 Glucocorticoid receptors 318 GnRH agonists 84 Gonadal function 294 Gonadotrophin-independent puberty 44 Growth 6 – factors 243 – hormone 92 – – deficiency 44, 53, 60 – – treatment 120 – retardation 126

Hair follicle 243, 259, 296 – loss 281 Height 126, 164 – velocity 126 hGHBP 203 Hirsutism 251, 281, 322 Hormone activity 230 – metabolism 230 – receptors 230 – response elements 301 – synthesis 230 Human chorionic gonadotrophin 44 – – gonadotropin 157 – hair growth 243 – skin 92 Hydroxy-ceramides 318 Hyperandrogenia 322 Hyperandrogenism 281 Hypergonadotropic hypogonadism 294 Hypertrichosis score 20 Hypoglycemia 49 IA2 181 Idiopathic short stature 60 IGFBP-2 60 IGFBP-3 60 IGF-I 60 IL-4 275 Immunoglobulin 49 Inhibin A 84 – B 84 iNOS 275 Insulin 169, 174, 181, 192 – resistance 322 – secretion 186 Insulin-like growth factor II 198 Intracrinology 218 Intrauterine growth retardation 60, 120 K2-EDTA 26 20K hGH 136, 203 22K hGH 136, 203 Keratinocyte(s) 296, 306, 312, 318 – proliferation 263 Lactate 26 Lentigines 334 Leptin 26, 69, 120, 169, 174, 192, 203 Leydig cells 157 Li-heparinate 26 Lipolysis 203 Liver metastasis 74

Macrophages 275 Melanocortin(s) 287 – receptors 287 ·-Melanocyte-stimulating hormone 287, 316 Merotaine 318 MIB-1 antibody 198 Microdialysis 312 Mouse 296 – epidermis 263 MSG-obese mice 186 Müllerian duct malformation 149 Multiple neoplasia syndromes 334 Needle biopsy 101 Neurosarcoidosis 98 Nuclear receptors 275

Pituitary 107 – adenoma 198 – development 107 – stalk thickening 98 Plasma 26 PPAR gene expression 263 PPAR-Á 275 Precocious pubarche 192 Prepubertal hypertrichosis 20 PRKAR1A gene 334 Procollagen 318 Progesterone receptor isoforms 32 Proliferating cell nuclear antigen 198 Protein kinase A 334 Pseudohypoaldosteronism 149 PTEN gene 334 de Quervain thyroiditis 101

Octreotide 74 Oestrogen receptor-ß 259 Opossum kidney cells 38 Organogenesis 107 Osteopenia 131 Osteoporosis 131 Overgrowth syndrome 1 p57KIP2 gene 1 11p15 region 1 Pancreatic cancer 74 – islets 186 Peutz-Jeghers syndrome 334 Phosphate transport 38

Subject Index

Rat GH receptor 136 Regulators, renal 38 Renal agenesis 149 Retinoic acid 301 SAHA syndrome 251 Scatchard plot 49 Sebaceous gland(s) 218, 259, 269, 306 Seborrhoea 251 Serum 26 Sex differentiation 327 Short children 120

Horm Res Vol. 54, 2000

Skin 218, 259 – wound healing 263 Smoking 169 Somatic mutagenesis 296 Somatotropin-releasing hormone 53 STAT-6 275 Stem cell factor 243 Steroid production 157 Stiffness 92 STK11 gene 334 Syrian hamster 74 T3 78 T4 78 3T3-L1-hGHR adipocytes 203 Tamoxifen 74, 296 Thyroid function 14, 294 Thyroid-stimulating hormone 14, 78 Thyroiditis, suppurative, subacute 101 Thyroxine 14 Transcription factors 107 Transfection 306 Triiodothyronine 14 Tubular insufficiency 294 Turner syndrome 60, 131 Uterovaginal anomalies 149 UVB 312 Vitamin D 301 Vitamin D3 312 Vitiligo 316

349

Contents Vol. 54, 2000

No. 1

No. 2

Original Papers 1

Original Papers

p57KIP2

Assessment of Gene Mutation in BeckwithWiedemann Syndrome

53

Gaston, V.; Le Bouc, Y.; Soupre, V.; Vazquez, M.P.; Gicquel, C. (Paris)

6

14

Schmiegelow, M.; Lassen, S.; Poulsen, H.S.; Feldt-Rasmussen, U.; Schmiegelow, K.; Hertz, H.; Müller, J. (Copenhagen)

Carpal Bone Maturation Assessment by Image Analysis from Computed Tomography Scans Canovas, F.; Banegas, F.; Cyteval, C.; Jaeger, M.; DiMéglio, A.; Bonnel, F.; Sultan, Ch. (Montpellier)

