VOLUME 172
SERIES EDITORS Geoffrey H. Bourne James F. Danielli Kwang W. Jeon Martin Friedlander Jonathan Jarvik
1949-1 988 1949-1 984 19671984-1 992 1993-1 995
EDITORIAL ADVISORY BOARD Aimee Bakken Eve Ida Barak Howard A. Bern Robert A. Bloodgood Dean Bok Stanley Cohen Rene Couteaux Marie A. DiBerardino Charles J. Flickinger Nicholas Gillham Elizabeth D. Hay P. Mark Hogarth Anthony P. Mahowald M. Melkonian Keith E. Mostov Audrey L. Muggleton-Harris
Andreas Oksche Muriel J. Ord Vladimir R. Pantic Thomas D. Pollard L. Evans Roth Jozef St. Schell Manfred Schliwa Hiroh Shibaoka Wilfred D. Stein Ralph M. Steinman M. Tazawa Yoshio Watanabe Donald P. Weeks Robin Wright Alexander L. Yudin
Edited by
Kwang W. Jeon Department of Zoology University of Tennessee Knoxville, Tennessee
VOLUME 172
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3 2 1
CONTENTS
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
ix
Transport of Glucose across the Blood-Tissue Barriers Kuniaki Takata, Hiroshi Hirano, and Michihiro Kasahara I. 11. 111. IV. V. VI.
Introduction . . . . . ............................ ............ Glucose Transporte .............. .................... Blood-Tissue Barriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Glucose Transport in the Blood-Tissue Barriers . . . . . . . . . . . . . . . Regulation of Glucose Transporter Expression in Blood-Tissue Barriers . . . . . . . . . . . . Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. ...... References
1 2 5 9 33 37 38
The Role of Suppressors in Determining Host-Parasite Specificities in Plant Cells Tomonori Shiraishi, Tetsuji Yamada, Yuki Ichinose, Akinori Kiba, and Kazuhiro Toyoda I. 11. II. IV. V. VI. VII.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Suppressors of Defense Response Produced by Phytopathogens .. Specific Production and Accessibility-Inducing Activity of Suppressors . . . . . . . . . . . . . Specific Suppression of the Establishment of Chemical Barriers . . . . . . . . . . . . . . . . . . Mode of Action of the Suppressors ........ Species-Specific Suppression of Cell Wall Function by the Suppressor . . . . . . . . . . . . Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
............. V
55 59 62 65 67 76 81 85
vi
CONTENTS
Distribution and Functions of Platelet-Derived Growth factors and Their Receptors during Embryogenesis Paris Ataliotis and Mark Mercola I. II. 111, IV.
Introduction Biochemistry of PDGFs and Their Receptors Distributionof PDGFs during Embryogenesis Functional Studies on the Effects of PDGF V. Concluding Remarks References
95 96 104 110 117 119
Adaptive Crystal Formation in Normal and Pathological Calcifications in Synthetic Calcium Phosphate and Related Biomaterials G. Daculsi, J.-M. Bouler, and R. Z. LeGeros I. 11. 111. IV. V.
VI. VH. VIII. IX.
Introduction Analytical Techniques Crystal Formation, Composition, and Properties in Normal Calcifications Crystal Formation, Composition, and Properties in Pathological Calcifications Factors Affecting Crystal Formation and Properties of Biologically Relevant Calcium Phosphates Calcium Phosphates and Related Bone Graft Biomaterials Comparative Properties of Bone and Calcium Phosphate Materials Bone/BiomaterialInterface Summary and Conclusion References
129 135 139 145 154 159 165 167 175 177
The Biogenesis, Traffic, and Function of Cystic Fibrosis Transmembrane Conductance Regulator Tamas Jilling and Kevin L. Kirk I. Introduction II. Cystic Fibrosis Transmembrane Conductance Regulator Ill. CFTR Mutations That Cause Disease IV. Physiological Role of CFTR as an Apical CI' Channel in Epithelial Tissues V. Regulation of CFTR Function by the Cytoskeleton VI. CFTR as a Regulator of Other Channels VII. Itinerary of CFTR Traffic within Epithelial Cells
193 195 200 208 212 213 215
vii
CONTENTS
VIII. Regulation of Membrane Traffic by CFTR? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IX. Summary and Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
220 231 232
Regulation of Phenylpropanoid Metabolism in Relation to Lignin Biosynthesis in Plants Mark S. Barber and Heidi J. Mitchell I. II. 111. IV. V. VI.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . General PhenylpropanoidPathway . . . . . . . . . . . . . . . . . . . . . . . Lignin Branch Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Storage and Transport of Monolignols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PolymerizationProcess . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding Remarks,. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
....................................................
243 247 262 269 271 273 273
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
295
This Page Intentionally Left Blank
Numbers in parentheses indicate the pages on which the authors' contributions begin.
Paris Ataliotis (95), Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02 1 15 Mark S. Barber (243),School of Biological Sciences, University of Southampton, Southampton SO 16 7PX, United Kingdom J,-M. Bouler (129), Laboratoire Recherche surles Tissus Calcifies et les Biomateriaux, Faculte de Chirurgie Dentaire, 44042 Nantes Cedex 01, France G. Daculsi (129), Laboratoire Recherche sur les Tissus Calcifies et les Biomateriaux, Faculte de Chirurgie Dentaire, 44042 Nanfes Cedex 01, France Hiroshi Hirano (1 ), Depaltment of Anatomy, Kyorin University School of Medicine, Mitaka, Tokyo 181, Japan Yuki lchinose (55), Laboratory of Plant Pathology and Genetic Engineering, College of Agriculture, Okayama University, Okayarna 700, Japan Tamas Jilling (193), Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, Alabama Michihiro Kasahara ( 1), Laboratory of Biophysics, School of Medicine, Teikyo University, Hachioji, Tokyo 192-03,Japan Akinori Kiba (55),Laboratory of Plant Pathology and Genetic Engineering, College of Agriculture, Okayama University, Okayama 700, Japan Kevin L. Kirk (193), Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, Alabama 35294
R. 2. LeGeros (129), Laboratoire Recherche sur les Tissus Calcifies et les Biomateriaux, Faculte de Chirurgie Dentaire, 44042 Nantes Cedex 01, France ix
X
CONTRIBUTORS
Mark Mercola (93, Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 021 15 Heidi J. Mitchell (243), Research School of Biological Sciences, Australian National University, Canberra ACT 0200, Australia Tomonori Shiraishi (55), Laboratory of Plant Pathology and Genetic ,Engineering, College of Agriculture, Okayama University, Okayama 700, Japan Kuniaki Takata (i), Laboratory of Molecular and Cellular Morphology, lnstifute for Molecular and Cellular Regulafion, Gunma University, Maebashi, Gunma 371, Japan KazuhiroToyoda (55),Laboratory of Plant Pathologyand Genetic€ngineering,College of Agriculture, Okayama University, Okayama 700, Japan Tetsuji Yamada (55),Laboratory of Plant Pathology and Genetic €ngineering, College of Agriculture, Okayama University, Okayama 700, Japan
Transport of Glucose across the Blood-Tissue Barriers Kuniaki Takata,* Hiroshi Hirano,t and Michihiro Kasaharat “Laboratory of Molecular and Cellular Morphology, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Gunma 371, Japan; ?Department of Anatomy, Kyorin University School of Medicine, Mitaka, Tokyo 181, Japan; and $Laboratory of Biophysics, School of Medicine, Teikyo University, Hachioji, Tokyo 192-03, Japan
In specialized parts of the body, free exchange of substances between blood and tissue cells is hindered by the presence of a barrier cell layer@).Specialized milieu of the compartments provided by these “blood-tissue barriers” seems to be important for specific functions of the tissue cells guarded by the barriers. In blood-tissue barriers, such as the blood-brain barrier, blood-cerebrospinal fluid barrier, blood-nerve barrier, blood-retinal barrier, blood-aqueous barrier, blood-perilymph barrier, and placental barrier, endothelial or epithelial cells sealed by tight junctions, or a syncytial cell layer@), serve as a structural basis of the barrier. A selective transport system localized in the cells of the barrier provides substances needed by the cells inside the barrier. GLUTl , an isoform of facilitated-diffusionglucose transporters, is abundant in cells of the barrier. GLUT1 is concentrated at the critical plasma membranes of cells of the barriers and thereby constitutes the major machinery for the transport of glucose across these barriers where transport occurs by a transcellular mechanism. In the barrier composed of doubleepithelial layers, such as the epithelium of the ciliary body in the case of the blood-aqueous barrier, gap junctions appear to play an important role in addition to GLUT1 for the transfer of glucose across the barrier. KEY WORDS: Glucose transporter, GLUTl , Gap junction, Blood-tissue barriers, Placental barrier, Epithelial cells, Endothelial cells.
1. Introduction The mammalian body is a conglomerate of highly differentiated organs. The organs are made of specialized tissue-specific cells, blood vessels, nerves, etc. lnrernarional Review of cyfO/Ogy, V d I72 (H174-769hiY7 $25.00
1
Copyright 0 1997 hy Academic Press All rights of reproduction i n any form reserved.
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KUNlAKl TAKATA ET AL.
The normal function of these tissue cells is maintained by an uninterrupted supply of nutrients and oxygen and concomitant removal of metabolites and carbon dioxide via the circulating blood. In some of the tissues and organs, free exchange of substances between blood and tissue cells is hindered by the presence of barrier cell layers. A specialized environment provided by these “blood-tissue barriers” seems to be important for specific functions of the tissue cells enclosed by the barrier. Tracer experiments have demonstrated that either cndothelial or epithelial cells are the anatomical basis of the barrier. Specific transport mechanism across such bloodtissuc barriers must exist for a number of substances. In this chapter, we focus on the cellular and molecular basis for the transport of glucose across the blood-tissue barriers. II. Glucose Transporters Glucose is onc of the most important sources of energy as well as a substrate for a variety of cellular molecules. Cellular membranes, whose basic structure is made of phospholipid bilayers, are practically impermeable to small polar molecules such as glucose. Glucose transporters are integral membrane proteins that mediate the transport of glucose and related substances across the cellular membranes (Wheeler and Hinkle, 1985; Baly and Horuk, 1988; Carruthers, 1990; Silverman, 1991; Baldwin, 1993). Two types of glucose transporters have been discovered in animal cells: facilitateddiffusion glucose transporters and Na+-dependentactive glucose transporters (cotransporters) (Kasahara et al., 1985; Baly and Horuk, 1988; Nikaido and Saier, 1992). Facilitatcd-diffusion glucose transporter was first identiticd by Kasahara and Hinkle (1977) in human erythrocyte ghosts as a zone 4.5 protein. The gene was cloned and sequenced from a HepG2 human hepatoma cell cDNA library (Mueckler et al., 1985) and from a rat brain library (Birnbaum rt a/., 1986), and later termed GLUT1 (Bell et al., 1990). The N a ’ -dependent glucosc transporter is a cotransporter that mediates the active uptake of glucose across the membrane driven by the influx of Nat according to its chemical gradient. The gene was first cloned from a rabbit intestinal cDNA library by the expression cloning method using Xenopus oocytes (Hedigcr et al., 1987) and termed SGLTl (Hediger et al., 1989). Each transporter constitutes a family, and several isoforms have been identitied and characterized.
A. Facilitated-Diffusion Glucose Transporter Family Six isoforms of facilitated-diffusion glucose transporter GLUT family have been cloned in mammalian cells (Table 1) (Bell etal., 1990,1993; Silvcrman,
TABLE I GLUT Family Transporters in Animal Cells
Gene
Species
Major site of expression
GLUT1
Human
Blood-tissue barriers. erythrocyte. fetal tissues
Transport substrate
K,,, (mM)"
Number of amino acids
17
492
Glucose
492 492 492 492 Fragment 490
Rat Mouse Bovine Rabbit Pig Chicken
GLUT2
Human
Liver. pancreatic 6-cell. small intestine, kidney
42
Glucose, fructose
Rat Mouse Chicken
GLUT3
503145 X16986. XI5684 222932
Dog Chicken Sheep
495 496 494
Rat
GLUT4
522 523
Mouse
11
Glucose
Human Rat
Adipocyte, muscle
4
Glucose
510
Mouse
GLUT5
Human Rat
GLUT7
Rat
509 509
Small intestine
Fructose
Liver microsome
Glucose
6
Rabbit ~
501 502
Human chromosomal location
JCqofor cytochalasin B (PM) 0.1
1
0.4
M13979, M22063 M22998. M23384 M60448 M21747 X17058 LO7300
~03810
533
Brain
K0319S
524
496 493 493
Human
GenBank accession number
M20681
3
7
12
0.1-2
D13962, U17978 X61093, U11853, M75135 L35267 M37785 L39214 M20747, M91463 D28561.504524, X14771. M25482 M23383
486
M55531 D13871, D28562, LO5195 D26482
528
X66031
17 0.3
1
No inhibition
~
* K,, for 3-0-methyl glucose transport under equilibrium exchange conditions, except for GLUT5 in which fructose is the substrate.
4
KUNlAKl TAKATA ET AL.
1991; Gould and Bell, 1990; Lienhard et al., 1992; Pessin and Bell, 1992; Thomas el al., 1992; Baldwin, 1993; Gould and Holman, 1993; Marger and Saier, 1993; Takata et al., 1993a; Mueckler, 1994). Analyses of the amino acid sequences of the transporters indicate that GLUT glucose transporters are transmembrane proteins that span the lipid bilayer 12 times, with both the amino and carboxyl termini facing the cytoplasmic side (Mueckler rt al., 1985). GLUT transporters are a member of thc major facilitator superfamily which possesses a common structural motif of 12 transmembrane-spanning a-helical segments (Marger and Saier, 1993). The distribution of GLUT transporters in organs, tissues, and cclls has bccn determined by Northern blotting, immunoblotting, in situ hybridization, and immunohistochemical staining. GLUT transporters are produced in a tissue- and cell-specific manner and seem to be closely related to a variety of cellular activities, ubiquitous and specific (Gould and Bell, 1990; Takata et al., 19931; Gould and Holman, 1993; Mucckler, 1994). GLUTl is a widely distributed transporter isoform whose synthesis begins at a very early stage during development and is responsible for the uptake of glucose for the basal cellular activities. The most characteristic feature of GLUTl is its abundance in the blood-tissue barriers. GLUT2 is a low-affinity, highcapacity transporter produced in liver, intestine, kidney, and pancreatic /3 cells (Thorens et al., 1988, 19YOa,b; Fukumoto et al., 1988a; Johnson et af., 1990; Orci et al., 1989; Thorens, 1992). This transporter, in combination with other molecules such as glucokinase, is considered to play an important role in the sensing of blood glucose level in pancreatic /3 cclls (Orci et u1.,1989; Thorens et a/., 1990c; Matschinsky, 1990; Unger, 1991; Thorens, 1992; Vionnct et al., 1992). GLUT3 is predominantly produced in the brain and is considered to be a glucose transporter of neurons (Kayano et af., 1988; Nagamatsu et d., 1992). GLUT4 is an isoform produced in insulinsensitive cells such as adipocytes and skeletal and cardiac muscle cells (Birnbaum, 1989, 1992; James et al., 1989; Kaestner et al., 1989; Charron et al., 1989; Fukumoto et a/., 1989). This transporter is prefercntially localized in the intracellular compartments such as endosomes and the transGolgi reticulum (Slot et al., 1990, 1991a,b; Friedman et al., 1991; Smith et al., 1991; Takata et al., 1992a). Upon insulin stimulation, cytoplasmic vesicles containing GLUT4 fuse with the plasma membrane (Suzuki and Kono, 1980; Cushman and Wardzala, 1980; Kono et al., 1982; Ezaki et al., 1986) and thereby the number of surface GLUT4 molecules increases. Such translocation of GLUT4 molecules from the intracellular pool to the cell surface mainly accounts for the insulin-stimulated increase in glucose transport activity and serves as a mechanism for the homeostasis of blood glucose level (Birnbaum, 1992). GLUT5 is a fructose transporter expressed in the small intestine and is responsiblc for the dietary absorption of fructose (Kayano et al., 1990; Burant et al., 1992; Davidson et al., 1992). GLUT6 is
TRANSPORT OF GLUCOSE ACROSS BLOOD-TISSUE BARRIERS
5
a pseudogene (Kayano etal., 1990). GLUT7 was isolated from a liver cDNA library and has a homology to GLUT2 (Waddell et af.,1992). It is a glucose transporter of the microsomal membrane and seems to play an important role in the exit from the endoplasmic reticulum of glucose formed by the action of glucose-6-phosphatase (Burchell et af., 1994).
B. Nat-Dependent Glucose Transporter Family Transport of glucose by Na+-dependent glucose transporter SGLTl is dependent on extracellular Na', and therefore it can mediate the uphill transport of glucose. SGLTl does not have sequence homology to transporters of the GLUT family (Hediger etaf., 1989). Rather, it is a member of a distinct Na+-dependent cotransporter SGLTl family (Wright, 1993; Hediger and Rhoads, 1994; Hediger et uf., 199.5). Four different models have been proposed so far for the configuration of the molecule in the lipid bilayer (Hediger et al., 1987; Wright et al., 1991; Lee et af., 1994; Turk et al., 1996). SGLTl is a high-affinity glucose cotransporter with a Na+/glucose coupling ratio of 2: 1 (Lee et af., 1994), and it is present in both the kidney and the small intestine (Takata et af., 1991a,b, 1992b; Hwang et al., 1991; Yoshida et af., 199.5). SGLTl plays a pivotal role in the absorption of glucose in the small intestine because mutation of SGLTl resulted in a severe dysfunction of glucose absorption in the small intestine (Turk et al., 1991). SGLTl in combination with GLUT2 and GLUT1 serves in the active transepithelial transport of glucose (Thorens, 1993; Takata, 1996). Recently, two low-affinity, Na+-dependent glucose transporters, SGLT2 (Kanai et af., 1994) and pSGLT2 (SAAT1) (Kong et al., 1993; Hediger et af., 199.5), were identified. SGLT2 exhibited a Na+/glucose coupling ratio of 1: 1 and seems to be responsible for the reabsorption of glucose from the urinary filtrate in the kidney S1 proximal tubules. Na+-dependent rnyoinositol cotransporter SMIT (Kwon et al., 1992) and nucleoside cotransporter SNSTl (Pajor and Wright, 1992) are also members of this family.
111. Blood-Tissue Barriers A. Dietary Absorption of Glucose Dietary carbohydrates are hydrolyzed to monosaccharides and absorbed in the alimentary tract. In the small intestine, glucose is absorbed through the absorptive epithelial cells lining the surface of the villi. Nat-dependent glucose cotransporter SGLTl is localized at the microvillous apical plasma
ti
KUNlAKl TAKATA ET AL.
membrane of the absorptive epithelial cells (Takata et al., 1992b; Yoshida et nl., 1995). Glucose in the intestinal lumen is actively transported into the cytoplasm of the absvrptive epithelial cells by SGLTl driven by the chemical gradient of Na+ across the plasma membrane, which is maintained by the action of Na'/K+-ATPase at the expense of ATP (Wright 1993; Wright rt al., 1991, 1994; Hediger and Rhoads, 1994). Glucose leaves the absorptive epithelial cells by the action of facilitated-diffusion glucose transporter GLUT2 localized at the basolateral plasma membrane (Thorens et of., 1990a; Thorens, 1992, 1993). It then enters the capillaries in the core of the villi and, after passing through the liver via the portal vein, it is distributed throughout the body via the vast network of blood vessels (Unger, 1991; Takata et al., 1993a).