60

Clinical Assessment and Serum Hormonal Profile in Prepubertal Hypertrichosis Gryngarten, M.; Bedecarràs, P.; Ayuso, S.; Bergadà, C.; Campo, S.; Escobar, M.E. (Buenos Aires)

26

Variability of Leptin Values Measured from Different Sample Matrices Gröschl, M.; Wagner, R.; Dörr, H.G. (Erlangen-Nürnberg); Blum, W. (Bad Homburg); Rascher, W.; Dötsch, J. (Erlangen-Nürnberg)

32

Progesterone Receptor Isoforms, PR-B and PR-A, in Breast Cancer: Correlations with Clinicopathologic Tumor Parameters and Expression of AP-1 Factors Bamberger, A.-M.; Milde-Langosch, K.; Schulte, H.M.; Löning, T. (Hamburg)

38

Hormonal Regulation of Sodium-Dependent Phosphate Transport in Opossum Kidney Cells Silverstein, D.M.; Spitzer, A.; Barac-Nieto, M. (Bronx, N.Y.)

Significance of Basal IGF-I, IGFBP-3 and IGFBP-2 Measurements in the Diagnostics of Short Stature in Children Ranke, M.B.; Schweizer, R.; Elmlinger, M.W.; Weber, K.; Binder, G.; Schwarze, C.P.; Wollmann, H.A. (Tübingen)

Thyroid Function and Obesity in Children and Adolescents Stichel, H.; l’Allemand, D.; Grüters, A. (Berlin)

20

Growth Hormone Response to a Growth Hormone-Releasing Hormone Stimulation Test in a Population-Based Study following Cranial Irradiation of Childhood Brain Tumors

69

Glucocorticoid Effects on the Diurnal Rhythm of Circulating Leptin Levels Nishiyama, M.; Makino, S. (Kochi); Suemaru, S. (Hiroshima); Nanamiya, W.; Asaba, K.; Kaneda, T.; Mimoto, T.; Nishioka, T.; Takao, T.; Hashimoto, K. (Kochi)

74 Influence of Octreotide and Tamoxifen on Tumor Growth and

Liver Metastasis in N-Nitrosobis-(2-oxopropyl)amine-Induced Pancreatic Cancer in Syrian Hamsters Wenger, F.A.; Kilian, M.; Mautsch, I.; Jacobi, C.A.; Schimke, I.; Saul, G.J.; Guski, H.; Müller, J.M. (Berlin)

78 Effect of Excitatory Amino Acids on Serum TSH and Thyroid

Hormone Levels in Freely Moving Rats Alfonso, M.; Durán, R.; Arufe, M.C. (Vigo)

84 Serum Inhibin A and Inhibin B in Central Precocious Puberty

before and during Treatment with GnRH Agonists Sehested, A.; Andersson, A.M.; Müller, J.; Skakkebaek, N.E. (Copenhagen)

92 Evaluation of Cutaneous Modifications in Seventy-Seven 44

Case Reports

Growth Hormone-Deficient Children

Gonadotrophin-Independent Puberty in a Boy with a ␤-HCGSecreting Brain Tumour

Conte, F. (Toulouse/Guyancourt/Stockholm); Diridollou, S.; Jouret, B.; Turlier, V.; Charveron, M.; Gall, Y. (Toulouse); Rochiccioli, P.; Bieth, E.; Tauber, M. (Toulouse/Guyancourt/ Stockholm)

Marx, M.; Beck, J.D.; Grabenbauer, G.G.; Fahlbusch, R.; Dörr, H.G. (Erlangen)

49

Identification of Monoclonal Insulin Autoantibodies in Insulin Autoimmune Syndrome Associated with HLA-DRB1*0401 Murakami, M.; Mizuide, M.; Kashima, K.; Kojima, A. (Maebashi); Tomioka, S. (Numata); Kohama, T.; Araki, O.; Ogiwara, T.; Mizuma, H.; Mori, M. (Maebashi)

25

Case Reports 98 Central Diabetes insipidus as the First Manifestation of

Neurosarcoidosis in a 10-Year-Old Girl Konrad, D.; Gartenmann, M.; Martin, E.; Schoenle, E.J. (Zürich)

101 Acute Thyroiditis in a Child: Misleading Result of Fine-Needle

Aspiration Biopsy

Congress Calendar

Tomasi, P.A.; Piga, S.; Tecleme, P.; Fanciulli, G.; Delitala, G. (Sassari)

104 Book Review 105 Congress Calendar

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No. 3

186 Insulin Secretion and Acetylcholinesterase Activity in

Monosodium L-Glutamate-Induced Obese Mice Balbo, S.L.; Gravena, C.; Bonfleur, M.L.; de Freitas Mathias, P.C. (Maringá)

Review 107 Transcription Factors in Pituitary Gland Development and Their

Clinical Impact on Phenotype Mullis, P.E. (Bern)

Original Papers 120 Serum Leptin in Short Children Born Small for Gestational Age:

Dose-Dependent Effect of Growth Hormone Treatment Boguszewski, M.C.S. (Göteborg/Curitiba); de Zegher, F. (Leuven); Albertsson-Wikland, K. (Göteborg); on behalf of the Nordic Study Group for Growth Hormone Treatment in SGA Children and the Belgian Study for Pediatric Endocrinology

192 Increased Bone Mineral Density and Serum Leptin in

Non-Obese Girls with Precocious Pubarche: Relation to Low Birthweight and Hyperinsulinism Ibáñez, L.; Potau, N. (Barcelona); Ong, K.; Dunger, D.B. (Cambridge); De Zegher, F. (Leuven)

Case Report 198 Significant Gene Expression of Insulin-Like Growth Factor II

and Proliferating Cell Nuclear Antigen in a Rapidly Growing Recurrent Pituitary ACTH-Secreting Adenoma Hashimoto, K.; Nagano, I.; Asaba, K.; Inoue, M.; Nishioka, T.; Takao, T.; Nakajo, T.; Mori, T.; Kurisaka, M. (Nankoku); Otsuka, F. (Okayama)

126 Progressive Deceleration in Growth as an Early Sign of Delayed

Puberty in Boys Du Caju, M.V.L.; Op De Beeck, L.; Sys, S.U.; Hagendorens, M.M.; Rooman, R.P.A. (Antwerp)

131 Low Areal Bone Mineral Density Values in Adolescents and

Young Adult Turner Syndrome Patients Increase after Long-Term Transdermal Estradiol Therapy Gussinyé, M.; Terrades, P.; Yeste, D.; Vicens-Calvet, E.; Carrascosa, A. (Barcelona)

Short Communication 203 Effects of 22K or 20K Human Growth Hormone on Lipolysis,

Leptin Production in Adipocytes in the Presence and Absence of Human Growth Hormone Binding Protein Asada, N.; Takahashi, Y.; Honjo, M. (Chiba)

208 Congress Calendar

136 Cellular Activities of 20K- and 22K-hGH Do Not Necessarily

Correlate with Their Binding Affinities for Rat GH Receptor Ikeda, M.; Matsumoto, K.; Uchida, H.; Naito, N.; Tsunekawa, B.; Wada, M.; Honjo, M. (Mobara)

No. 5–6

143 Clomiphene, an Ovulation-Inducing Agent, Mobilizes

Intracellular Ca2+ and Causes Extracellular Ca2+ Influx in Bladder Female Transitional Carcinoma Cells

Basic Research in Endocrine Dermatology 3rd Teupitzer Colloquium 2000, Berlin, September 17–20, 2000

Jan, C.-R.; Yu, C.-C.; Huang, J.-K. (Taipei)

Edited by C.C. Zouboulis, Berlin

Case Report 149 Transient Pseudohypoaldosteronism with Complex

Malformation of Internal Genitalia. A Case Report Iliev, D.I.; Petruch, U.R.; Ranke, M.B.; Binder, G. (Tübingen); Leriche, C. (Nuremberg); Strotbek, G. (Waiblingen); Wollmann, H.A. (Tübingen)

153 ESPE Bulletin Board 155 Congress Calendar

No. 4

217 Preface 218 Intracrinology and The Skin Labrie, F.; Luu-The, V.; Labrie, C.; Pelletier, G.; El-Alfy, M. (Québec City) 230 Human Skin: An Independent Peripheral Endocrine Organ Zouboulis, C.C. (Berlin) 243 The Hair Follicle: A Paradoxical Androgen Target Organ Randall, V.A.; Hibberts, N.A.; Thornton, M.J. (Bradford); Hamada, K. (Odawara City); Merrick, A.E. (Bradford); Kato, S. (Tokushima); Jenner, T.J.; De Oliveira, I. (Bradford); Messenger, A.G. (Sheffield) 251 The SAHA Syndrome Orfanos, C.E.; Adler, Y.D.; Zouboulis, C.C. (Berlin) 259 Oestrogen Receptor Beta Is Not Present in the Pilosebaceous

Original Papers 157 Effect of Human Chorionic Gonadotropin Derivatives on Leydig

Cell Function Ronco, A.M.; Llanos, M.N. (Santiago)

164 Height, Bone Mineral Density and Bone Markers in Congenital

Adrenal Hyperplasia Paganini, C.; Radetti, G. (Bolzano); Livieri, C.; Braga, V.; Migliavacca, D. (Pavia); Adami, S. (Mantua)

169 Relationship between the Serum Level of Leptin and Life-Style

Habits in Japanese Men Togo, M.; Hashimoto, Y.; Futamura, A.; Tsukamoto, K.; Satoh, H.; Hara, M.; Watanabe, T.; Nakarai, H.; Nakahara, K.; Kimura, S. (Tokyo)