6. Blood-Tissue Barriers: Endothelium Type and Epithelium Type In most parts of the body, relatively free exchange of substances including glucose occurs between blood and tissue cells (parenchymal cells). In the specialized tissues and organs, in which a specific microenvironment is of great importance for their specific functions, such free exchange is hindered by the presence of barrier cell layers (Fig. 1). The barrier property of the tissues was well demonstrated by tracer experiments: Intravenously administered cytochemical tracers, such as dyes or horseradish peroxidase, b
a tissue cells
C
tissue cells
tissue cells
FIG. 1 Blood-tissue barriers. (a) The absence of blood-tissue barricrs. In most lissues, eudolhelial walls of blood vessels are highly permeable, and free exchange of a variety of substances occurs bctwcen blood and tissue cells. (b) Blood-tissue barrier of the cndothelium type. The endothelium is impermeable and constitutcs a barrier layer. (c) Blood-tissue barrier of the epithelium type. Although the blood vessels are permeable, the epithelial barricr layer prcvcnts the exchange of substances between blood constitucnts and tissue cclls.
7
TRANSPORT OF GLUCOSE ACROSS BLOOD-TISSUE BARRIERS
failed to penetrate into these tissues. Such impermeable barriers, as summarized in Table 11, are found in the brain (blood-brain barrier and bloodcerebrospinal fluid barrier), the eye (blood-ocular barrier: i.e., bloodretinal barrier and blood-aqueous barrier), the inner ear (blood-perilymph barrier and blood-endolymph barrier), the peripheral nerves (blood-nerve
TABLE II Blood-Tissue Barriers and Glucose Transporters"
Barrier ~
Site of the barrier
~
~
Major glucose transporter in the barrier ~~
Blood-brain barrier
Endothelial cells (brain microvessels)
GLUT^ *
Blood-cerebrospinal fluid barrier
Endothelial cells (brain rnicrovessels)
GLUTl
Epithelial cells (choroid plexus)
GLUT1'
Blood-retinal barrier
Endothelial cells Pigment epithelial cell
GLUTl GLUTl
Blood-aqueous barrier
Epithelial cells (ciliary body) Endothelial cells (iris)
GLUTI"
Blood-perilymph barrier
Endothelial cells (inner ear)
GLUTl
Blood-endolymph barrier
Basal cells (stria vascularis)
GLUTl'
Blood-nerve barrier
Perineurial cells (perineurium) Endothelial cells (endoneurial blood vessels)
GLUTl GLUTl
Placental barrier (human)
Syncytiotrophoblast layer
Placental barrier (rat)
Syncytiotrophoblast layers
GLUTIK
Blood-testis barrier
Sertoli cells
NDhJ
Blood-thymus barrier (cortex)
Endothelial cells'
N D~
CLUTlf
See text for references. In addition to GLUTl, the presence of GLUT3 or GLUT4 was suggested. GLUTl is localized at the basolateral membrane. 'IGap junctions (connexin 43), in combination with GLUTl, serve as a transport machinery. GLUTl in the basal cells may also serve in the transport of glucose across the perilymphendolymph barrier. 'In addition to GLUTl, the presence of GLUT3 was suggested. Gap junctions (connexin 26), in combination with GLUTl, serve as a transport machinery of glucose. GLUT3 is also present. Not detected. ' GLUTl is localized in the endothelial cells of the blood vessels surrounding seminiferous tubules. 1 Contribution of macrophages is also suggested.
''
8
KUNIAKI TAKATA E r AL.
barrier) , the placenta (placental barrier), the testis (blood-testis barrier), and the thymus (blood-thymus barrier). These barriers are collectively called the blood-tissue barriers (Takata et al., 1990a,b, 1993a). Histological and ultrastruct ural examinations have revealed that endothelial cells sealed by tight junctions serve as an anatomical basis for the barrier when blood vessels are the barrier (Fig. l b ) (Takata et al., 1990b). In another case, the blood vessels are highly permeable, but the adjacent epithelial cells sealed by tight junctions function as an impermeable barrier (Fig. lc). In special cases, a syncytial cell layer works as a barrier layer instead of cells sealed by tight junctions. Specific transport machinery located in the barrier layer may ensure the supply of substances needed by the cells enclosed by the barrier layer. Glucose is a ubiquitous source of cellular metabolism in the mammalian body. Glucose transporters located in the cells of the barrier play a pivotal role in the passage of glucose across the barrier for the nourishment of the cells inside the barrier. Among the isoforms of glucose transporters, GLUTl is abundant in the cells of blood-tissue barriers and serves as the major glucose transporter isoform in these barriers (Figs. 2 and 3) (Takata et al., 1990a,b, 1993a; Harik et al., 1990a,b). In some cases, connexins of gap junctions in combination with GLUTl seem to be involved in the passage of glucose through the barrier (Takata et al., 1991c, 1994; Shin et ul., 1996a,b).
continuous capillary
fenestrated epithelial barrier
FIG. 2 GLUTI, a glucose transporter isoform, in blood-tissue barriers. The two basic types of blood-tissue barriers are illustrated. TJ, tight junction. (a) Endothelium type. A continuous capillary sealed by tight junctions serves as the structural basis for the barrier. Glucose transfer across the barrier is carried out by the transendothelial transport of glucose via plasma membrane GLUTI. Note that GLUTl is present at both the luminal and contraluminal domains of the plasma membrane. (b) Epithelium type. Fenestrated capillarics are highly permeable, An epithelium sealed by tight junctions, or a syncytial epithclial cell (not shown), serves as the structural basis for this barrier. Glucose transfer across the barrier is carried o u t by the transepithelial transport of glucose via plasma membrane GLUT1. Note that GLU'TI is present at both the apical and basolateral domains of the plasma membrane.
9
TRANSPORT OF GLUCOSE ACROSS BLOOD-TISSUE BARRIERS
-200K -116K - 97K
-
66K
45K
FIG. 3 Immunoblotting with anti-GLUT1 antibody of rat tissues having blood-tissue barriers. Cell homogenates (10pgprotein each) were analyzed. GLUTl is detected in brain microvessels (brain vessel, 50 kDa), inner portion of the retina (retina, 43 kDa), pigment epithelium of the retina (pig. epithelium, 46 kDa), ciliary body and iris of the eye (ciliary B. & Iris, 46 kDa), and in the placenta (44 kDa). No comparable amount of GLUTl was found in testis and thymus. Reproduced from Takata et al. (1990b) with permission from Academic Press.
IV. Glucose Transport in the Blood-Tissue Barriers A. Blood-Brain Barrier 1. Structure Among the blood-tissue barriers, the blood-brain barrier has been extensively studied (Fishman, 1980; Betz and Goldstein, 1986; Cserr, 1986; Strand, 1988; Dermietzel and Krause, 1991). In the blood-brain barrier, tight junctions between endothelial cells are responsible for preventing the free passage of substances across the barrier (Reese and Karnovsky, 1967; Stewart et al., 1994). Intravenous injection of tracers such as horseradish peroxidase demonstrated that the tracers remained in the lumen of the blood vessels in the brain and were not found beyond the vascular endothelium (Reese and Karnovsky, 1967). A similar tracer experiment also established the blood-cerebrospinal fluid barrier, in which the epithelium of the choroid plexus, in addition to the cerebral blood vessels, functions as the structural basis for this barrier (Brightman and Reese, 1969).
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KUNlAKl TAKATA ET AL.
2. GLUTl in the Blood-Brain Barrier Crone (1965) measured the transfer of glucose from the blood into brain tissues by the intracarotid injection of labeled glucose in dogs. A carriermediated. facilitated passage of glucose across the blood-brain barrier was observed. Based on these findings, Crone (1965) proposed a possible mechanism involving endolhelial cells in the brain capillaries for the transport of glucose across the blood-brain barrier. Dick et al. (1984) demonstrated that cerebral microvessels were rich in a 53-kDa glucose transporter by the binding of cytochalasin B, a specific ligand for glucose transporter, and by anti-human erythrocyte glucose transporter (GLUTl) antibody labeling. The abundance of GLUTl in the blood-brain barrier was confirmed in brain capillary specimens by immunoblotting (Fig. 3 ) (Kalaria et al., 1988; Gerhart et al., 1989; Pardridge et al., 1990; Takata et al., 1990b), Northern blotting (Flier et al., 1987; Boado and Pardridge, 1990), and binding of cytochalasin B (Kalaria et al., 1988). Comparison of quantitative immunoblotting for GLUT1 and cytochalasin B binding indicated that GLlJTl is the principal glucose transporter isoforni mediating glucose transport across the blood-brain barrier (Pardridge et al., 1990; Dwyer and Pardridge, 1993). GLUTl was shown to be localized in the endothelial cells of the brain microvessels by immunohistochemical staining (Fig. 4) (Kalaria et al., 1988; Gerhart et al., 1989; Kasanicki et al., 1989; Harik et al., 1990a; Pardridge et al., 1990; Takata et al., 1990b) and by in situ hybridization (Pardridge et aL, 1990). GLUTl localized at both the luminal and contraluminal plasma membranes (Gerhart et al., 1989;Takata et al., 1990b;Farrell and
FIG. 4 GLCJTI in the blood-brain barrier. GLUTl is localized at hoth the luminal (arrowheads) and contraluniinal (arrows) plasma membranes of microvessel cndothclial cells. Immunofluorescence (a) and corresponding Nomarski differential interference contrast (b) images arc shown. Bar = 10 Fm.
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11
Pardridge, 1991) is thought to be the molecular basis of the transendothelial transfer of glucose in the blood-brain barrier. In an immunogold electron microscopic study, Gerhart et al. (1989) showed that GLUTl was equally abundant on the luminal and contraluminal membranes of endothelial cells of canine brain microvessels. Most of the label was restricted to the plasma membrane, and less than 10%of the GLUTl was found in the cytoplasm. Farrell and Pardridge (1991), on the other hand, showed GLUTl to be asymmetrically distributed on endothelial cells of the rat brain capillaries: There was a fourfold greater abundance on the contraluminal membrane compared with the density on the luminal membrane. Similar results were obtained for rabbit brain endothelial cells (Cornford f’t al., 1993). The difference observed between canine and rat/ rabbit blood vessels with respect to the distribution of GLUT1 is not clear but may be attributed to species differences or the method used. The asymmetrical distribution of GLUTl might create a faster rate of glucose transport across the contraluminal membrane compared with that across the luminal membrane, thereby minimizing the phosphorylation of glucose in the cytoplasm and maximizing the transfer of glucose across the barrier. In addition. in this case more than 40% of the GLUTl was observed within the cytoplasm of the endothelial cells (Farrell and Pardridge, 1991). This cytoplasmic pool of GLUT1 might serve as a reservoir and could be in a possible translocation machinery as seen for GLUT4 in the insulin-sensitive cells (Birnbaum, 1992). GLUTl is also expressed in astrocytes (Devaskar et al., 1991; Lee and Bondy, 1993; Morgello et al., 1995), epithelial cells of the choroid plexus (Kalaria et al., 1988; Bagley et al., 1989; Takata et al., 1990b; Farrell et al., 1992a), ependymal epithelial cells lining the ventricular wall (Farrell et al., 1992a), and in tanycytes (Young and Wang, 1990) in the brain. Differences in apparent molecular mass of GLUTl were observed following SDSpolyacrylamide gel electrophoresis: Whereas GLUTl of microvessels showed a molecular mass of 54 or 55 kDa, that of astrocytes and choroid plexus exhibited a lower one of 42-45 kDa (Maher et al., 1993, 1994; Vannucci, 1994; Kumagai et al., 1994a; Morgello et al., 1995). The apparent difference is caused by differential N-linked glycosylation (Kumagai et al., 1994a), the functional significance of which remains to be clarified. GLUT1 was not expressed in the endothelial cells devoid of barrier properties in the brain, nor was it detected in the blood vessels of the median eminence and area postrema, both of which lack barrier characteristics (Kalaria rt al., 1988; Young and Wang, 1990). The adenohypophysis was devoid of GLUT1, whereas some blood vessels were GLUTl positive in the neurohypohysis (Gerhart et al., 1989). Endothelial cells in the choroid plexus have numerous fenestrae and are thus highly permeable. GLUTl was not found in these cells. Instead, epithelial cells of the choroid plexus
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were strongly positive for GLUTl (Kalaria et al., 1988; Bagley et al., 1989; Takata et al., 1990b). 3. Glucose Transporters Other Than GLUTl
GLUT3 is expressed in the brain (Kayano et al., 1988; Bell et al., 1990; Nagamatsu et al., 1992; Maher et af., 1994). Northern blotting and in situ hybridization as well as immunoblotting and immunohistochemical staining showed that GLUT3 is specifically localized in neurons in the rodent brain (Nagamatsu el al., 1992,1993a). Although GLUTl is the prime isoform of glucose transporters in the cerebral microvessels and astrocytes, and GLUT3 in the neurons, the contribution of transporter isoforms other than GLUT1 in the blood-brain barrier has been suggested. Using anti-peptide antibody raised against the carboxyl terminal end of human GLUT3 protein, Gerhart et al. (1992) detected GLUT3 in the blood-brain barrier in frozen sections from dogs and rats. Immunohistochemical staining demonstrated that neurons and microvessels were positively stained. Becausc the amino acid sequencc of the carboxyl terminus of GLUT3 differs considerably between human and rodent GLUT3 (Kayano et al., 1985; Nagamatsu et al., l992), some of these positive reactions may not represent GLUT3. In fact, antibody specific for rodent GLUT3 failed to result in positive staining in rat brain microvessels (Gerhart et nl., 1995). In the dog brain, however, endothelial cells in the blood brain barrier were positively stained with an antibody against canine GLUT3 (Gerhart et al., 1995). In the human brain, although GLUT3 is primarily expressed in neurons, the presence of GLIJT3 in the microvascular endothelial cells was reported (Mantych et nl., 1992). However, GLUT3 was not detected in isolated human or rat microvessels (Maher et al., 1993). The previously mentioned inconsistency in results with regard to the presence of GLUT3 in brain microvessels may be due to spccies differences andlor to the variation in carboxyl terminal sequences used to generate the antibodies, which would result in differences in the cross-reactivity of the antibodies as well as in nonspecific binding of the antibody to other molecules (Shepherd et al., 1992a). Although further studies are needed, GLUTl is clearly the major transporter in the bloodbrain barrier, and the contribution of GLUT3 to the transfer of glucose across the barrier is probably minimal, if any (Maher et al., 1993, 1994; Vannucci 1994). Fructose transporter GLUTS was detected in the human brain by immunoblotting (Shepherd et aL, 1992b; Mantych et al., 1993a). Immunohistochemical analysis demonstrated that only some of the brain microvascular endothelial cells were positive for GLUTS, although all the vessels were positive for GLUT1 and factor VlIl staining (Mantych et al., 1993a). Because fructose is not used as a source of nutrient in the brain, it is unlikely
TRANSPORT OF GLUCOSE ACROSS BLOOD-TISSUE BARRIERS
13
that GLUTS carries fructose through the blood-brain barrier. The possible role of GLUT5 remains obscure. In addition to the facilitated-diffusion transfer mechanism, immunohistochemical and immunoblotting studies on bovine cortical vessels suggested the presence of a Na+-dependentglucose cotransporter of the SGLT family in these vessels (Nishizaki et af.,1995). Further examination will be needed to determine the possible involvement of a Na'-dependent system in the blood-brain barrier.
6 . Blood-Cerebrospinal Fluid Barrier
1. Structure Cerebrospinal fluid, which fills the cranial cavity, is an important determinant of the extracellular fluid that bathes neurons and glia in the central nervous system (Fishman, 1980; Wood, 1980; Cutler, 1980; Rowland et af., 1991). It is secreted mainly by the choroid plexus in the lateral ventricle and is absorbed through the arachnoid villi. Another source of the cerebrospinal fluid is parenchymal blood vessels. The fluid from the blood vessels enters the ventricular system through the ependymal cell layer. The permeability barrier between blood and cerebrospinal fluid is called the bloodcerebrospinal fluid barrier. The choroid plexus is made of cuboidal to columnar epithelial cells connected by tight junctions. Capillaries located underneath the choroid plexus epithelium are of the fenestrated type. Tracer experiments using horseradish peroxidase and lanthanum demonstrated that endothelial cells in the brain microvessels and epithelial cells of the choroid plexus, both of which are sealed by tight junctions, serve as the anatomical basis for the blood-cerebrospinal fluid barrier (Brightman and Reese, 1969).
2. Glucose Transport A carrier-mediated glucose transport system across the barrier was shown by the intravenous or intracisternal administration of sugars (Fishman, 1964) and by the ventriculocisternal perfusion method (Bradbury and Davson, 1964). The epithelial cells of the choroid plexus are rich in GLUT1, which is localized at the basolateral plasma membranes (Kalaria et al., 1988; Bagley et al., 1989; Takata et nf., 1990b; Farrell et al., 1992a). A 45- to 47-kDa form of GLUT1 was detected in the choroid plexus (Kumagai et al., 1994a; Vannucci, 1994). The apparent difference in M , of GLUT1 from that of brain microvessels is due to the differential glycosylation of GLUT1 in the choroid plexus (Kumagai et al., 1994a). Such differential glycosylation
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might contribute to the basolateral targeting of GLUTl molecules in the choroid plexus. Glucose transporters in the apical plasma membrane of the epithelial cells in the choroid plexus have not been demonstrated so far. Because the disposition of glucose transporters at both the apical and basolateral plasma membranes is a prerequisite for the successful transepithelial transport of glucose (Thorens, 1993; Takata, 1996), the choroid plexus is not likely to be responsible for the secretion of glucose into the cerebrospinal fluid. Rather, GLUT1 at the basolateral membrane may contribute to the uptake of glucose to fuel the epithelial cells, which are very active in the transport of various substances in regulating the composition of the cerebrospinal fluid (Spector and Johanson, 1989). A similar basolateral localization of GLUTl was observed in the S3 segment of the kidney proximal tubules (Thorens et ul., 1990b; Takata et d.,1991b), where it may serve to supply glucose to these metabolically active cells.
C. Blood-Ocular Barrier The eyes develop from the neural tube and are often considered to be an extension of the central nervous system. The blood-ocular barrier (bloodeye barrier) therefore has similarities to the blood-brain barrier but has various unique aspects of its own as well. It consists of the blood-retinal barrier and blood-aqueous barrier (Raviola, 1977).
D. Blood-Retinal Barrier 1. Structure
The retina originates from the neural tube and develops a unique photoreception system. It is nourished from both inside and outside. In the former case, nutrients and oxygen are supplied directly from the blood vcsscls distributed inside the retina, which are branches of the central retinal blood vessels in the optic nerve. The blood vessels of the retina have a barrier property as is seen in the brain (Raviola, 1977; Bill et al,, 1980). When a tracer, such as thorium dioxide (Shakib and Cunha-Vaz, 1966), horseradish peroxidase (Shiose, 1970; Raviola, 1977), or microperoxidase (Smith and Rudt, 1975), was injected into the bloodstream, it failed to penetrate the endothelial cell layer of the capillaries in the retina. Similar results were observed in the permeability of the capillaries to endogenous serum proteins such as albumin and immunoglobulin (Pino and Thouron, 1983).Ultrastructural examination showed that retinal capillaries are of the continuous type,
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OF GLUCOSE ACROSS BLOOD-TISSUE BARRIERS
15
i.e., the endothelial cells are devoid of fenestrae and are connected by tight junctions (Raviola, 1977). The retina is surrounded by the highly vascularized choroid layer. Blood vessels in the choroid adjacent to the retinal pigment epithelium have numerous fenestrae in their endothelial cells (Bernstein and Hollenberg, 1965; Raviola, 1977) and are highly permeable (Bill et al., 1980). Intravenously injected tracers easily escape from the blood vessels (Shiose, 1970; Raviola, 1977). Retinal pigment epithelial cells, which constitute the outermost layer of the retina, are connected by well-developed tight junctions and gap junctions (Hudspeth and Yee, 1973; Raviola, 1977). Observation of freeze-fracture replicas demonstrated elaborate arrays of intramembranous particles of tight junctions (Hudspeth and Yee, 1973). Intravenous administration of horseradish peroxidase demonstrated that the retinal pigment epithelium constitutes a permeability barrier, i.e., horseradish peroxidase was effectively blocked by the tight junctions connecting the retinal pigment epithelial cells (Shiose, 1970; Smith and Rudt, 1975; Raviola, 1977; Caldwell and McLaughlin, 1983). 2. Glucose Transport
The existence of the barriers in the inner and outer parts of the retina suggests the presence of selective machineries for the transport of nutrients and metabolites across the barrier, which would be crucial for the maintenance of retinal function. In fact, transport of glucose (Zadunaisky and Degnan, 1976; Pascuzzo et al., 1980; Masterson and Chader, 1981; Crosson and Pautler, 1982; Stramm and Pautler, 1982; DiMattio and Streitman, 1986; Miceli et al., 1990) as well as of ions (DiMattio et al., 1983; Kennedy, 1990), retinoids (Ottonello et al., 1987; Bok, 1990), myo-inositol (Khatami, 1988), amino acids (Pautler and Tengerdy, 1986; Sellner, 1986), and ascorbate (Khatami et al., 1986) was observed to occur in the retinal pigment epithelial cells. In the inner blood-retinal barrier, GLUTl is abundant in the endothelial cells of the blood vessels, the site of the barrier (Takata et al., 1990b, 1992c; Harik et al., 1990b; Mantych et al., 1993b). Electron microscopic immunohistochemistry revealed that both the luminal and contraluminal plasma membranes were positive for GLUTl together with cytoplasmic staining (Takata et al., 1992c; Kumagai et al., 1996). These observations indicate that glucose passes through the continuous capillaries via GLUTl localized at both the luminal and contraluminal plasma membranes of the endothelial cells (Fig. 5). Immunoblotting of the homogenate of rat retinal pigment epithelium demonstrated abundant GLUTl protein of 46 kDa, which was also in abundance in the ciliary body (Fig. 3) (Takata et al., 1990b, 1992~).A
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KUNlAKl TAKATA ET AL.
FIG.5 Blood-retinal barrier and GLUTl. Both the cndothelium- and epithelium-type barriers are present in the retina. GLUTl plays a pivotal role in the transfer of glucose across the barrier in both cascs. RPE, rctinaf pigment epithelium; TJ, tight junction.
50-kDa protein was detected in the human retina with anti-GLUT1 antibody (Mantych et al., 1993b). Immunohistochemical staining showed that the retinal pigment epithelium is rich in GLUTl (Takata et al., 199Ob, 1992~; Harik et al., 199Ob; Mantych et al., 1993b). GLUTl was present along all aspects of the plasma membrane of the cell, i.e., it was found in the basolateral domain with well-developed infoldings as well as in the microvillous apical membrane facing the outer segments of the photoreceptor cells. The endothelial cells of the adjacent choriocapillaries were negative for GLUTl (Takata et al., 1992~).Taking into account the ultrastructure of the choriocapillaries and of the retinal pigment epithelium as well as the localization of GLUT1, we have proposed that glucose passes the outer blood-retinal barrier in a transepithelial manner as follows (Takata et a/., 1992~):Glucose (i) leaves the choriocapillary through the fenestrae into the extracellular matrix, (ii) is transported into the cytoplasm of the retinal pigment epithelial cell via GLUTl at the infolded basal plasma membrane, (iii) leaves the pigment epithelial cell via GLUTl at the microvillous apical plasma membrane (Fig. 5). The reverse transcriptase-polymerase chain reaction (RT-PCR) and immunoblotting demonstrated the expression and thc presence of the lowaffinity glucose transporter GLUT2 in the retina (Watanabe et af., 1994). Immunofluorescence staining showed that GLUT2 was localized in the boundary between the outer nuclear layer and the photoreceptor layer, corresponding to the location of the external limiting membrane. Immunoelectron microscopic examination showed that the microvilli extending from the apical ends of the Miiller cells were densely stained €or GLUT2 with less intense staining in their nonprojecting apical ends. Other structures including photoreceptor cells were negative for GLUTZ. Polarized distribution of GLUT2 was observed in pancreatic /3 cells (Orci er ul., 1989) and enterocytes (Thorens et al., 1990a; Thorens, 1992). Miiller cclls are located between two independent blood-retinal barrier systems: the inner system, composed of blood vessels inside the retina, and the outer system, conipris-
TRANSPORT OF GLUCOSE ACROSS BLOOD-TISSUE BARRIERS
17
ing the retinal pigment epithelium and the choriocapillaries. Whereas GLUT1 is instrumental in transporting glucose across these barriers (Takata et al., 1990b, 1992c; Harik et al., 1990b), GLUT2 in the apices of Miiller cells may be involved in the transport of glucose into the anterior part of the retina. Because GLIJT2 is a low-affinity glucose transporter and is considered to be a part of the sensing machinery in pancreatic fl cells for the regulation of the blood glucose level (Unger, 1991; Thorens, 1992), GLUT2 in the Miiller cells might play a regulatory role in the intraretinal glucose homeostasis (Watanabe et al., 1994). In addition, GLUT3 was detected, but neither GLUT4 or GLUT5 was expressed, in the human retina (Mantych et al., 1993b). GLUT3 was restricted to the inner synaptic layer and therefore does not participate in the transport of glucose across the barrier.
E. Blood-Aqueous Barrier 1. Structure The aqueous humor is a transparent fluid filling the anterior and posterior chambers of the eye. It nourishes the lens and cornea, both of which are devoid of vascularization. This fluid is continuously produced by the ciliary body, flows from the posterior chamber into the anterior chamber, and drains into the venules through the canal of Schlemm (Stamper, 1979; Sears, 1981). The ciliary body epithelium is the site of aqueous humor production and is mainly responsible for the determination of the constituents of the aqueous humor. When compared with the composition of plasma, that of the aqueous humor is characteristic in having a low concentration of serum protein and a high concentration of ascorbic acid (Bito, 1977; Cole, 1984). The barrier between the blood and the aqueous humor is called the bloodaqueous barrier. The glucose level of the aqueous humor is comparable to that of blood, suggesting a specific transport machinery in the epithelium of the ciliary body (Bito, 1977; Cole, 1984). The ciliary body epithelium is made of two cell layers: the nonpigmented epithelial cell layer and the pigmented epithelial cell layer (Fig. 6a). These two layers originate from the two layers of the invaginated optic cup in the embryo. The bases of nonpigmented epithelial cells face the posterior chamber, whereas the bases of pigmented epithelial cells rest on the ciliary body stroma (Raviola, 1977). Thus, the nonpigmented and pigmented epithelial cells oppose with each other at their apical surfaces. The pigmented epithelial cell layer is a continuation of the pigmented epithelium of the retina posteriorly and of the anterior layer of the iridial epithelium. The nonpigmented epithelial cell layer is the continuation of the neural retina
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b
a aqueous humor
C C Y t maternal blood glucose
FIG. 6 Glucose transfer across the blood-tissue barrier of a double-layered epithelium. (a) Ciliary body of the eye. Glucose leaves the capillary through fenestrae and enters the pigmented epithelial cells via GLUTl. Then glucose enters the nonpigmentcd epithelial cells through gap junctions made o€connexin 43 (Cx43) connecting pigmented and nonpigmented epithelial cells and is finally transferred to the aqueous humor via GLUTl in the pigmented epithelial cells. (b) Rat placenta. Glucose in the maternal blood easily crosses the cytotrophoblast layer through pores; glucose then enters the syncytiotrophoblast I layer via GLUTl. Next, the sugar mows into the syncytiotrophoblast 11 layer through gap junctions made ofconnexin 26 (Cx26). Glucose leaves the syncytiotrophoblast 11via GLUTl and finally enters the fetal blood through the fenestrae of thc endothelial cells. Note that in both cases, GLUTI, connexin of gap junctions, and GLIJTI located in series serve as the glucose transfer machinery across the barrier. The specificity of the transport of the system is determined by the specific transporter molccules assigned for the entry into and exit from each end of the double-layered epithelium. TJ, tight junction; PE, pigmented epithelial cells; NPE, nonpigmented epithelial cells; Cyt, cytotrophoblast; Syn 1, syncytiotrophoblast I; Syn 11, syncytiotrophoblast 11.
posteriorly and of the posterior layer of the iridial epithelium (Raviola, 1977). Ultrastructural examination revealed that tight junctions are formed between nonpigmented epithelial cells (Smith and Rudt, 1973; Raviola, 1977; Raviola and Raviola, 1978; Freddo, 1987). Endothelial cells lining the capillary wall have many fenestrations and are highly permeable (Bill et al., 1980). The barrier property of the ciliary body was analyzed by the intravascular administration of tracers (Shiose. 1970; Vegge, 1971; Smith and Rudt, 1973, 1975; Raviola, 1974, 1977). The injected horseradish peroxidase easily escaped from the fenestrated capillaries and filled the intercellular space between pigmented epithelial cells and that between pigmented and nonpigmented epithelial cells including the ciliary channels. The further penetration of the tracer was blocked by the tight junctions between nonpigmented epithelial cells, and the aqueous humor was free of the tracer. Similar results were obtained when the fluorescent dye acriflavine neutral was used as a tracer (Rodriguez-Peralta, 1975). The barrier function of the tight
TRANSPORT OF GLUCOSE ACROSS BLOOD-TISSUE BARRIERS
19
junctions between nonpigmented epithelial cells was also demonstrated by the administration of colloidal lanthanum from the aqueous side (Raviola, 1977). These observations show that capillary walls in the ciliary body stroma are highly permeable and that nonpigmented epithelial cells connected by tight junctions are the principal site of the blood-aqueous barrier (Fig. 6a). Capillaries in the iris stroma are of the nonfenestrated continuous type connected by tight junctions (Freddo and Raviola, 1982a,b) and constitute another site of blood-aqueous barrier (Raviola, 1974, 1977; Smith and Rudt, 1975; Freddo and Raviola, 1982a). Intravascularly administered tracers failed to pass through the endothelial cells of these capillaries (Vegge, 1971; Raviola, 1974; Smith and Rudt, 1975; Freddo and Raviola, 1982a).
2. Glucose Transport in the Ciliary Body The concentration of glucose in the aqueous humor is maintained at a level simiIar to that found in the plasma (Bito, 1977; Cole, 1984). Thus, the existence of a glucose transporter system in the blood-aqueous barrier, i.e., in the ciliary body epithelium, was suggested (Bito, 1977; Cole, 1984). A high level of 46- or 47-kDa GLUTl protein was detected in the rat ciliary body specimens (Fig. 3) (Takata et al., 1990b, 1991~).Light microscopic immunohistochemistry revealed that GLUTl is abundant in the epithelial cells of the ciliary body and the iris (Figs. 7a and 7b) (Takata et al., 1990b, , Mantych et al., 1993b). Blood vessels in the ciliary 1991~; Harik et ~ l . 1990b; body stroma were negative for GLUT1, whereas those in the iris stroma, another site of blood-aqueous barrier, were positive for GLUTl (Takata et al., 1990b, 1991c; Harik et al., 1990b). Ultrastructural examination revealed that GLUTl is abundant in both the pigmented and nonpigmented epithelial cells (Figs. 7c and 7d) (Takata et al., 1990b, 1991~).Fenestrated endothelial cells beneath the pigmented epithelial cells were negative for GLUTl. In pigmented epithelial cells, GLUTl is present along the entire surface except at gap junctions and desmosomes. Well-developed basal infoldings are present, which drastically increase the surface area of the cells. Most GLUTl transporters, therefore, are localized along these basal infoldings. In the nonpigmented epithelial cells, GLUTl is also present along the entire surface except at junctional regions. Because basal infoldings facing the posterior chamber constitute the majority of the plasma membrane, most of the GLUTl molecules are present in these basal infoldings. Semiquantitative analysis of the colloidal gold label for GLUTl in ultrathin sections revealed an approximately twofold higher labeling density for GLUTl in the basal infoldings of the pigmented epithelial cells than in the basal infoldings of the nonpigmented epithelial cells (Takata et af., 1991~).
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FIG. 7 GLUTl in the blood-aqueous barrier ofthe rat. (a, b) ImmunoHuorescencc localization of GLUTl in the ciliary body. GLUTl is present in the pigmented (PE) and nonpigmented
(NPE) epithelial cells (a). Corresponding Nomarski diffcrcntial interference contrast image is also shown (b). In NPE cells, QLIJTI is abundant along the posterior chamber (P). In PE cclls, strong positive labcling for GLUTl is seen along the connective tissue stroma. Only a
TRANSPORT OF GLUCOSE ACROSS BLOOD-TISSUE BARRIERS
21
Because the pigmented and nonpigmented epithelial cells have a similar surface area of their respective basal infoldings (Okami er aL, 1989), these observations indicate that pigmented epithelial cells have approximately twofold more GLUTl than d o nonpigmented epithelial cells. Such asymmetrical distribution of GLUTl may be attributed to the consumption of glucose by the nonpigmented epithelial cells themselves rather than simply to the transfer of glucose. Another explanation is a possible kinetic asymmetry of the GLUTl molecule for the transport of glucose (Lowe and Walmsley, 1986; Carruthers, 1990). Asymmetric distribution of GLUT1 in the blood tissue barriers was observed in the endothelial cells of the blood vessels in the rat brain when immunogold labeling of ultrathin sections was carried out (Farrell and Pardridge, 1991). The luminal plasma membrane, which is the site of entry into the barrier, exhibited less labeling compared with the contraluminal membrane, the site of exit from the barrier cell. When considering the direction of the transport of glucose, this observation is in contrast to that in the ciliary body, where the site of entry into the barrier layer has more GLUTl.
3. Gap Junctions Gap junctions develop between pigmented epithelial cells, between pigmented and nonpigmented epithelial cells, and between nonpigmented epithelial cells (Bairati and Orzalesi, 1966; Smith and Rudt, 1973; Kogon and Pappas, 1975; Raviola and Raviola, 1978; Freddo, 1987), among which rows of gap junctions between pigmented and nonpigmented epithelial cells are prominent. Such junctions are hydrophilic channels connecting the cytoplasm of adjacent cells. They are made of transmembrane proteins named connexins (Beyer, 1993; Dermietzel and Spray, 1993). Spherical molecules as large as 900-1000 Da are allowed to pass the gap junction channels (Spray and Bennett, 1985;Pitts and Finbow, 1986).Fluorescencelabeled glucose was demonstrated to pass through gap junctions (Loewenstein, 1979), showing that cells connected by gap junctions are metabolically coupled. Because the pigmented and nonpigmented epithelial cells are connected by well-developed gap junctions, the cytoplasms of these
small amount of GLUTl is seen between PE and NPE cells. Bar = 10 pm. (c, d) Immunogold labeling of ultrathin frozen sections for GLUT1 . In NPE cells of the ciliary body (c), GLUTl is localized along the infolded plasma membrane (arrowheads) facing the posterior chamber (P). In PE cell (d), GLUTl is localized along the infolded plasma membrane (arrowheads). Endothelium (E) of the adjacent capillary is not labeled for GLUTl. Bars = 0.5 pm. Figure 7c was reproduced from Takata et al. (1990b) with permission from Academic Press.
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KUNlAKl TAKATA ET AL.
cells are equivalent to a single cell cytoplasm as far as the cytoplasmic diffusion of glucose is concerned. How does glucose pass the blood-aqueous barrier during the course of the production of the aqueous humor in the ciliary body epithelium? Taking into account the presence of tight and gap junctions, and the localization of GLUT1, we suggested the following major pathway for the transport of glucose across the ciliary body epithelium (Takata et al., 1991c)(Fig. 6a): Glucose freely passes through the fenestrae of the endothelial cells and is then transported into the cytoplasm of the pigmented epithelial cells via GLUTl localized in the basal infoldings. Next, glucose enters the cytoplasm of nonpigmented epithelial cells by passing through arrays of gap junctions connecting the apposing apical plasma membranes of pigmented and nonpigmented epithelial cells. Finally, glucose leaves the cytoplasm of nonpigmented epithelial cells via GLUTl located in their infolded basal plasma membrane and thus passes into the aqueous humor. The existence of gap junctions in the epithelium of the ciliary body makes the double-layered epithelium functionally a single-layered epithelium. In this model, the specificity of the transport is determined by the specific transporters located at both ends of the system: Transport through the barrier is mediated by a combination of a specific transporter for the entry into the barrier, nonspecific gap junction channel for transfer between two cells, and a specific transporter for the exit from the barrier. Immunoblotting and immunohistochemistry revealed that the gap junction protein connexin 43 is concentrated in the gap junctions connecting the pigmented and nonpigmented epithelial cells (Coca-Prados etal., 1992; Shin et al., 1996a). In summary, the combined sequential action of GLUTl ,connexin 43, and GLUTl could be key in the transport of glucose across the blood-aqueous barrier and play a pivotal role in supplying glucose to the anterior and posterior chambers, thereby nourishing the lens and cornea. 4. Glucose Transport in the Iris
Another part of the blood-aqueous barrier is the endothelial cells of the blood vessels in the iridial stroma. Heavy labeling for GLUTl was observed in both the luminal and contraluminal plasma membranes of these endothel i d cells (Takata et al., 1991~).As occurs in the capillaries in other bloodtissue barriers, glucose may pass the endothelial cell layer by using GLUTl for entry into and exit from the cytoplasm of the endothelial cells. These capillaries may be substantial in nourishing the cells of the iris. The iridial epithelium, which is a continuation of the ciliary body epithelium (Raviola, 1977), is also rich in GLUT1. This epithelium is similar to that of the ciliary body, connected by tight and gap junctions, and a tracer experiment showed the barrier characteristics (Freddo, 1984). Ultrastruc-
TRANSPORT OF GLUCOSE ACROSS BLOOD-TISSUE BARRIERS
23
turd examination revealed that GLUTl is abundant along the plasma membranes of these cells (Takata et al., 1991~).GLUT1 in the iridial epithelium may serve to facilitate the transfer of glucose between the anterior and posterior chambers.