174 Time Relationship between Circadian Variation of Serum Levels

of Leptin, Insulin and Cortisol in Healthy Subjects Wagner, R.; Oberste-Berghaus, C.; Herpertz, S. (Essen); Blum, W.F. (Giessen/Bad Homburg); Pelz, B. (Essen); Hebebrand, J. (Marburg); Senf, W.; Mann, K. (Essen); Albers, N. (Bonn)

181 Influence of Sex and Age at Onset on Autoantibodies against

Insulin, GAD65 and IA2 in Recent Onset Type 1 Diabetic Patients Bilbao, J.R.; Rica, I.; Vázquez, J.A.; Busturia, M.A.; Castaño, L. (Barakaldo)

Contents

Unit of Red Deer Skin during the Non-Breeding Season Thornton, M.J. (Bradford); Taylor, A.H.; Mulligan, K.; Al-Azzawi, F. (Leicester)

263 Nuclear Hormone Receptors and Mouse Skin Homeostasis:

␤ Implication of PPAR␤

Michalik, L.; Desvergne, B. (Lausanne); Basu-Modak, S. (Lausanne/Bath); Tan, N.S.; Wahli, W. (Lausanne)

269 Peroxisome Proliferator-Activated Receptors and Skin

Development Rosenfield, R.L.; Deplewski, D.; Greene, M.E. (Chicago, Ill.)

275 Regulation of Macrophage Gene Expression by the Peroxisome

␥ Proliferator-Activated Receptor-␥

Ricote, M.; Welch, J.S.; Glass, C.K. (La Jolla, Calif.)

281 Neuroimmunoregulation of Androgens in the Adrenal Gland

and the Skin Alesci, S.; Bornstein, S.R. (Bethesda, Md.)

287 The Role of Melanocortins in Skin Homeostasis Böhm, M.; Luger, T.A. (Münster) 294 Prevalence of Endocrine Dysfunction in HIV-Infected Men Brockmeyer, N.H.; Kreuter, A.; Bader, A.; Seemann, U.; Reimann, G. (Bochum)

Hormone Research Vol. 54, 2000

III

296 Targeted Somatic Mutagenesis in Mouse Epidermis Indra, A.K.; Li, M.; Brocard, J.; Warot, X.; Bornert, J.-M.; Gérard, C.; Messaddeq, N.; Chambon, P.; Metzger, D. (Illkirch)

Suppl. 1

301 Interaction of Vitamin D and Retinoid Receptors on Regulation

Analytical Methods in Clinical Osteology

of Gene Expression

Editors: Cowell, C.T. (Parramatta); Wüster, C. (Heidelberg/Wiesbaden)

Jimenez-Lara, A.M. (Illkirch); Aranda, A. (Madrid)

306 Development of Efficient Transient Transfection Systems for

Introducing Antisense Oligonucleotides into Human Epithelial Skin Cells Fimmel, S.; Saborowski, A.; Orfanos, C.E.; Zouboulis, C.C. (Berlin)

312 A Novel Pathway for Hormonally Active Calcitriol Lehmann, B.; Knuschke, P.; Meurer, M. (Dresden) 316 Successful Treatment of Non-Segmental Vitiligo: Systemic

Therapy with Sex Hormone-Thyroid Powder Mixture Nagai, K.; Ichimiya, M.; Yokoyama, K.; Hamamoto, Y.; Muto, M. (Ube)

318 Liposomal Ursolic Acid (Merotaine) Increases Ceramides and

Collagen in Human Skin Yarosh, D.B.; Both, D.; Brown, D. (Freeport, N.Y.)

322 Association of Insulin Resistance with Hyperandrogenia in

Women Pugeat, M.; Ducluzeau, P.H.; Mallion-Donadieu, M. (Lyon)

327 The Molecular Basis of Androgen Insensitivity Nitsche, E.M.; Hiort, O. (Lübeck) 334 Genetics of Peutz-Jeghers Syndrome, Carney Complex and

Other Familial Lentiginoses Stratakis, C.A. (Bethesda, Md.)

344 Author Index Vol. 54, No. 5–6, 2000 345 Subject Index Vol. 54, No. 5–6, 2000 346 Author Index Vol. 54, 2000 347 Subject Index Vol. 54, 2000

S. Karger Medical and Scientific Publishers Basel 폷 Freiburg 폷 Paris 폷 London New York 폷 New Delhi 폷 Bangkok Singapore 폷 Tokyo 폷 Sydney

IV

Drug Dosage The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any change in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.

Hormone Research Vol. 54, 2000

All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher or, in the case of photocopying, direct payment of a specified fee to the Copyright Clearance Center (see ‘General Information’). © Copyright 2001 by S. Karger AG, P.O. Box, CH–4009 Basel (Switzerland) Printed in Switzerland on acid-free paper by Reinhardt Druck, Basel

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