F. Blood-Perilymph and Blood-Endolymph Barriers in the Inner Ear The mammalian inner ear is an enclosed compartment containing transparent Auids called perilymph and endolymph, both of which have a composition distinct from that of blood. Tracers administered via the bloodstream failed to enter these fluids (Duvall et al., 1971; Winther, 1971a,b; Gorgas and Jahnke. 1974; Santos-Sacchi and Marovitz, 1980). The barrier in the inner ear is composed of the blood-perilymph and blood-endolymph (blood-strial) barriers. Ferrary et ul. (1987) showed the transport of glucose by facilitated diffusion across the blood-perilymph barrier. Immunohistochemical staining revealed that GLUT1 is present in the microvascular endothelial cells in the soft tissues of the labyrinth (It0 et al., 1993). In the vestibular system, strong staining for GLUT1 was found in the capillaries in the crista ampullaris. The staining of the vascular network is similar to that seen in the blood-brain barrier. GLUT1 in these blood vessels serves to nourish cells in the inner ear while the barrier effectively prevents the nonspecific entry of other blood constituents including blood cells. In the cochlea, the stria vascularis is responsible for the production of the endolymph. Blood vessels are present inside the stria. The strial basal cells are connected by tight junctions, forming a barrier toward the perilymph (Winther, 1971b; Reale et al., 1975). The strial marginal cells are also sealed by tight junctions, forming a barrier toward the endolymph of the cochlear duct (Winther, 1971b; Reale et ul., 1975). In guinea pig ears, intravenously administered horseradish peroxidase easily penetrated the strial blood vessels but was blocked by these cells (Duvall et al., 1971; Winther, 1971b; Gorgas and Jahnke, 1974). Therefore, the basal and marginal cells of the stria vascularis are the anatomical basis for the bloodperilymph and blood-endolymph barriers, respectively. In the mouse, intravenously administered ferritin and iron dextran were blocked by the endothelial cells of the stria vascularis, demonstrating a blood-strial barrier (Santos-Sacchi and Marovitz, 1980). Strong staining for GLUT1 was observed in these endothelial cells, indicating that GLUTl is responsible for the transport of glucose across the blood-stria1 barrier (It0 et aZ., 1993). The apparent differential permeability of the intrastrial blood vessel wall between guinea pig and mouse may be attributed to the species difference or to the tracers used. In any event, at least strial cells (basal and marginal
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cells) connected by tight junctions function as a barrier layer. GLUTl is present in the strial basal cells. GLUTl in these cells may serve in the selective uptake of glucose from the interstitial fluid and its transport across the basal cell layer, thus transferring glucose across the perilymphendolymph barrier. In summary, GLUT1 in the inner ear, whether it is in the endothelial cells or strial epithelial cells, may play an important role in the transfer of glucose across the barriers including blood-perilymph, blood-endolymph, blood-strial, and perilymph-endolymph barriers.
G. Blood-Nerve Barrier 1. Structure Peripheral nerves are a continuation of the central nervous system, which is protected from the free access of blood constituents by the blood-brain barrier. The outermost part of the nerve fibers are surrounded by an epithelial cell-likecell layer called the perineurium (Shanthaveerappa and Bourne, 1962). Ultrastructural examination revealed that cells of the perineurium are connected by tight junctions (Thomas, 1963). When horseradish peroxidase was injected as a tracer into the endoneural space of peripheral nerves, it was blocked by this perineurial sheath (BGck and Hanak, 1971; Olsson and Reese, 1971). Peroxidase injected locally around the sciatic nerve was prevented from diffusing into the nerve by the perineurium as well (Olsson and Reese, 1971).These observation show that perineurium sealed by tight junctions serves as a permeability barrier. Thick nerves have blood vessels inside them. These microvessels show barrier characteristics similar to those found in the brain microvessels. Intravascularly injected tracers, such as Ruorescence-labeled albumin and horseradish peroxidase, failed to pass through the endothelium of the blood vessels within the nerve (Olsson, 1966; Bock and Hanak, 1971; Olsson and Reese, 1971). These results clearly show that nerve fibers are separated from the bloodstream from both inside and outside. The blood-nerve barrier was also demonstrated for inorganic ions (Welch and Davson, 1972; Weerasuriya et ul., 1980). 2. Glucose Transport An in situ perfusion experiment with labeled glucose showed that saturable and stereospecific glucose transport machinery is present in the rat peripheral nerve (Rechthand et ul., 1985). Abundant GLUTl was demonstrated in the perineurial sheaths (Froehner et al., 1988;Takata et af.,1990b;Gerhart and Drewes, 1990; Harik et ul., 1990a; Handberg et al., 1992). lmmunogold
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electron microscopy also demonstrated that GLUTl is localized at the plasma membrane of the perineurial cells (Gerhart and Drewes, 1990). GLUTl was also found to be localized in the endoneurial blood vessels (Froehner et af., 1988; Takata et al., 1990b; Gerhart and Drewes, 1990; Handberg el al., 1992). These results, obtained from both rats and dogs, show that peripheral nerve fibers are nourished from both inside and outside by the action of GLUTl located at the plasma membrane of the cells of the blood-nerve barrier. In the adult human sciatic nerve, the perineurium is rich in GLUTl, whereas only a few endoneurial capillaries stained positively for it (Muona et af., 1993). The expression of GLUTl in the human sciatic nerve during development was investigated (Muona et af.,1993). At 14 weeks of gestation the perineurial cells were negative for GLUTl , whereas endoneurial and epineurial blood vessels were intensely positive for it. During the course of development, positive staining for GLUTl in the endoneurial capillaries became reduced, and in the adult only a few of them were positive. In marked contrast, the intensity of positive staining for GLUTl in the perineurium increased with the maturation of the barrier properties of the perineurium. In the adult human nerves, GLUTl in the perineurium seems to play a major role in nourishing the nerve fibers. GLUT3, which is abundant in the brain, was not detected in the peripheral nerves (Haber et al., 1993; Muona et af., 1992). Other transporter isoforms, such as GLUT2 or GLUT4, were not detected in the rat peripheral nerve (Muona et al., 1992). In summary, GLUTl is concentrated at the sites of the blood-nerve barrier and plays an important role in supplying glucose to the nerve fibers and associated cells.
H. Placental Barrier The placenta is an organ in which exchange of gases, nutrients, and metabolites occurs between maternal blood and fetal blood, in the absence of any mixing of the two. The structure of the placenta differs considerably from species to species, and hence the structure of the barrier differs accordingly (Wimsatt, 1962; Enders, 1965a,b; Faber and Thornburg, 1983; Benirschke and Kaufmann, 1995a). The glucose transport mechanism across the placental barrier in humans and rats is reviewed. I. Human Placental Barrier
1. Structure In the human placenta, villous trees are directly surrounded by the maternal blood (Benirschke and Kaufmann, 1995b; Castellucci and Kaufmann, 1995).
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In the first trimester, the human placenta is hemodichorial, i. e., the maternal blood faces two layers of trophoblasts. The superficial layer facing the maternal blood is syncytial and called the syncytiotrophoblast, underneath which are cytotrophoblasts (Langhans cells). Cytotrophoblasts, by fusing with the adjacent syncytiotrophoblast layer, serve as a source for the syncytium (Enders, 196Sb). At term, only a small number of the cytotrophoblasts remains, and a single syncytiotrophoblast layer lines the surface of the villous tree, thus forming the hemomonochorial placenta (Rhodin and Terzakis, 1962; Enders, 1965b; Benirschke and Kaufmann, 1995b). The structural basis of the human placental barrier is attributed to the syncytiotrophoblast layer (Benirschke and Kaufmann, 199%). The syncytiotrophoblast is a single continuous cell layer separating the maternal and fetal circulations. In the term placenta, from the maternal blood side to the fetal blood side, the syncytiotrophoblast directly faces the maternal blood. Next comes the cytotrophoblast sporadically found underneath the syncytiotrophoblast. The fetal blood is enclosed by the endothelial cells of the fetal capillaries, which are of the continuous type and develop tight junctions (Heinrich et al., 1976). The permeability of the fetal capillaries in the term human placenta resembles that of skeletal muscle (Eaton et al., 1993). Detailed examination revealed that tight junctions are not continuous, suggesting the contribution of the paracellular pathway across the capillary wall in the human placenta (Leach and Firth, 1992). 2. Glucose Transport
The continuous syncytiotrophoblast layer is the prime barrier layer in the human placenta and therefore must have the machinery for the exchange of various substances between mother and fetus. Glucose is a major nutrient for the fetal development and is supplied from the maternal blood through the placenta (Dancis, 1962; Smith et al., 1992). Glucose somehow must pass this syncytial layer. Facilitated diffusion is the main process for the transplacental transfer of glucose (Carstensen et al., 1977: Morris and Boyd, 1988). Carrier-mediated glucose uptake was observed in vesicles prepared from the apical microvillous (Johnson and Smith, 1980; Bissonnette et al., 1981, 1982) and basal (Johnson and Smith, 1985) plasma membranes of the syncytiotrophoblast of the human placenta. The uptake was inhibited by cytochalasin B and phloretin. Photoaffinity labeling of microvillous membrane with cytochalasin B revealed D-glucose-sensitive cytochalasin Bbinding proteins of 52 kDa (Johnson and Smith, 1982) and 60 kDa (Ingermann et al., 1983). A protein of 42-68 kDa was also detected by the photoaffinity labeling of the human placental microsomes with cytochalasin B (Wessling and Pilch, 1984).
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Northern blot analysis showed that GLUT1 mRNA is abundant in the human placenta (Fukumoto et al., 1988b; Bell et al., 1990). GLUTl mRNA is detectable at an early stage of placental development and is developmentally regulated with mRNA levels fivefold higher in the first trimester than at term (Hauguel-De Mouzon et al., 1994). In situ hybridization demonstrated that GLUT1 is highly expressed in the syncytiotrophoblast, suggesting its involvement in transplacental glucose transport ( Jansson et al., 1994). GLUT1 was detected in the human term placenta by immunoblotting (Takata et al., 1992d), and the results agreed with those of photoaffinity labeling with cytochalasin B (Johnson and Smith, 1982; Ingermann et aL., 1983; Wessling and Pilch, 1984). Immunohistochemical staining revealed that GLUT1 is localized in the syncytiotrophoblast layer (Takata et al., 1992d). Immunofluorescence (Takata et al., 1992d) and immunoperoxidase (Jansson et al., 1993; Hahn et al., 1995) as well as immunogold electron (Takata et al., 1992d) microscopy demonstrated that GLUTl is localized at both the microvillous apical and infolded basal plasma membranes of the syncytiotrophoblast. Immunohistochemical staining of sections of the villi showed that the apical microvillous membrane is more intensely labeled for GLUTl (Jansson et al., 1993), although GLUTl was reported to be associated with the basal membrane by electron microscopic analysis (Arnott et al., 1994). lmmunoblotting of microvillous and basal membranes confirmed that GLUTl is present in both membranes but in different amounts: the amount of GLUTl in the microvillous membrane is about 20-fold larger than that in the basal membrane (Jansson et al., 1993). Such semipolarized distribution of GLUTl, together with its abundance, in the syncytiotrophoblast may be important in the efficient transfer of glucose across the barrier as well as in nourishing the placental cells. GLUTl is present at the plasma membrane of the cytotrophoblasts as well (Takata et al., 1992d; Hahn et al., 1995). This observation shows that a high level of GLUTl expression begins prior to the fusion to form the syncytiotrophoblast layer. Positive staining for GLUTl was also observed in the endothelial cells of the capillaries in the core of the villi (Takata et al., 1992d; Hahn et al., 1995). Because capillaries are relatively permeable as noted, GLUTl may serve for the uptake of glucose for the endothelial cells as well as contribute to the transendothelial transfer of glucose into the fetal circulation. In addition to the villi, GLUTl was detected in the fetal membranes, which also constitute the barrier in the term placenta (Wolf and Desoye, 1993). In the amnion epithelial cells, GLUTl is predominantly localized at their apical membrane and may cover their basal glucose requirement from the amniotic fluid. Aside from GLUTl, Northern blot analysis also revealed that a high level of GLUT3 is also expressed in the human placenta (Kayano et al.,
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1988; Bell ef al., 1990). I n situ hybridization showed that GLUT3 mRNA is evenly distributed between syncytial and other placental cells at a low level ( Jansson et ul., 1994). Immunofluorescence staining showed that GLUT3 is localized at the apical membrane of the syncytiotrophoblast (Arnott et al., 1994). However, immunoblot analyses detected only a small amount of GLUT3 protein (Shepherd et al., 1992a; Haber et al., 1993) or failed to detect any significant amount of GLUT3 protein in the human placenta (Maher etaf., 1992;Jansson et al., 1993). These observations suggest that GLUT3 protein is not present, or at least is not as abundant as in the brain, in the human placenta. The inconsistent results may be attributed to the specificity of the anti-GLUT3 antibodies used (Shepherd rt al., 1992a; Jansson et al., 1993) as well as to the methods employed. The discrepancy between the abundance of GLUT3 mRNA and paucity of GLUT3 protein may be due to the possible blocking of the translation of GLUT3 mRNA in the placenta, or alternatively, to cross hybridization of the cDNA probe with other mRNA species (Haber ~t al., 3993). The human placenta is rich in insulin receptors (Siege1 et al., 1081; FujitaYamaguchi et al., 1983). Glucose transport in the human placenta, however, is insensitive to insulin (Johnson and Smith, 1980 Challier et d., 1986). GLUT4, an isoform of insulin-regulatable glucose transporter, is mainly rcsponsiblc for the insulin action in adipocytes and skeletal muscle cells. Northern blot analysis showed that only a very low level of GLUT4, if any, is expressed in the human placenta throughout pregnancy (Fukumoto et ul., 1989; Hauguel-De Mouzon et al., 1994). Immunohistochemical staining of human term placental tissues failed t o detect GLUT4 (Takata et al., 1992d). These results show that the GLUT4 insulin-regulatable glucose transporter is unlikely to participate in the transfer of glucose across the human placental barrier. A possible major route for transplacental glucose transfer across the human placental barrier may be envisioned as follows: Glucose in the maternal blood enters the cytoplasm of the syncytiotrophoblast via GLUTl localized at its microvillous apical membrane and the sugar leaves the syncytiotrophoblast via GLUTl localized at its basal membranc (Takata, 1994, 1996; Takata and Hirano, 1996). Contribution of GLUT1 to the entry into and exit from the barrier cell layer is basically the same as that seen in other blood-tissue barriers. The glucose concentration in the umbilical artery is about 80% of that in the maternal vein in the human placenta (Economides and Nicolaides, 1989). This concentration gradient serves as a driving force for the glucose transfer by facilitated diffusion across the placental barrier. In mid-gestation, there were fetuses whose glucose concentration in the umbilical vein exceeded the maternal concentration (Bozzetti et af., 1988). A similar reversal of glucose concentration gradient was observed in other species (Anand
TRANSPORT OF GLUCOSE ACROSS BLOOD-TISSUE BARRIERS
29
et al., 1979; Thomas et al., 1990). These observations suggest a possible backtransfer of glucose to the trophoblast or to the maternal blood. In fact, the dually perfused isolated lobule of the human placenta indicates the bidirectional glucose transfer across the placental barrier (Reiber et al., 1991). However, the high efficiency of the transfer of glucose from the maternal to fetal circulation compared with the transfer in the reverse direction suggests a possible protective measure against glucose loss of the fetus under maternal hypoglycemia (Reiber et al., 1991). J. Rat Placental Barrier
1. Structure The rat develops hemochorial placentae where maternal and fetal blood flows are separated by the trophoblastic layers and endothelial cells. Instead of the villi in the human placenta, a complex of maternal and fetal circulation routes, the labyrinth, is formed in the rat placenta (Wimsatt, 1962; Enders, 1965a; Faber and Thornburg, 1983; Benirschke and Kaufmann, 1995a). Between maternal and fetal bloodstreams lie a single cytotrophoblast, two syncytiotrophoblastic layers (hereafter termed from the maternal blood side as syncytiotrophoblasts I and II), basal lamina, and the endothelial cells of the fetal capillary (Jollie, 1964, 1976; Enders, 1965a; Metz et al., 1976a,b;Metz, 1980 Faber and Thornburg, 1983). Ultrastructural examination showed that the cytotrophoblast, which lies next to the maternal blood, has numerous fenestrations in its thin cytoplasm. Tracers, such as horseradish peroxidase and lanthanum chloride, administered via the maternal circulation easily penetrated the cytotrophoblast layer through the fenestrations (Metz et a[., 1978). They failed to penetrate the syncytiotrophoblast I, demonstrating that syncytiotrophoblast I is the structural basis for the placental barrier from the maternal side. When tracers were injected into the umbilical artery, they rapidly traversed the capillary endothelium, where fenestration and pinocytotic vesicles and probably leaky junctions provided the pathway for the tracers (Aoki et al., 1978).The penetration of the tracers was blocked by the syncytiotrophoblast 11, showing that syncytiotrophoblast I1 is the structural basis for the placental barrier from the fetal side. These tracer experiments showed that two syncytial layers, syncytiotrophoblast layers 1 and 11, serve as a barrier between maternal and fetal circulations (Fig. 6b).
2. Glucose Transporters In situ hybridization showed that both GLUT1 and GLUT3 are expressed in the rat placental labyrinth (Zhou and Bondy, 1993). The level of GLUT3
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KUNlAKl TAKATA ET AL
mRNA remained constant from mid-gestation through term, whereas a reduction in that of GLUTl mRNA was observed during this period. In addition, GLUTl expression was found in the junctional zone, where the highest uptake of 2-deoxyglucose was observed. Zhou and Bondy (1993) suggested that GLUT3 is important for glucose transfer to the embryo, whcreas GLUT1 is responsiblc for supplying glucose for use as a placental fuel. By the immunoblotting of the labyrinthine spccimens, abundant GLUTl (Fig. 3) (Takata et al., 1990b, 1994) and GLUT3 (Boileau et d, 1995) were detected. Two syncytial layers serve as the barrier, in which four layers of plasma membranes are the principal barrier. How do hydrophilic molecules such as glucose pass through these lipid bilayers? Immunohistochemical labeling showed that the interhemal membrane or the labyrinthine wall is rich in GLUTl (Fig. 8) (Takata et al., 1990b, 1993b, 1994; Takata, 1994; Hahn et al., 1995; Boileau et al., 1995). GLUT1 is abundant in the syncytiotrophoblast layers I and 11, whereas it is not detected in the cytotrophoblasts or endothelial cells of the blood vessels (Takata et al., 1990b, 1993b, 1994; Takata, 1994). In the syncytiotrophoblast layer I, GLUT1 is abundant along the highly infolded plasma membrane facing the cytotrophoblasts. The basal plasma membrane of syncytiotrophoblast layer I1 is also rich in GLUTl. The apposing plasma membranes of syncytiotrophoblasts I and 11, which have straight contour, exhibited poor labeling for GLUT1 (Takata et al., 1994).
FIG. 8 GLUT1 and gap junction protein connexin 26 in the rat placenta. Double irnrnunofluorcscence staining. (a) GLUT1 . (b) Connexin 26. (c) Noniarski differential interference contrast image. Note that GLlJTl (arrowheads), connexin 26 (arrows), and GLUT1 (double arrowheads) arc present in this order in the placcntal barricr from the maternal blood (M) sidc to the fctal blood (F) side. Bar = 10 pni.
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Immunofluorescence staining showed that the expression of GLUT3 protein is restricted to the labyrinthine zone, whereas GLUTl protein is ubiquitously distributed in both the junctional and labyrinthine zones (Boileau et af., 1995). The expression of GLUT3 was stimulated under hyperglycemic conditions and seems to play an important role in the case of diabetic pregnancies (Boileau et al., 1995). The precise determination of the location of GLUT3 at the cellular level should shed light on the role of GLUT3 and GLUTl in rat placental function.
3. Gap Junctions Ultrastructural examination revealed the presence of numerous gap junctions between syncytiotrophoblast layers I and I1 (Forssmann et al., 1975; Heinrich et al., 1976; Metz et al., 1976a; Metz, 1980). These gap junctions can thus function as a channel for the glucose transfer between these two syncytial layers (Takata et af.,1993b, 1994; Takata, 1994) in a way similar to that proposed in the double epithelial cell layers in the ciliary body of the eye (Takata et al., 1991c, 1993a; Shin et al., 1996a). A high expression level of connexin 26, an isoform of gap junction proteins, was observed in the rat placenta, suggesting that connexin 26 may constitute a major fetomaternal exchange route (Risek and Gilula, 1991). Double immunofluorescence microscopy for GLUTl and connexin 26 demonstrated that connexin 26 is abundant and localized in between syncytiotrophoblasts I and I1 (Fig. 8) (Shin et al., 1996b). Such spatial distribution of GLUTl and connexin 26 indicates the transfer of glucose across the rat placental barrier as follows (Fig. 6b) (Takata, 1994; Takata and Hirano, 1996; Takata et al., 1994; Shin et al., 1996b): Glucose in the maternal blood passes through the cytotrophoblast via numerous pores penetrating the cytoplasm and is then transported into the cytoplasm of the syncytiotrophoblast I via GLUTl localized at the plasma membrane of the cytotrophoblastic side. Next, glucose passes the gap junction channels of connexin 26 connecting syncytiotrophoblasts I and I1 and enters the cytoplasm of the syncytiotrophoblast 11.The sugar leaves the cytoplasm of the syncytiotrophoblast I1 via GLUTl localized at the basal plasma membrane and finally enters the fetal circulation by passing through the fenestration of the endothelial cells of the capillary wall. A similar mechanism may possibly be at work for the transport of hydrophilic small molecules other than glucose.
K. Blood-Testis Barrier Germinal cells differentiate to become sperm inside the seminiferous tubules of the testis. The interior of the tubule is separated from the exterior
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KUNIAKI TAKATA E r AL.
by well-developed arrays of tight junctions between Sertoli cells (Nicander, 1967; Dym, 1973; Ross, 1977; Russell, 1978; Nagano and Suzuki, 1983), thereby preventing the exposure of sperm antigen to the immune system as well as providing a favorable environment for sperm differentiation. The blood-testis barrier was demonstrated by tracer experiments: Lanthanum nitrate and intravascularly injected horseradish peroxidase were blocked by these tight junctions from gaining cntrance to the interior of the seminiferous tubules (Dym, 1973; Ross, 1977; Russell, 1978). Although cultured Sertoli cells express GLUTl (Ulisse et al., 1992), immunohistochemical staining revealed that Sertoli cells, the critical barrier cell layer, was not positive for GLUT1. Instead, GLUTl was concentrated in the endothelial cells of the blood vessels surrounding the seminiferous tubules (Takata et al., 1990b; Harik et al., 1990a; Holash et al., 1993). The capillaries surrounding the tubules are of the nonfenestrated continuous type. Holash et al. (1993) compared the blood-testis barrier with the bloodbrain barrier and found that P-glycoprotein (Cordon-Cardo et al., 1989; 1990) and y-glutamyl transpeptidase (Orlowski et al., 1974), both of which are markers of barrier properties of brain microvessels, are present in the testis rnicrovessels. Transferrin receptor, another marker of brain microvessels ( Jefferies etal., 1984), however, was absent in the testis vessels. Interestingly, intertubular Leydig cells, adjacent to the blood vessels, expressed the astrocyte marker proteins such as the glial fibrillary acidic protein, glutamine synthetase, and S-100 protein (Michetti et al., 1985; Holash ef al., 1YY3), suggesting a similarity between the blood-testis and blood-brain barriers (Holash et al., 1993). These observations lead to the idea that, in the testis, Sertoli cells are equivalent to astrocytes in the blood-brain barrier and possibly induce and maintain the brain microvessel-like characteristics of the endothelial cells in the testicular microvessels. Endothelial cells and Sertoli cells may constitute “in series” the blood-testis barrier to achieve a favorable environment for spermatogenesis inside the seminiferous tubule (Holash et al., 1993).The transport mechanism of nutrients including sugars across the Sertoli cell layer, however, remains to be clarified.
L. Blood-Thymus Barrier Tracer experiments showed that horseradish peroxidase, cytochrome c, catalase, ferritin, and lanthanum are retained in the lumen of capillaries in the cortex of the mouse thymus (Raviola and Karnovsky, 1972). These tracers failed to penetrate the endothelial cells connected by tight junctions, indicating the presence of the blood-thymus barrier, which would provide a favorable environment for differentiating lymphocytes. The barrier does not seem to be maintained solely by endothelial
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cells, however. Tracers penetrating the endothelial cells were promptly sequestered by macrophages stretched out in a continuous row along the cortical capillaries (Raviola and Karnovsky, 1972). In the medulla of the thymus, on the other hand, blood vessels including postcapillary venules are leaky, and the tracers easily escaped from the lumen of the blood vessels. These results show that blood-thymus barrier is present but is limited to the cortex. Glucose, however, does not seem to be prevented from entering the cortex by the barrier maintained by the phagocytic macrophages, and hence a sufficient amount of glucose may pass the blood-thymus barrier. Immunoblotting and immunohistochemical staining so far have failed to demonstrate an abundance of GLUTl or of other glucose transporters in the blood-thymus barrier (Takata, 1990b). The apparent lack of glucose transporters in the barrier may also be due to the relatively low metabolic activity of the thymic cortex. Another possibility is that the cortex is nourished by glucose that diffuses from the medulla.
V. Regulation of Glucose Transporter Expression in Blood-Tissue Barriers
A. Developmental Regulation During mouse development, GLUT1 is detected as early as in the oocyte (Aghayan et al., 1992). GLUTl remained expressed in both the inner cell mass and the trophoectoderm throughout the preimplantation development (Hogan et al., 1991; Aghayan et al., 1992), suggesting that GLUTl is a basic glucose transporter in mammalian cells. The expression of GLUTl and GLUT3 is developmentally regulated (Devaskar et al., 1991, 1992; Bondy et al., 1992; Cornford et al., 1993, 3994; Dwyer and Pardridge, 1993; Harik et al., 1993; Vannucci, 1994; Vannucci et al., 1994; Nagamatsu et al., 1994; Bauer et al., 1995). In the course of the brain development, GLUTl is present in both the capillary and the neuroectoderm at first, and later GLUT1 expression is upregulated and mainly restricted to the endothelium of the blood vessels. During the development of the mouse telencephalon, GLUTl immunoreactivity in the intramural blood vessels is associated with the formation of the blood-brain barrier, as measured by the impermeability of the intravenously administered tracers such as trypan blue and horseradish peroxidase, and with the concomitant loss of GLUTl in the neuroectoderm cells (Bauer et al., 1995). In the human newborn brain, GLUTl is associated with the microvascular endothelium (Mantych et al., 1993~).The tightness of the cerebral endothelium, as demonstrated by
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KUNlAKl TAKATA ET AL.
the exclusion of the intravascularly applied horseradish peroxidase and fluorescence-labeled dextrans, is accompanied by a reduction in the amount of GLUTl in neuroepithelial cells and confinement of the transporter to the endothelium during rat brain development (Dermietzel et al., 1992). Immunogold electron microscopy also revealed that GLUTl density in the capillary walls increases during the postnatal period (Cornford et al., 1993, 1994). These observations suggest that the development of the blood-brain barrier and the GLUTl expression in the brain capillaries seems to be closely related. During the postnatal development of rabbit brain, GLUT1 protein undergoes marked upregulation, whereas mRNA level remains unchanged, suggesting a posttranscriptional mechanism of regulation for GLUT1 gene expression (Dwyer and Pardridge, 1993).
6 . Induction of GLUT1 in Blood Vessels Stewart and Wiley (1981) demonstrated with a transplantation experiment that the blood-brain barrier is induced by the environment of the central nervous systcm. When astrocytes were injected into the anterior chamber of the eye, nonleaky endothelial cells were induced, whereas meningeal cells failed to induce nonleaky properties (Janzer and Raff, 1987). This result might suggest that astrocytes, which surround the microvessels in the brain, could be responsible for the induction of the barrier properties of the microvessels (Janzer and Raff, 1987; Maxwell et aE., 1987; Goldstein, 1988). The brain capillary is characteristic in its nonfenestrated continuous wall and high expression level of GLUTl. In cultured bovine brain capillary endothelial cells, GLUT1 expression is markedly downregulated ( Farrell eta/., 1992b). A bovine brain homogenate induced GLUTl at the transcriptional level, suggesting the aclion of a brain-derived trophic factor(s) for the expression of GLUTl (Boado et al., 1994b). Tumor necrosis factor a: partially mimicked this effect. Because astrocytes are closely situated around brain microvessels, they have been thought to be responsible for the induction of the characteristics of the blood-brain barrier. An increase in the mRNA level of GLUTl was observed by treatment with phorbol estcrs and serum (Farrell et al., 1992b).Hurwitz et al. (1993) cultured human astrocytes and brain endothelial cells on the opposite sides of a permeable membrane. The endothelial cells expressed GLUTl and y-glutamyl transpeptidase, markcrs of the blood-brain barrier. Such expression was dependent on the endothelial cells being in close apposition to or in direct contact with the astrocytes, suggesting that characteristics of brain microvessels including GLUTl expression are regulated by astrocytes.
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C. Effect of Glucose and Diabetes
Expression of glucose transporters is regulated by glucose (Klip et al., 1994). It was proposed that GLUT1 belongs to the glucose-regulated protein family of stress-inducible proteins (Wertheimer et al., 1991). Low glucose treatment induced an increase in hexose uptake in primary cultures of bovine brain microvessel endothelial cells (Takakura et al., 1991). Deprivation of glucose in cultured bovine brain capillary endothelial cells resulted in an increase in GLUTl mRNA via its stabilization (Boado and Pardridge, 1993). In experimental chronic hypoglycemia in the rat, an approximately 50% increase in both mRNA and protein of GLUTl in the brain microvessels was observed (Kumagai et a!., 1995). The elevation of the GLUTl level in the endothelial cells in the blood-brain barrier upon hypoglycemia suggests that the increase occurs to compensate for the low blood glucose level so that proper nourishment of the central nervous system can be maintained. High glucose treatment, on the other hand, had no significant effect on hexose uptake. Moreover, in experiments using the intracarotid injection method, the blood-brain glucose transport was downregulated in the hyperglycemic mouse (Cornford et al., 1995). In the human diabetic retina, neovascular endothelium of proliferative retinopathy did not stain for GLUT1, showing that the loss of barrier function is associated with an absence of GLUTl (Kumagai et al., 1994b). In the diabetic retina with minimal or no clinical retinopathy, drastic localized upregulation of GLUTl in the retinal blood vessels was observed by quantitative immunoelectron microscopy (Kumagai et al., 1996). Such focal increase in the amount of GLUTl in the blood vessels may amplify the toxic effects of hyperglycemia, thus leading to the focal retinopathy encountered in diabetes (Kumagai et al., 1996).
D. Effect of Ischemia and Hypoxia In the ischemic hippocampus of the rat, the amount of GLUT3 decreased, possibly related to the loss of ischemically damaged neurons (McCall et af., 1995). Kinetic analysis suggested that ischemia downregulates the glucose transporter in the blood-brain barrier in the rat brain (Suzuki et al., 1994). In the gerbil brain, however, ischemia upregulated GLUTl in brain microvessels as well as GLUT3 in neurons (Gerhard et a/., 1994). In situ hybridization studies showed that expression of GLUTl increased in response to an ischemic insult in microvessels, astrocytes, and some neurons in the rat brain (Lee and Bondy, 1993). In cultured bovine aortic and human umbilical vein endothelial cells, hypoxia induced the expression of GLUTl
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(Loike et ul., 1992). In the brain under chronic hypoxia, an increase in the amount of GLUTl mRNA was seen in fetal and developing rats, whereas a decrease was observed in the adult animals (Xia et uZ., 1995).
E. Degenerative Disease
In Alzheimer’s disease, GLUT1 in the brain microvessel endothelium was significantly reduced compared with its amount in age-matched normal brains, suggesting decreased glucose availability to the brain (Kalaria and Harik, 1989; Harik, 1992; Horwood and Davies, 1994; Simpson ef al., 1994). However, these was no change in the density of GLUT1 in erythrocytes, suggesting that the decrease is the result rather than the cause of the disease (Harik, 1992). In Huntington’s disease, a drastic decrease in GLUTl and GLUT3 proteins was observed (Gamberino and Brennan, 1994). These observations indicate that the amount of GLUTl in the blood-brain barrier is regulated by the existence and/or activity of neurons and the subsequent consumption of glucose.
F. Tumors Altered glucose transporter expression, especially in the induction of GLUT3, was observed in human brain tumors (Nishioka et aZ., 1992; Nagamatsu et ul., 1993b; Boado et al., 1994a). The blood vessels inside a brain tumor, whether the tumor was primary or the result of metastasis, usually lost GLUTl immunoreactivity (Harik and Roessmann, 1991). Expression of GLUTl depends on the type of tumors and not on the permeability of the vessels (Guerin et al., 1992a). In the rat intracerebral9L glioma model, dexamethasone treatment reduced the vascular permeability of tumor vessels as measured by Evans blue, with a concomitant increase in the number of GLUT1-positive blood vessels (Guerin et al., 1992b). This observation suggests that GLUTl in the brain tumor may be used to identify the aggressiveness of the tumor. The expression of GLUT1 in tumor blood vessels is also influenced by the location of the tumor, because positive GLUTl staining seen in the intracerebral rat 9L and F98 glioma was virtually lost in the subcutaneous implants of the same tumor (Arosarena et al., 1994). The glioma cells by themselves are not sufficient to induce the expression of GLUTl in the blood vessels. The induction of GLUTl in the microvascular endothelial cells inside the brain tumor may be closely related to the property of the tumor cells, which usually have a glial origin. Because astrocytes are in close apposition to the endothelial cells and
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possibly affect the expression of GLUT1 in the blood-brain barrier, the ability of tumor cells to influence the endothelial cells to express GLUTl may be closely related to the loss of normal cell characteristics in the tumor cells. In addition, expression of GLUT3, whose expression is normally restricted to nerve cells, was observed in the tumor vessels in glioblastomas (Nishioka et al., 1992).
G. Defective Glucose Transporters Low cytochalasin B binding and decreased hexose uptake together with the loss of immunoreactivity to anti-GLUT1 antibody was observed in the erythrocytes of patients with persistent hypoglycorrhachia (low concentration of glucose in cerebrospinal fluid), seizures, and developmental delay (De Vivo et al., 1991; Harik, 1992). Because GLUT1 is responsible for the transport of glucose across the blood-brain barrier, reduced glucose transport activity across the barrier by the defective GLUTl may be responsible for these phenomena.
VI. Concluding Remarks Glucose transporters are one of the most extensively studied transporter molecules in mammalian cellular membranes. We proposed that GLUTl is the glucose transporter isoform of blood-tissue barriers (Takata et al., 1990a,b). Accumulating evidence has confirmed that GLUT1 is highly produced in the cells of blood-tissue barriers. GLUTl is present at the sites of both entry into and exit from the cells of the barrier, although semipolarized distribution is sometimes encountered. An abundance of GLUT1 at the critical plasma membranes of the cells of the blood-tissue barrier ensures a sufficient supply of glucose to cells isolated from the general circulation. Among the six isoforms, GLUT1 appears to serve as the main glucose transporter for the blood-tissue barriers. Transport of glucose via GLUTl is little affected by the regulatory mechanism under physiological conditions, which makes a marked contrast to the transport via GLUT2 or GLUT4. Such steady characteristics of GLUTl, together with its high affinity to glucose, may be ideal as a glucose transport machinery in the blood-tissue barriers in which a constant and stable supply of glucose is crucial. Further analysis of the glucose transport system across the blood-tissue barriers, along with comparative and developmental studies, will lead to a more detailed characterization of these barriers.
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Acknowledgments We thank S. Tsukui and M. Kanai for secretarial assistance. This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan. and Japan Private School Promotion Foundation.
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The Role of Suppressors in Determining Host-Parasite Specificities in Plant Cells Tomonori Shiraishi, Tetsuji Yamada, Yuki Ichinose, Akinori Kiba, and Kazuhiro Toyoda Laboratory of Plant Pathology and Genetic Engineering, College of Agriculture, Okayama University, Okayama 700, Japan
Plant pathogens secrete the compounds that delay or prevent defense responses only of the host plants, with resultant conditioning of host cells such that they become susceptible even to avirulent microorganisms. The principles, which are called suppressors, have been characterized as glycoproteins, glycopeptides, peptides, or anionic and nonanionic glucans. Suppressors do not evoke drastic and visible damages of plant cells and, thus, they can be distinguished from host-specific toxins produced by several fungal species almost belonging to genera Helminfhosporiumand Alfernaria. The mode of action of these suppressors has been found to disturb fundamental functions of host cells. The suppressor from a pea pathogen, Mycosphaerelh pinodes, inhibits both the ATPase activity and the polyphosphoinositidemetabolism in pea plasma membranes, causing the temporary suppression of the signal transduction pathway leading to the expression of defense genes encoding key enzymes in the biosynthetic pathway to phytoalexin. Moreover, it affects the function of cell wall in a strict species-specific manner even in vitro. In this chapter, evidence for the role of suppressors in the determination of plant host-parasite specificity is summarized. KEY WORDS: Defense responses, Determinants of specificity, Suppressor, Susceptibility induction, Transmembrane signaling, Cell wall.
1. Introduction In nature, plants as well as other organisms are resistant or immune to the vast majority of pathogens. In other words, the number of pathogens that Inrernarif~n,nnlH e b ~ i c wof’ L:vrolqy, V n l 172 n 0 7 4 - 7 m i ~ 7$ZS.MI
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Copyright (0 1YY7 by Aczidemic Press All rights of reproduction in any form rrservcd.
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have the potential to attack a given plant species is extremely limited. For example, rice plants are severely diseased by only a few species of 50 fungal pathogens of rice plants among 8000 phytopathogenic fungi in a few hundred thousands species of mycota (Agrios, 1978). This phenomenon is universally observed and is called “host-parasite specificity,” of which the determining mechanism is one of the most intriguing issues. Thus, even in plant-parasite interactions, resistance is the rule and susceptibility is the exception (Oku, 1994). Plant resistance is physiologically classified as static and active. Both resistances are thought to protect plants phasedly and synergistically against the attack by pathogens. Static resistance includes preformed properties such as the strength of cell surfaces and the presence of constitutive antimicrobial compounds. On the other hand, active (induced) resistance involves the formation of chemical and physical barriers, such as phytoalexins, infection inhibitors, active oxygen species, pathogenesis-related (PR) proteins, lignin. callose, and hydroxyproline-rich glycoprotein (Lamb et al., 1989; Ouchi, 1991). The latter is thought to be more essential for the resistance mechanism because suppression of active resistance by treatment with certain metabolic inhibitors, by high temperature, or by inoculation with virulent (compatible) fungi allows avirulent pathogens to infect even nonhost plants. The idea for the molecular mechanism in the active resistance was first presented as “phytoalexin theory” by Miiller and Borger (1940) who hypothesi~edthat certain antimicrobial substances may be accumulated in the potato tissues based on the double inoculation with an incompatible and a compatible race of the late blight fungus Phytophthora in,festans.The following are their conclusions: 1. A principle, designated as “phytoalexin,” which inhibits the development of the fungus in a hypersensitive tissue, is formed or activated only when the host cells come into contact with the parasite. 2. The defensive reaction occurs only in living cells. 3. The inhibitory material is a chemical substance and may be regarded as the product of necrobiosis of the host cell. 4. This phytoalexin is nonspecific in its toxicity toward fungi; however, fungal species may be differentially sensitive to it. 5. The basic response that occurs in resistant and susceptible hosts is similar. The basis of differentiation between resistant and susceptible hosts is the speed of formation of the phytoalexin. 6. The defense reaction is confined to the tissue colonized by the fungus and its immediate neighborhood. 7. The resistant state is not inherited. It is developed after the fungus has attempted to infect. The sensitivity of the host cell that determines the speed of the host reaction is specific and genotypically determined.
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If the term “phytoalexin” is put in different words such as “newly and inductively formed chemical and physical barriers,” it seems to be more suitable to explain the mechanism in active resistance. A factor inducing active resistance was at first isolated from the mycelia of Monilinia fructicola and it was partly characterized as a peptide named monilicolin A, which induced a bean phytoalexin, phaseollin (Cruickshank and Perrin, 1968). Substances that induced phytoalexin production were named “elicitors” by Keen (1975). Later, the term elicitor has been used in a more wide sense for the substances that are able to induce active resistance in plants (DeWit, 1986). The term “inducers” has been also used for these resistance-inducing substances (Hayami el al., 1982). The elicitors were prepared from the culture filtrate, hyphal cell walls, or spore germination fluid of pathogenic fungi and they have been characterized as polysaccharide, glucan, chitin, chitosan, glycoprotein, peptide, lipid, and so on (Darvill and Albersheim, 1984; DeWit, 1986; Ralton et al., 1986). The significance of elicitors in plant-parasite interactions and their mode of action on induction of active resistance were reviewed by Lamb et al. (1989) and Yoshikawa et al. (1993). Compared to the amount of information on the mechanism of plant resistance, little is known about those of susceptibility or accessibility (local susceptibility; Ouchi et al., 1974a). However, an important phenomenon was reported that plant tissues, which had been preliminary infected by virulent or compatible pathogens, became accessible even to avirulent or hypovirulent pathogens (Yarwood, 19.59; Ouchi and Oku, 1981). Preliminary inoculation with a compatible pathogen (or race) was reported to predispose potato to an incompatible race of the late blight fungus (Tomiyama, 1966), barley to powdery mildew fungi (Moseman and Greeley, 1964; Tsuchiya and Hirata, 1973; Ouchi et al., 1974a,b; Kunoh et al., 1985), and oat to an incompatible race of crown rust (Tani et al., 1975). Tsuchiya and Hirata (1973) found that 45 of 51 powdery mildew fungi were able to infect mildewed barley leaves and 30 of the 45 species formed conidia. Ouchi et af. (1974a,b) demonstrated that, within 18 h after inoculation, a compatible race of Erysiphe graminis hordei conditioned barley leaves to be accessible not only to incompatible races of the fungus but also to the wheat and melon powdery mildew fungi. On the other hand, barley leaves that had been inoculated with an incompatible race became inaccessible even to a compatible race of barley fungus within 12 h. Thus, apparently such a phenomenon is inducible. They also clarified that both accessibility- and inaccessibility-induced areas in barley leaves were found to localize near the sites of the primary inoculation with fungi (Ouchi eta!., 1979) and that both cellular conditionings were indeed irreversible. Kunoh et al. (1985, 1988) clarified the timing of establishments of accessibility and inaccessibility by inoculating barley coleoptiles with a compatible race of E. graminis
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and an avirulent pathogen, E. pisi, respectively. That is, E. pisi alone never infected the barley coleoptile, but 30% of conidia established their infection on the cell whcre E. graminis penetrated 1 hr earlier than did E. pisi. The molecular mechanism in accessibility induction is also partly included in the phytoalexin theory. Their conclusions clearly indicate that active defense is delayed in the compatible combination. In fact, the delaying of defense responses was caused by inoculation with compatible races or virulent fungi (Bell et al., 1986; Cuypers et al., 1988; Oku et a!., 1975a,b; Yoshikawa et al., 1978; Yoshioka et al., 1995). This indicates two possible mechanisms: (i) compatible races or virulent pathogens do not produce elicitors that are effective only on the host plants at least during an early stage of infection, and (ii) the pathogens have an aggressive ability to suppress the active resistance in the hosts. If the former case was true, the challenging incompatible pathogens could not infect the plants preinoculated (predisposed) with compatible pathogens because the active resistance must be induced by the effective elicitors that are produced by the challenging pathogens themselves. However, even the incompatible challenger is able to establish itself on the predisposed plants as described. Furthermore, as far as we know, there is no pathogen that does not produce an elicitor. For example, the fragments of common compounds, such as P-glucans and chitin, constitutively present in the hyphal cell walls of many pathogenic fungi can act as nonspecific elicitors (Darvill and Albersheim, 1984). It was also reported that some polysaccharide or glycopeptide elicitors, which were secreted in spore germination fluid of pathogens at the infection sites, induced active resistance even in their own hosts (Hayami et ul., 1982; Shiraishi et al., 1978b; Toyoda et al., 1993b: Yamamoto et al., 1984, 1986; Yoshioka et a[., 1992b). Once the chemical and physical barriers have been established in plant tissues, the penetration, growth, and/or reproduction of the pathogens is crucially inhibited (Oku et al., 1976;Shiraishi et al., 1978a; Yamamoto et al., 1986). Thus, the rejection reaction is indeed irreversible. Therefore, the ability to overcome the host’s resistance is essential for the establishment of infection and colonization by pathogens (Oku, 1980). In other words, the specificity cannot be explained solely by the production of elicitors but is rather determined by the substances that are able to circumvent or negate the active resistance of host plants (Heath, 1981; Oku, 1980; Ouchi and Oku, 1981; Shiraishi et al., 1994). In medical science, nontoxic bacterial factors that inhibit the defense mechanism of multicellular organisms are called “aggressin” or “impedin.” The latter concept was originally presented by Torikata in 1917 (see Ouchi and Oku, 1981) who found that certain bacteria did not produce toxic compounds but produced nontoxic substances, disturbing the host’s immunoreaction. In immunological literature, Glynn (1972) recommended usage of thc term impedin because the term aggressin literally gave an impression
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59
that it damaged host cells (Ouchi and Oku, 1981). Furthermore, the term aggressin was already used by M. C. Chou to define the factor from pollen that induced expanding or aggressive lesions with Botrytis cinerea on Vicia faba (Warren, 1972). A few plant pathologists proposed to extend “the impedin concept” to plant host-parasite interactions (Mahadevan, 1979; Ouchi and Oku, 1981). Ouchi and Oku (1981) further defined the term impedin as “the factor that is produced by a pathogen and actively or passively suppresses the defense reaction of host cells or tissues, enabling the producer or other potential pathogens to establish infection.” At that time, however, the term “suppressor” had been already used for such a substance that had no visible toxicity but suppressed phytoalexin production induced by elicitors or inoculation with avirulent pathogens and conditioned plant tissues to be accessible to avirulent fungi (Oku et al., 1977; Shiraishi et al., 1978b). In this chapter, therefore, available physiological information on the production, the mode of action, and the significant role in determination of specificity of these substances from plant pathogenic fungi, in particular those from M . pinodes, will be introduced by using the term suppressor.
II. Suppressors of Defense Response Produced by Phytopathogens Several phytopathogens were found to produce the metabolites to suppress plant active resistance that is induced by elicitors or avirulent pathogens in a strict species-specific or a race cultivar-specific manner as shown in Table I, in which toxins, endogenous suppressors, and suppressors in infected plants were excluded. Doke (1975) reported that zoospore constituents of a compatible race of Phytophthora infestuns blocked or delayed the hypersensitive cell death of potato tissues induced by inoculation with an incompatible race, but those of an incompatible race showed less activity. The substance was reported to be composed of 17-23 glucose units and contained /3-1,3- and /3-1,6-glycosidic linkages (Doke et al., 1979). Similar activity, which was released from germinating sporangia of P. infestans but not from mycelia, suppressed the hypersensitive reaction of tomato (Storti et al., 1988). The cause of pea Mycosphaerella blight, M . pinodes, secreted a high-molecular-weight elicitor and a low-rnolecular-weight peptidecontaining suppressor for biosynthesis of pea phytoalexin, pisatin, into the spore germination fluid (Oku et al., 1977; Shiraishi et al., 1978b). Later, similar substances in culture filtrate mycelia, or spore germination fluid of several pathogenic fungi were found to suppress NADPH-dependent generation of superoxides (Doke, 1983a), the accumulation of phytoalexins (Doke et al., 1979; Kessmann and Barz, 1986; Ziegler and Pontzen, 1982)
TABLE I Suppressors from Phytopathogenic Fungi
Fungus
Origin
Chemical nature
Host plant
Defense suppressed
Specificity
Accessibility
Site of action
Reference
?
?
Kessmann and Bar7 (1986)
Ascochyfa rahiei
Culture filtrate
Glycoprotein
Chick pea
Race cultivar
PA
Borrytis sp
Germination Huid
Peptide +l
Alliiini spp.
Genus (species)
General'?
Induced
MycoAphaerella ligulicola
Germination Huid
Glycopeptide?
Chrysanthemum
Species
General?
Induced
?
O k u er al. (1987)
M . melonif
Germination fluid
Genus (species)
General'?
Induced
?
Oku er ul. (1987)
Induced
CW, PM (ATPase, PI metabolism)
Oku r f nl. (1 977)
Kodama rt ol. (198Y)
PM?
Germination Huid
Glycopeptide
Pea
Species
General'? 1.1. PA. PR proteins
Phytophthora cupsici
Mycelia
Glucan
Sweet pepper. tomato
Species
HR, AOS
1
PM"
Sanchez era/. (1995)
P. riicotiana
Mycelia
Glucan
Tobacco. tomato
Species
HR, AOS
?
PMI
Sanchez e f nl. (1995)
P. infestans
Zoospore Mycelia
Glucan Phosphoglucan
Potato Tomato
Specie\ Race cultivnr
AOS. HR. PA
?
PM(Ca*+. NADPHoxidase)
Doke (1975)
P. infestans
Zoospore
Glucan?
Tomato
Race cultivar?
HR
?
?
Start1 e r a / . (1988)
P. glycinra
Culture filtrate
Mannan glycoprotein (invertase)
Soybean
Race cultivar
PA
f
?
Ziegler and Pontzcn (1982)
Uromyces pliaseoli
Infection structure
Kidney bean
Species
General? silicon deposits
'?
Heath (1981)
'?
Induced?
Note. AOS, active oxygen species; CW, cell wall; HR, hypersensitive reaction; I.I., infection-inhibitor; PA, phytoalexin; PI metabolism, polyphosphoinositide metabolism; PM, plasma membrane; PR proteins. pathogenesis-related proteins such as endochitinase and P-13gIucanase.
ROLE OF SUPPRESSORS IN HOST-PARASITE SPECIFICITIES
61
and of infection inhibitors (Yamamoto et al., 1986), and the deposition of silicon-containing compounds (Heath, 1980). A part of these metabolites was clarified to cause plant tissues to become accessible even to avirulent pathogens (Kodama et al., 1989; Oku et al., 1980, 1987; Shiraishi et al., 1978b). Furthermore, it was also demonstrated that M . pinodes substances affect both the ATPase and transmembrane signaling of host cells (Kato et al., 1993; Shiraishi et al., 1991a; Toyoda et al., 1992, 1993a; Yoshioka et al., 1990, 1992a,b,). In plant-bacterial pathogen interactions, the artificial administration of an exotoxin, phaseolotoxin, which was specifically produced by the cause of halo blight of bean-Pseudomonas syringae pathoverphaseolicola-only in the susceptible cultivar, was reported to result in suppression of both the hypersensitive reaction and the accumulation of bean phytoalexins and in stimulation of bacterial multiplication even in the resistant cultivar (Patil and Gnanamanickam, 1976). On the other hand, the substance(s) that was able to suppress or delay the production of pea phytoalexin in a race cultiver-specific manner was found in the culture filtrate and the water exudate of the cause of bacterial blight of pea-P. syringae pathovar pisi (Yamada et al., 1994). According to previous reports (Oku, 1980; Oku et al., 1980, 1987), suppressors were defined as “determinants for pathogenicity (specificity) without apparent phytotoxicity.” In detail, (i) they are produced by pathogens at the site of infection; (ii) they participate in suppression of general resistance and in induction of local susceptibility (accessibility) in host plants; (iii) they are host specific; and (iv) they are not toxic to plants. Although the host-specific or selective toxins (HSTs) were first discovered to be the substances that cause necrosis of host cells or tissues, suppressors did not cause any visible damage of the host tissues or protoplasts as far as has been examined. Thus, the suppressors should be distinguished from HSTs. However, the significant role of HSTs in determining specificity has been determined to be the function of suppressing the defense responses of their own hosts (Kohmoto et al., 1987; Hayami et al., 1982; Yamamoto et al., 1984) and conditioning host cells to be accessible to pathogens (Comstock and Scheffer, 1973; Otani et al., 1975; Yoder and Scheffer, 1969), as do the suppressors. The chemical nature of suppressors was determined to be a water-soluble glucan, phosphoglucan, glycopeptide, glycoprotein (such as invertase), or peptide, but unlike HSTs the structure of suppressors was unknown for a long time. However, the structures of two mucin-type suppressors, supprescins A and B, isolated from a pea pathogen. M. pinodes, were determined as GalNAc-0-Ser-Ser-Gly and Gal(P-1,4)GalNAc-O-Ser-Ser-Gly-AspGlu-Thr, respectively (Shiraishi et al., 1992).
62
TOMONORI SHIRAISHI E r AL.
111. Specific Production and Accessibility-Inducing Activity of Suppressors If the fungal metabolites, which show highly biological activities, do not exist at the site ol infection, their significance in host-parasite interactions or in the determination of specificity is little. The initial interaction between plants and pathogens is considered to be mediated by substances that are secreted into spore germination fluids because the majority of phytopathogenic fungi commonly infest and infect through their conidiospores. As mentioned previously, cystospores of P. infestans secrete anionic and nonanionic water-soluble glucans into the germination fluid, and the amounts of both types of glucans increase during incubation. These glucan suppressed, in a race cultivar-specific manner, hypersensitive cell death in and the production of phytoalexin by potato tubers that were induced by an incompatible race of the fungus or by treatment with hyphal cell wall components (elicitors) of the fungus (Doke et al., 1979, 1980). Two infection-inducing factors were isolated from spore germination fluid of Botrytis sp., the cause of scallion bulb rot (Kodama et al., 1989). The infection hyphae from conidiospores of a saprophytic or nonpathogenic strain of Alfernaria afternufawere formed at significant levels on plants in t h e genus Alliurn, such as scallion, onion, wakegi (Alliurn ~ ~ ~ t ~ L.), ~ l ~ ~ . s u Chinese chive, and garlic, that had been treated with the spore germination fluid or the factors from Botrytis sp., whereas the fluid was unable to inducc susccptibility on nonhosts such as strawberry, tomato, and Japanese pear (Kodama et al., 1989). It was also reported that these active substances ( M , <5000) contained peptide moieties, which may be essential for the activity, but did not induce necrosis in host plants (Kodama et al., 1989). Similar nontoxic substances, which induced accessibility and contained peptides, were found in the low-molecular-weight fractions from the pycnospore germination (suspension) fluids of M . ligulicofa, the cause of chrysanthemum ray blight, and M. mefonis, the cause of gummy stem blight of genera Cucumis and Lagenaria (Oku et al., 1987). A pea pathogen, M . pinodes secretes a glycopeptide elicitor ( M , >70,000) and glycopeptide suppressors ( M , <5000) of the defense responses of pea plants in its pycnospore germination fluid (Matsubara and Kuroda, 1987; Oku et al., 1977; Shiraishi er al., 1978a; Thanutong et af., 1982; Shiraishi et al., 1992). The spore germination fluid from a virulent strain (OMP-I) and a hypovirulent strain (OMP-X76) of M . pinodes and from an avirulent fungus, M . ligulicofa (strain OML), was collected at the times indicated in Fig. 1. The filtrate was separated into high- and low-molecular-weight fractions and the activities of elicitor and supprcssor in the respective fractions were dctcrmined by quantitative analysis of the accumulated pi-
63
ROLE OF SUPPRESSORS IN HOST-PARASITE SPECIFICITIES
0
6
12
18
24
0
6
12
18
24
Time after suspending (h) FIG. 1 The activity of elicitor (a) and suppressor (b) produced by pycnospores of Mycosphaerella pinodes and M . ligulicola. The elicitor and suppressor were obtained separately from the spore germination fluid of a virulent strain (OMP-1; 0 )and a hypovirulent strain (OMPX-76; 0) of M . pinodes and from that of a chrysanthemum pathogen, M. ligulicola strain OML (O),by ultrafiltration as described previously (Shiraishi e t a / . 1978).
satin. As shown in Fig. 1, the elicitor activity was detected 3 h after spores had been suspended in sterilized water and the activity increased for 12 h, whereas the elicitor activity produced by these fungi was almost similar. By contrast, the suppressor activity, which was determined as activity to reduce pisatin accumulation induced by the OMP-1 elicitor, was found immediately after suspending OMP-1 spores. It increased up to 9 h. The secretion of suppressor from hypovirulent OMP-X76 spores was lower than that from virulent OMP-1, and avirulent OML secreted only a negligible amount of suppressor over the course of 24 h. The same results were obtained when spores of OML and OMP-1 were placed on pea leaves. Spores of OML were able to infect pea leaves that had been treated with the spore germination fluid from OMP-1 but not with the fluid from OML (T. Shiraishi and Y. Yamamoto, unpublished results). The results are summarized as follows: (i) the elicitor activity was produced even by a virulent fungus in the spore germination fluid, (ii) the secretion of suppressor that was indeed specific and more rapid than the elicitor reflects the order of aggresiveness or virulence of the pathogens, and (iii) the activity of the suppressor mainly reveals even in the simultaneous presence of the elicitor. Thus, it is suggested that the specificity at least in species-species combinations is determined by the production of suppressor and that plant cells may be conditioned to be accessible before penetration by the virulent pathogens (Shiraishi et al., 1991b).
64
TOMONORI SHlRAlSHl ET AL
Treatment of pea tissues with the suppressor from M. pinodes allowed infection by many avirulent pathogens tested, such as Stemphyliurn sarcinaeforme, M. melonis, A . alternatu, M. ligulicola, and so on. Alternaria alternata and S. sarcinaeforme were able to colonize and the former formed conidiospores on the suppressor-treated tissues, whereas they were unable to colonize on untreated tissues (Oku rt al., 1987). Thus, the suppressor from M. pinodes conditioned pea plants to accept even avirulent fungi under natural conditions. The relationship between the biological activity of the suppressor of M. pinodes and the host range of the fungus is shown in Table 11. Alternaria alterriafa could not infect any of 12 leguminous plants tested, but, in the presence of the suppressor, it infected 5 leguminous species, which were infected to varying degrees by M . pinodes (Oku et al., 1980). Thus, the biological specificity of the suppressor of M. pinodes coincides with the host specificity of the fungus. Together with our previous paper (Oku et al., 1987),these results suggest that the suppressors from Botrytis sp., M. pinodes, M. ligulicnla, and M. melonis may act as determinants of specificity of the respective fungi to
TABLE II Effect of the Suppressor from Mycosphaerella pinodes on the Infection by Akernaria alfernataof 12 Leguminous Species
Formation of infection hyphae A . alterriatu
M . pinodes
A. rrlrernata
+ supprcssor
0
0
0
0- 1
n
0
0
2
L. hicolor
2 0
0
0
Lolits cornicrrkilus var. jnponicus
0
0
0
Medicago sativu
0
1
0
2
Pisiim sarivrtm
I 2 4
0
4
Trqoliuni prutrme
1
0
1
Leguminous species Aracliis hypogaea Glycine rn(~x'
Lrspedera birergeri
Milletria japotzicn
1: repens
0
0
0
Viria fabn
0
0
0
V i g m sinensis
0
0
0
Note. Nunihers represent the rate of formation of infection hyphae: 0, no hyphae formed; 4, abundant hyphae formed (Oku r / al., 1980). The sporulation was observed.
ROLE OF SUPPRESSORS IN HOST-PARASITE SPECIFICITIES
65
suppress the general resistance only of host plants and to establish basic compatibility in the respective combinations (Heath, 1981).
IV. Specific Suppression of the Establishment of Chemical Barriers As briefly mentioned previously, active defenses in host plants are inhibited or delayed by suppressors. The hypersensitive reaction in potato tissues or protoplasts that is induced by an incompatible race or by hyphal wall components (a nonspecific elicitor) of P. infestans was prevented or delayed by water-soluble glucans from zoospores of the compatible race (Doke, 1975; Doke et al., 1980; Doke and Tomiyama, 1977). The transient accumulation of phenylalanine ammonia lyase (PAL) transcript, which was induced in potato tuber slices within 30 min after treatment with hyphal wall components, was markedly suppressed by the glucan suppressor only from the compatible race of the fungus (Yoshioka et al., 1995). It was also shown that the NADPH-dependent superoxide generation, which was induced within a few minutes by the hyphal wall fraction, prior to hypersensitive cell death and production of phytoalexins, was suppressed by the watersoluble glucans in a race cultivar-specific manner (Doke, 1983a,b, 1985). Furthermore, water-soluble glucans, which were prepared from mycelia of P. capsici, P. nicotianae var. nicotianae, and P. infestans, also blocked the hypersensitive death of suspension-cultured cells of sweet pepper, tobacco, and tomato, respectively, caused by hyphal cell wall components (elicitors) from Phytophthora spp., indicating that these glucan suppressors acted specifically only on typical host species of the producer (Sanchez et al., 1992). Thus, the glucan suppressors seem to participate in determining species specificity as well as race cultivar specificity. A n extracellular mannan glycoprotein (invertase) of P. megasperma f. sp. glycinea inhibited the accumulation of a soyben phytoalexin, glyceollin, in a race cultivar-specific manner. The invertase from race 1 suppressed glyceollin accumulation in wounded cotyledons of two compatible cultivars induced by a nonspecific glucan elicitor from the cell walls of the same fungus but did not in the incompatible cultivar (Ziegler and Pontzen, 1982). Table I11 shows a summary of the chemical nature and activity of the elicitor and suppressor from M . pinodes. The glycopeptide elicitor induces defense responses in pea such as the accumulation of pisatin, a major pea phytoalexin, and an as yet unidentified infection inhibitor (Yamamoto et al., 1986), as well as enhancement of the activities of endochitinase and endo-P-1,3-glucanase (Yoshioka et al., 1992b). However, these defense responses are markedly suppressed by the concomitant presence of the
66
TOMONORI SHlRAlSHl ET AL.
TABLE 111 Several Propelties and Biological Activities of the Suppressor and Elicitor from Mycosphaerella pinodes Elicitor
Suppressor
Origin
Pycnospore
Pycnospore
Time of secretion
4h
Oh
Coinposition
Man, Glc, Ser
Molecular weight
>70 kDa
Gal, GalNAc, Ser, Gly, Asp, Glu, 'Ihr 4 kDa
Induce Enhance Induce Nonspecific Inhihit ND
Delay Delay Inhibit Species specific Inducc (spccies specific) ND
Stimulate Enhance Unknown ND Induce Enhance (nonspecific) Enhance (nonspecific)
Inhibit Inhihit Inhibit Inhibit Inhibit Inhibii Inhibit
Biological activity Active resistance Pisatin PR protein Infection inhibitor Speciticily Infection Toxicity Action site in cell PI metabolism in PM PM-ATPase (in vilro) ATPase in cell (in vivo) H '-pumping activity (in virro) Na+,K+ efflux Ccll wall-bound ATPase (in vitro) Cell wall-hound peroxidase
(nonspecific) (species specific)
(species specific) (species specific)
Note. ND. not detected; PM,plasma membrane; PI metabolism, polyphosphoinsitide metahdim; PR protein, pathogenesis-related protein.
suppressor with the elicitor. For example, the elicitor induced the accumulation of pisatin within 9 h; an increase of the activity of PAL, a key enzyme in the biosynthetic pathway of pisatin, within 3 h; and thc accumulation of transcripts of PAL and chalcone synthase (CHS) genes within 1 h, whereas the concomitant presence of the suppressor delayed these defense responses for 3-6 h (Yamada et al., 1989). The result indicates that the suppressor tcmporarily intcrruptcd the strcam from the recognition of the elicitor to the transcriptional activation of genes encoding PAL and CHS and also that the suppressor does not cause any drastic damage of pea cells. These findings may coincide with delaying of defense responses caused by inoculation with compatible races or virulent fungi as mentioned prcviously (Bell el ul., 1986; Cuypers et ul., 1988; Oku et ul., 1975a,b; Yoshikawa rt ul., 1978; Yoshioka et al., 1995). The extent of accumulation of pisatin was also significantly reduced, even when the suppressor of M . pinodes was applied to pea tissues 9- 12 h after the start ol the treatment with elicitor (Yoshioka et al., 1990). This finding
ROLE OF SUPPRESSORS IN HOST-PARASITE SPECIFICITIES
67
suggests that the suppressors do not necessarily compete with the elicitors for binding at receptor sites on plants and that the suppressors might affect more fundamental or multiple functions in host cells. Therefore, the mode of action of M. pinodes suppressor does not fit the model that suppressors prevent elicitors from binding to receptors so that defenses are not triggered (Bushnell and Rowell, 1981; Basse et al., 1992). The suppressor of M. pinodes also blocks the accumulation of two phytoalexins, medicarpin and maackiain, in red clover, or which M. pinodes is able to establish its infection (see Table II), but it cannot inhibit the accumulation of glyceollin of soybean and phaseollin of kidney bean (Shiraishi et al., 1991b). The suppressor of M . pinodes also suppresses the activation of proteins, such as endochitinase and @1,3-glucanase, in pea tissues but not in soybean and kidney bean. In the latter case, these PR proteins are rather activated by treatment with the suppressor alone as well as by treatment with the M. pinodes elicitor (Yoshioka et a/., 1992b). These results clearly demonstrate that a suppressor affects defense responses in a species-specific manner and that, for the nonhost of M. pinodes, the suppressor inversely elicits defense reaction.
V. Mode of Action of the Suppressors
A. Effects of Suppressors on the ATPase It has been thought that elicitors are recognized initially by putative receptors on plasma membranes and that they induce changes in membrane functions that are linked to defense responses. The binding proteins for the P-glucan elicitors from P. megnsperma glycinea were found in a membrane fraction of soybean (Schmidt and Ebel, 1987; Yoshikawa et al., 1983; for review, see Roller, 1995). However, their functions are unknown. It has been reported that the suppressor M. pinodes inhibited the ATPase in pea plasma membranes both in vitro and in vivo. The suppressor inhibited the ATPase activity in isolated pea plasma membranes in an uncompetitive manner similar to orthovanadate (Yoshioka et al., 1990), but the plasma membrane ATPases of nonhost plants, such as kidney bean, soybean, cowpea, and barley, were also significantly inhibited in vitro to varying extents by the suppressor in a nonspecific fashion (Shiraishi et al., 1991a). Nevertheless, a cytochemical analysis by TEM and EDX showed that the suppressor placed on leaf surface severely inhibited the ATPase activity in pea cells only (out of five plant species tested), whereas an effective inhibitor of Ptype ATPase, orthovanadate, inhibited the ATPase activity in all plant species tested. At the site of infection, the ATPase activity was also inhibited
68
TOMONORI SHlRAlSHl ET AL.
for at least 6 h by inoculation with M . pinodes but not with an avirulent pathogen of pea, M . ligulicofa (Fig. 2; Shiraishi et al., 1991a). Thus, the inhibition of ATPase activity by the suppressor in vivo coincides with the specific induction of susceptibility (Oku et al., 1980) and with the delaying of the defense responses in pea tissues caused by the suppressor (Yamada et af., 1989; Yoshioka et al., 1992b). It was also shown that orthovanadate nonspecifically suppresses the activation of endochitinase and endo-P-1,3glucanase in the tissues of pea, soybean, and kidney bean treated with the elicitor of M. pinodes (Yoshioka et al., 1992b). Orthovanadate also delayed the start of the accumulation of pisatin for 6 h and the mRNAs encoding PAL and CHS for 3 h (Yoshioka et al., 1990,1992a), as observed with the suppressor from M. pinodes. In addition, for the suspcnsion-cultured cells of red bean or peanut, orthovanadate elicited the activation of enzymes in biosynthetic pathway for phytoalexin (Hattori and Ohta, 1985; Steffens et
M. pinodes
M. ligulicola
FIG.2 ATPase activity in pea cells inoculated with a pea pathogen, Mycosphuerella pinodes, and an avirulent pathogen of pea, M. ligulicolu. The letters, P, L. CW. and PM indicate M . pinodes, M. ligdicola, cell wall, and plasma membrane, respectively. The bars = 2 Fm.High electron density particles show the lead deposit that was generated on the active ATPase by the method of Hall et nl. (1980) (in protoplasma 104, 193-200). Note that thc ATPase activity in the M . pinodes-inoculated cell was severely inhibited for 6 hr as the suppressor-treated pea cells (Shiraishi et rrL, 1991a).
ROLE OF SUPPRESSORS IN HOST-PARASITE SPECIFICITIES
69
af., 1989). In pea cultured cells, orthovanadate alone at concentrations of 0.01-1 mM also induces the accumulation of pisatin and slow cell death (T. Shiraishi, T. Yamada, Y. Ichinose, A. Kiba, and K. Toyoda, unpublished results). However, orthovanadate acts as a nonspecific suppressor when applied to differentiated cells as previously mentioned. Our data from experiments with orthovanadate tend to support the hypothesis that the plasma membrane ATPases, which carry out many fundamental cellular functions such as ion transport and regulation of intracellular pH (Serrano, 1989), must be closely correlated with the regulation of defense responses. In this connection, it was found that the suppressor also inhibited proton transport in proteoliposomes that had been reconstituted with pea plasma membrane ATPase (Amano et al., 1995). The mucin-type suppressors, supprescins A and B, which were purified from the germination fluid of M.pinodes (Shiraishi et al., 1992), inhibited the accumulation of pisatin in pea tissues, but only supprescin B inhibited the ATPase activity in isolated pea plasma membranes (Shiraishi et al., 1992). In vitro action of the peptide moieties in the supprescins on the ATPase activity was reported in detail by Kato etaf. (1993). The components of supprescins, such as galactose, N-acetylgalactosamine, SG, DE, and respective amino acids, did not affect the ATPase activity. However, eight synthesized peptides-SSGDET, SSG, SSGTED, TSGDET, SSGD, GDET, DET, and GDE-inhibited the ATPase activity. The kinetics of inhibition of the ATPase indicated that the inhibitors could be classified into three groups: I, competitive; 11, mixed competitive and noncompetitive; and 111, noncompetitive. The common sequences found in groups 1-111 were Ser-Ser-Gly, Gly-Asp, and Asp-Glu, respectively. Because SSGDET had the greatest inhibitory effect among the peptides tested, whereas SSGTED had only a small effect, the sequence of Gly-Asp in the peptide chain seems to play an important role in the inhibition of the ATPase. The proposed ATP binding domain in the P-type ATPase has a Gly-Asp sequence and the aspartic acid residue has been thought to bind to the phosphate or adenosine moiety of an ATP molecule (Serrano, 1989). Supprescin A did not inhibit the ATPase activity but reduced the inhibitory effect of supprescin B. However, the peptide moiety of supprescin A, SSG, inhibited the ATPase activity. These findings suggest that supprescin A has the potential ability to interact with the ATPase molecule. An examination on the action of supprescins and related peptides on acid phosphatase activity in pea plasma membranes with p-nitrophenylphosphate showed that the oligopeptides in group I11 and a part of group I1 GDET and SSGD, inhibited the activity, whereas supprescin A and other peptides did not show any inhibitory effects. With the exception of SSGD, the inhibitors of phosphatases share a common sequence. Asp-Glu. Thus, the peptide moiety of supprescin B, SSGDET, includes at least two active
70
TOMONORI SHlRAlSHl ET AL.
sequences, Ser-Ser-Gly and Asp-Glu. Because the former sequence inhibited ATPase activity in a competitive manner and the latter one deduced acid phosphatase activity, it is possible that SSGDET might interact with both the ATP-binding domain and the phosphatase one in the ATPase molecule (Kato et ul., 1993). In addition, SSGDET, SSG, GDE, and DET each suppressed the aceumulation of pisatin in pea tissues that is induced by the elicitor from M . pinodes (T. Shiraishi, T. Yamada, Y. Ichinose, A. Kiba, and K. Toyoda, unpublished results). Together with our previous results (Oku et ul., 1980), the data show that the peptide moiety is essential for the activity of the suppressor of M. pinodes. 6. Suppression of Transmembrane Signaling Related t o the Defense Response There are many reports that fungal elicitors induced rapid transcriptional activation of plural defense genes. As described, the elicitor of M . pinodes rapidly induces defense responses that include the remarkable accumulation of transcripts within 1 h of defense genes such as PAL and CHS in pea epicotyl tissues, whereas the concomitant presence of the suppressor delays these responses (Yamada er al., 1989). A further nuclear run-on assay revealed an increase in the transcriptional rate of PAL and CHS in nuclei isolated from pea epicotyls that were treated with the elicitor for at least 5 min (Kato et al., 1995); however, the M. pinodes suppressor rapidly deactivated such an elicitor-triggered transcription of these genes (Wada et at., 1095). This finding suggests that the fungal signals are recognized by certain molecules on the surface of pea cells and cause rapid changes in the functions related to transmembrane signaling. Here, we describe the pathway of signal transduction leading to plant defense responses.
1. Calcium Ion and Cyclic Nucleotides Calcium ions and CAMPwere reported to act as second messengers in the elicitation of defense responses of the suspension-cultured cells of carrot (Kurosaki et ai., 1987b) and soybean (Stab and Ebel, 1987). Kurosaki et al. (1987a) also found that the rapid increase in IP1 occurred in elicitortreated carrot cells. However, an earlier report indicated that PI metabolism was not involved in the signal transduction that leads to phytoalexin biosynthesis in cultured cells of parsley and soybean (Strasser et al., 1986). At tissue levels, treatment of potato tuber with EGTA, before inoculation with an incompatible race of Phytophthoru infesrans or elicitation by hyphal cell wall elicitors, interfered with the activation of O f generation that
ROLE OF SUPPRESSORS IN HOST-PARASITE SPECIFICITIES
71
seemed to be dependent on Ca2+(Doke, 1983b). The EGTA application after inoculation with an incompatible race also reduced the activation of NADPH-dependent 02-generation and resulted in suppression of both hypersensitive cell death and phytoalexin production (Doke, 1983a; Doke and Miura, 1995). Thus, Ca2+seems to be necessary for the activation of the NADPH-dependent Of-generating system and the subsequent hypersensitive reaction in potato tissues. The important role of Ca2' in this system was also reported in the plasma membrane fraction isolated from tuber tissues (Doke and Miura, 1995). In pea pod tissues, however, it was reported that the addition of exogenous Ca2+,EGTA, or calcium channel antagonists, such as diltiazem, nifedipine, and methoxyverapamil hydrochloride, did not affect the accumulation of pisatin induced by chitosan (Kendra and Hadwiger, 1987). These authors suggested that the response induced by chitosan might not be mediated by Ca2' or calmodulin. Similar data were presented with pea epicotyl tissues (Shiraishi et al., 1990). Treatment of pea epicotyls with LaCh, quinacrine, TMB-8, or ECTA did not suppress the elicitorinduced pisatin accumulation even if they were applied before treatment with the elicitor. Calmodulin inhibitors, such as amitryptyline and chlorpromazine, inversely induced pisatin accumulation in the epicotyls. Furthermore, the presence of dibutyryl cyclic AMP (CAMP) or dibutyryl cyclic GMP (cGMP) showed a negligible effect on the accumulation of pisatin in pea tissues despite the absence or presence of the elicitor and/or suppressor of M . pinodes, suggesting that CAMP and cGMP might not necessarily participate in the signal transduction pathway for defense responses in pea tissues (T. Shiraishi, T. Yamada, Y. Ichinose, A. Kiba, and K. Toyoda, unpublished results).
2. Polyphosphoinositide Metabolism The elicitor and/or suppressor from M . pinodes was reported to affect markedly the activities of phosphatidylinositol kinase (PtdIns kinase), phosphatidylinositol monophosphate kinase (PtdInsP kinase), and phospholipase C (PLC) in polyphosphoinositide (PI) metabolism in epicotyl tissues and isolated plasma membranes of pea plants (Toyoda et al., 1992,1993a). In elicitor-treated tissues, the rapid accumulation and turnover of phosphatidylinositol bisphosphate (PtdInsP2)were observed within 5 s, but the level of PtdInsP did not increase. From 5 s to 6 min after the start of treatment with the elicitor, a second increase and decrease in levels of [32P]PtdInsP2 were observed. However, in the concomitant presence of the suppressor, the first rapid increase in PtdInsPz was completely blocked, and the second increase was also suppressed. The elicitor also induced a biphasic and transient accumulation of inositol 1,4,5-triphosphate ( IP3) with peak levels
72
TOMONORI SHIRAISHI E r AL.
at 30 s and 7 min, whereas such an increase was also markedly inhibited by the concomitant presence of the suppressor. Neomycin, an effective inhibitor of PLC, also inhibited the elicitor-induced accumulation of IP3 and pisatin in pea tissues. The neomycin-treated area of pea tissues became susceptible to avirulent fungi (Toyoda et ul., 1993a). As shown in Fig. 3, PI metabolism in pea plasma membranes was also affected by the elicitor andlor suppressor from M . pinodes. A time-course study of phosphorylation of PtdIns and PtdInsP showed that the simultaneous incorporation of radioactivity from [y3*P]ATPinto both phospholipids
El k u
0 .6
0.0
0
2 4 6
8
16
Time (min) FIG.3 Changes in the levels of 32P-labeled PtdInsP and PtdlnsPa in isolated pea plasma membranes upon the addition of clicitor from M. pinodes alone (O),clicitor plus suppressor (0),supprcssor alone (A), o r distilled water (x). Isolated plasma membranes were incubated at 25°C with ly-”PJATP without exogenous phospholipids. The extcnt of incorporation of radioactivity from [y-j2P]ATPwas determined by photostimulatcd fluorography with a Bioimaging analyzer (Bas 2000; Fujix, Tokyo, Japan).
ROLE OF SUPPRESSORS IN HOST-PARASITE SPECIFICITIES
73
was detectable within 5 s after respective treatments and increased for up to 4 min. The elicitor enhanced such phosphorylation of the phospholipids but the concomitant presence of the suppressor markedly reduced it. In particular, the phosphorylation of PtdIns to PtdInsP seemed to be severely inhibited by the suppressor. The phosphorylation of PtdInsP was also inhibited by several other reagents, such as orthovanadate, K-252a (an effective inhibitor of protein kinases), and neomycin, that suppress the defense response of pea (Shiraishi et al., 1990; Toyoda et al., 1993a; Yoshioka et al., l990,1992a,b). The concomitant presence of these inhibitors markedly inhibited the incorporation of radioactivity into PtdInsP and/or PtdInsPz compared with the elicitor treatment. The elicitor and suppressor also affected the accumulation of IP3 in isolated plasma membranes of pea (Toyoda et al., 1993a). The elicitor induced a transient increase of IP3within 30 s. The increase of IP3 in the plasma membranes seems to coincide with the first increase of IP3 observed in pea tissues. No such increase in levels of IP3 was, however, observed in plasma membranes when the suppressor was also present. Thus, the elicitor and suppressor from M . pinodes regulate rapid changes in PI metabolism in both tissues and isolated plasma membranes of pea plants. Chen and Boss (1990) found that the rapid activation of kinases of inositol phospholipids was induced in plasma membranes from carrot cultured cells that had been pretreated with driselase and hemicellulase that induce the release of an endogenous elicitor from carrot cell walls. The subsequent steps in signal transduction might involve the degradation of PtdInsP2to IP? and diacylglycerol (DAG) by PLC (Einspahr and Thompson, 1990; Kurosaki et al., 1987a; Melin et al., 1987; Zbell and Walter-Back, 1988). Recently, it was reported that levels of IP2 but not IP3 increased in tobacco BY-2 cells within 4 min after the start of treatment with the elicitor from Phytophrhoru nicotianae (Kamada and Muto, 1994a). It was assumed that the elicitor activated PtdIns kinase and that its product, PtdInsP, was converted to IP2 by PLC in the cultured cells. Together with these results, our findings suggest that PtdIns kinase, PtdlnsP kinase, and PLC in pea plasma membranes, which respond immediately to fungal signals, are closely related to defense responses. In other words, PI metabolism seems to be indispensable for the initial elicitation of defense responses of plants, as is the case for responses to light (Memon and Boss, 1990; Morse et aL, 1987) or plant hormones (Connett and Hanke, 1987; Ettlinger and Lehle, 1988;Zbell and Walter-Back, 1988). The suppressor from M. pinodes disturbs such a transmembrane signaling essential for the defense responses described previously. 3. GTP-Binding Protein
In animal cells, PLC is assumed to be regulated by GTP-binding proteins (Cockcroft and Bar-Sagi, 1990) and the activity of PtdIns-4-kinase may be
74
TOMONORI SHlRAlSHI €7 AL.
regulated by guanine nucleotide regulatory proteins (Smith and Chan, 1989). Guanine nucleotides were reported to promote the release of derivatives of inositol from the plasma membranes isolated from suspension-cultured cells of Acr.rpsei.ldoplatanus (Dillenschneider etal., 1986). As a result of introduction of rgp-7-cncoding rice small GTP-binding protein into tobacco plants, Sano and Ohashi (1995) suggested that the signal transduction system for defense response includes a network of cytokinins, salicylic acid, small GTPbinding proteins, and so on. On the other hand, GTP was reported not to influence the activity of PLC in plasma membranes isolated from Triticum aestivum (Melin et al., 1987). In our system, GTP and GTPyS each slightly increased the phosphorylation of Ptdlns and PtdInsP in isolated plasma membranes and each scarcely affected the accumulation of pisatin in pea epicotyls compared to the level in the absence of GTP, regardless of the presence or absence of the elicitor and/or suppressor from M . pinodes (Toyoda efal.,1992; T. Shiraishi, T. Yamada, Y. Ichinose, A. Kiba, and K. Toyoda, unpublished results). In othcr words, the application of these nucleotides failed to prevent the elicitation or suppression of pea defense responses by the fungal signals. Thus, it remains uncertain as to whether GTP-binding proteins might crucially participate in regulation of thc signal transduction leading to defense responses in pea tissues.
4. Fatty Acids The activity of phospholipasc A (PLA) also seems to be regulated by these fungal signals because increases in 1ysoPtdInsP and lysoPA were induced by the elicitor from M . pinodes in pea plasma membranes within 5 s,whereas they were suppressed by the concomitant presence of the suppressor. In this connection, it is of interest that linoleic acid and linolenic acid, which are putative metabolites generated from phospholipids (PtdlnsP or PA) by the action of PLA, elicited the accumulation of pisatin (Toyoda et al., 1992; T. Shiraishi, T. Yamada, Y. Ichinose, A. Kiba, and K. Toyoda, unpublished results). These results suggest that fatty acids might also act as second messengers for defense responses in pea, as described in tomato tissues by Farmer and Ryan (1992).
5. Phosphorylation of Proteins A tomato gene, Pto, which confers resistance to races of Pseudomonas syringae pv. tomuto, was cloned and found to include a sequence similar to those found in serine-threonine protein kinases, suggesting a role for Pto in a signal transduction pathway (Martin et al., 1993). The possibility that protein kinases might participate in the transmembrane signaling of elicitors has been proposed in several reports. Elicitors rapidly stimulate
ROLE OF SUPPRESSORS IN HOST-PARASITE SPECIFICITIES
75
the phosphorylation of proteins in membranes (Farmer et al., 1989) and in suspension-cultured cells (Blowers et al., 1988; Dietrich et al., 1990; Felix et al,, 1991; Grab et al., 1989; Viard et al., 1994), and inhibitors of protein kinases affect defense responses (Conrath et al., 1991; Kamada and Muto, 1994b; Shiraishi et aL, 1990). Although K-252a was reported to enhance the synthesis of coumarin in cultured parsley cells that were induced by a fungal elicitor (Conrath et al., 1991), the inhibitor suppressed the production of pisatin in pea epicotyls (Shiraishi et al., 1990) and the induction of PAL in tobacco BY-2 cells (Kamada and Muto, 1994b). In pea epicotyls, K-252a inhibited the accumulation of pisatin only when K-252a was applied to the tissues prior to the treatment with the elicitor of M. pinodes. The accumulation of pisatin was inversely enhanced by the application of K252a 3-6 h after the start of treatment with the elicitor (Shiraishi et al., 1990) when the mRNAs encoding PAL and CHS had already accumulated (Yamada et al., 1989). This finding indicates that the phosphorylation of proteins occurs immediately after the elicitor has been recognized by the putative receptor, and such phosphorylation might also be required for the initiation of defense responses in the upstream of the transmembrane signaling. Our preliminary data show that the suppressor from M. pinodes markedly inhibits, only in the concomitant presence of Ca”, the incorporation of radioactivities from [ Y - ~ ~ P ] A T into P specific plasma membrane proteins of 78,62, and 42 kDa. Moreover, supprescin B and its peptide moieties, SSGDET, SSGD, GDET, SSG and DET, each suppress the activity of protein kinase C prepared from rat brain (T. Shiraishi, T. Yamada, Y. Ichinose, A. Kiba, and K. Toyoda, unpublished results). These results suggest the possibility that the suppressor might also be able to regulate the activity of a certain protein kinase(s) in the signal transduction cascade that leads to defense responses. Furthermore, it seems that protein phosphorylation is required not only for the elicitor-mediated signal transduction cascade in or around plasma membranes but also for the activation of defense genes in nuclei. Recently, cis-regulatory elements necessary for the elicitor-mediated defense gene activation were partly elucidated in pea plants. An AT-rich sequence exists as one of the cis-regulatory elements in a pea defense gene, PSCHSl, and it was found to enhance the elicitor-mediated transcription (Seki et al., 1996). Similar AT-rich sequences, which are present at the upstream region of the promoter in other CHS and PAL genes, were found to form a low mobility complex (LMC) with nuclear extracts by gel mobility shift assay (Kato et al., 1995; Seki et al., 1996). The LMC was markedly formed by addition of nuclear extract prepared from the elicitor-treated tissues (Kato et al., 1995; Seki et al., 1996) but it became undetectable with the addition of the nuclear extract that had been pretreated with alkaline phosphatase (Kato et al., 1995). Therefore, the phosphorylation of some nuclear proteins
76
TOMONORI SHlRAlSHl ET AL
seems to be essential for the activation of the defense genes. We found a prominent change in the formation of the complex of the PSPA L2 promoter region and a nuclear factor(s) that was prepared from pea tissues pretreated with the M . pinodes suppressor (Wada et a]., 1995). Although a nuclear factor(s) suppressing the defense gene expression has not yet been determined, the suppressor might affect the process of phosphorylation of some nuclear protein(s).
C. Possible “Cross-Talk” between PI Metabolism and ATPase in Plasma Membranes The suppressor of M. pinodes inhibits the ATPase activity and PI metabolism in pea plasma membranes both in vitro and in vivo (Shiraishi et al., 1991; Yoshioka et af., 1990; Toyoda et af., 1992, 1993a). Phospholipids in plasma membranes have been shown to be essential for maintenance and regulation of ATPase activity in highcr plants (Kasamo and Nouchi, 1987: Chen and Boss, 1991; Memon et af., 1989; Serrano, 1988). Furthermore, Memon and Boss (1990) demonstrated that light-induced inhibition of inositol phospholipid kinases in plasma membranes was closely correlated with a simultaneous inhibition of P-type ATPase activity, suggesting that surrounding phospholipids regulate the ATPase activity in pea plasma membranes. Phosphatidylinositol bisphosphate of six phospholipids tested enhanced the activity of pea plasma membrane ATPase about 1.8-fold at a concentration of 40 p M . By contrast, a trapper 01 PtdlnsP2, neomycin, inhibited the ATPase activity in a noncompetitive manner (T. Shiraishi, T. Yamada, Y. Ichinose, A. Kiba, and K. Toyoda, unpublished results). Therefore, some of the inhibition of the ATPase activity by the suppressor might result from a decrease in the levels of endogenous PtdInsPz and/ or PtdInsP. Orthovanadate inhibits PtdIns kinase, PtdInsP kinase, and PLC in pea plasma membranes (Toyoda etal., 1992; Shiraishi et af.,1994). These findings suggest that the inhibition of ATPase activity results in the suppression of PI metabolism. In this connection, we note that ouabain, an inhibitor of Na+/ K’-ATPase, has been reported to suppress the activation of lymphocytes via its action on phospholipid metabolism (Szamel and Resch, 1981). Taken together, the results indicate that there exists a functional association (crosstalk) between ATPase and PI metabolism.
VI. Species-Specific Suppression of Cell Wall Function by the Suppressor Cell wall, the most exterior organelle of plant cells, is thought to play an important role in the recognition and modification of external or internal
ROLE OF SUPPRESSORS IN HOST-PARASITE SPECIFICITIES
77
signals such as osmotic stress and plant hormones (Ralton et al., 1986; Showalter, 1993; Varner and Lin, 1989). In fact, plant hormone-binding proteins have been reported to exist in cell walls of maize and hyoscyamus (Jones and Herman, 1993;Macdonald etal., 1991).With respect to microbial stress, plant cell wall is thought to be the primary receptor site at which the secondary signals for defense responses are generated (Darvill and Albersheim, 1984; Ralton et al., 1986). Fragments of plant cell wall, such as pectic polysaccharides and oligosaccharides, have been reported to induce the accumulation of phytoalexins and/or the expression of defense responsive genes (Hahn et al., 1981; Darvill and Albersheim, 1984; Ryan, 1988). It was also reported that several cell wall-bound proteins, such as peroxidase (Apostol et al., 1989;Bradley, et al., 1992),proline-rich glycoprotein (Bradley et al., 1992; Brisson et al., 1994), P-fructosidase (Benhamou et af., 1991), P-1,3-glucanase (Benhamou et al., 1989; Mauch et al., 1989), and chitinase (Benhamou et al., 1990), might play important roles in plant defense responses. However, there has been no report that cell wall-bound proteins respond to suppressors and also that receptors exist in cell walls for fungal signals, in particular for suppressors.
A. Specific Suppression of Cell Wall-Bound ATPase
In an early study, it was demonstrated that activities of phosphatases, including ATPase, were associated with cell walls prepared from corn Coleoptiles (Kivilaan et af., 1961). However, the relationship between these phosphatases and external signals is unknown. As mentioned previously, the suppressor from M. pinodes inhibits the ATPase activity in both isolated pea plasma membranes and pea cells as well as did orthovanadate (Kato et al., 1993; Shiraishi et al., 1991a; Yoshioka et al., 1990). On the other hand, orthovanadate suppresses pea defense responses similarly to the suppressor (Yoshioka et al,, 1990, 1992b). These results suggest that inhibition of the ATPase is closely correlated with suppression of the defense responses of pea plants. However, the suppressor was found to inhibit nonspecifically the ATPase activities in plasma membranes isolated from seedlings even of nonhosts of M.pinodes, such as cowpea, kidney bean, soybean, and barley. The cytochemical observations, however, showed that the suppressor inhibited the ATPase activity only in pea cells from among these plant species tested (Shiraishi et al., 1991a). That is, the action of the suppressor appears to be strictly species specific in viva These findings suggested the possibility that cell walls include certain target molecules of the suppressor and that these putative molecules affect the function of plasma membrane, including ATPase and PI metabolism, and might play an important role in the determination of plant-parasite specificity.
TOMONORI SHlRAlSHl €7 AL
78
We have shown that certain phosphatases, such as nucleoside triphosphatascs, p-nitrophenolphosphatase, and pyrophosphatases, werc tightly bound to cell wall fractions prcpared from pea and cowpea seedlings. (Kiba et al., 1995, 1996b). As shown in Table IV, the ATP-hydrolyzing activity in pea cell wall fraction was considerably different in several properties, such as substrate spccificity, the dependence on both divalent cations and pH, and sensitivity to neomycin, from those in the isolated pea plasma membranes. Such as ATPase activity in cell wall was affected by the elicitor and suppressor from M . pinodes (Kiba et al., 1995, 1996b). That is, the elicitor enhanced nonspecifically the activities in cell walls of pea, cowpea, kidney bean, and soybean (Fig. 4). On the other hand, the suppressor inhibited the activity only in pea cell wall but inversely enhanced those of nonhost plants of M . pinudes (Fig. 4). The result clearly showed that the effect of the suppressor on cell wall-bound ATPase was strictly species specific even in vitro. In pea plants, even when the tissues were treated with the M . pinodes elicitor without prior injury, a local resistance was induced within 1 h that was accompanied by the production of an as yet unidentified infection inhibitor, whereas the suppressor also blocked this defense response (Yamamoto ef al., 1986) and conditioned pea cells to be susceptible to avirulent pathogens (Oku et al., 1980; Shiraishi et al., 1978). In this case, the suppressor and elicitor seemed to affect pea cells via pea cell walls. Orthovanadate, placed on the surface of cells, also inhibited the ATPase activities associated with all membrane systems in the epidermal cells of five plant spccics tested (Shiraishi ef al., 1991a). As mentioned above, the suppressor inhibited in vitro the ATPase activities in plasma membranes isolated from both host and nonhost plants of M. pinodes, whereas the
TABLE IV Several Properties of ATPases in Cell Walls and Plasma Membranes Prepared from Etiolated Pea Seedlings Plasma membrane
Cell wall
Optimum pFl
6-7 (6.5-6.7)
5-9 (6 and 8)
Substrate specificity
ATP B CTP > G'TP > UTP
IJTP
=
> PPi
CTP > GI'P > ATP = pNPP
Demand ol divalcnt cation
Mn2+,Mg2' (none; YO% loss)
Ca2+,Mn", Mg" (none; 20-4074) IOSS)
Inhibitor
Orthovanadate Ncomycin
Orthovanadate
In vitro action ofsupprcssor from Mycnsphu~wNu pinodes
Not specics-specific Inhi hit ion
Species-spccific Pea: inhibition Nonhosts: activation
79
ROLE OF SUPPRESSORS IN HOST-PARASITE SPECIFICITIES 7
8
6 3
5
6
3
k
7
.-
4
4
3
Y u
g
2
2
1
if
;-0
0
wc
S
E
wc
S
E
FIG. 4 Effects of the suppressor and elicitor from Mycosphaerella pinodes on ATPase activities in cell wall fractions from pea, cowpea, kidney bean, and soybean. The ATPase assay was carried out at 25°C for 20 min in 30 mM Tris-MES (pH 6 . 5 ) containing 3 mM Mg-ATP in the absence (water control; WC) or presence of 100 kg/ml elicitor alone (E) or 100 pg/ml suppressor alone (S) as described by Kiba et al. (1995). The protein contents of cell wall fractions from pea, cowpea, kidney bean, and soybean were 4.0, 14.6, 15.0, and 12.3 mgig dry wt respectively. Each value represents the mean with standard deviation (SD) of results from triplicate experiments. Note that the elicitor activated the ATPase activities in cell wall fractions of all species but that the suppressor inhibited the activity only in pea fraction and rather activated those of nonhosts of M. pinodes compared lo the water control.
ATPase activities of nonhost cells were never inhibited in vivo by the suppressor, and those of cell walls isolated from nonhosts in vitro were also not inhibited. Taken together with our previous reports, it is likely that the cell wall (or cell wall-bound ATPases) might affect or regulate the ATPases of other organella such as the plasma membrane and vacuole. In other words, inhibition of cell wall ATPases might result in a decrease in the activity of plasma membranes including ATPases with subsequent suppression of defense responses as described (Shiraishi et al., 1991b; Yoshioka et al., 1990, 1992a,b). If so, the cell walls might also participate in acceptance of a virulent pathogen as well as in rejection of an avirulent pathogen. Because tight connections between cell walls and cytosolic microtubles via plasma membranes were reported to exist (Akashi and Shibaoka,
80
TOMONORI SHlRAlSHl ET AL
1991; Shibaoka, 1993), the above concept may not bc distant from the facts. The finding that cell wall-bound ATPases are stimulated nonspecifically by the elicitor but are inhibited by the suppressor in a species-specific manner also indicates that the putative receptor for the fungal signals might bind tightly to and affect the cell wall-bound ATPases or that the cell wallbound ATPases might act as a receptor and/or a modifier to recognize and change these fungal signals. Alternatively, there remains the possibility that the cell wall-bound ATPasc itself might be the receptor for both signals from M. pinodrs.
B. Specific Suppression of 02-Generation It was reported that generation systems of H 2 0 2and 02-were contained in the cell walls of horseradish and tobacco, which may play an important role in lignin synthesis (Gross et af., 1977; Halliwell, 1978; Mader and Fussl, 1982). However, the relationship between such a gcncration system in cell wall and plant-microbe interactions has been obscure for a long time. We found that O2 was generated on the surface of uninjured lcavcs of pea and cowpea and in the fractions solubilized from cell walls of both plants (Kiba et al., 1996b). The elicitor from M. pinodes, which was placed on both leaves, induced the nitroblue tetrazolium-reducing activity sensitive to superoxide dismutase, whereas the concomitant presence of the M. pinodes suppressor markedly inhibited such a blue formazan formation on pea leaves but not on cowpea leaves. Moreover, thc formation of blue formazan on cowpca leaves was rather enhanced by the suppressor alone. A superoxide dismutase-sensitive NBT-reducing activity was also tound in the fraction of NaCl solubilized from cell walls, which were isolated from etiolated seedlings of pea and cowpea (Kiba et af., 1996a). The activity was NAD(P)H dependent and required manganese ion and p-CA as cofactors but was markedly reduced in the presence of a scavenger of H202,catalase, in a dose-dependent manner. The requirement of these cofactors and the inhibition by catalase intensively indicate that such an 02-generation system is sustained by a certain cell wall-bound peroxidase(s) as described by Halliwell (1 97X) with horseradish peroxidase. Inhibitors of NADPH oxidase, quinacrine and imidazole, which are bound to flavoprotein (Cross and Jones, 1991) and b-type cytochrome (Iizuka et al., 19X5), respectively, generation in the fraction solubilixed did not inhibit or scarcely affected 02from cell walls of both plants. On the other hand, SHAM, an inhibitor of pcroxidase, remarkably inhibited the 02-generation in both fractions. This result also supports the idea that cell wall-bound peroxidase(s) may mainly participate in the NADH-dependent O2 generation in cell wall fractions, whereas a NAD(P)H oxidase(s) might not. Such an hypothesis that cell
ROLE OF SUPPRESSORS IN HOST-PARASITE SPECIFICITIES
81
wall peroxidase(s) catalyzed the 02-production by a complex pathway involving NADH, NAD-, and NADt has been presented by several groups (Gross et al., 1977; Halliwell, 1978; Mader and Amberg-Ficher, 1982). Neomycin did not affect 02-generation in both fractions. On the other hand, orthovanadate markedly inhibited the activity of the fractions from both plants. Effects of both inhibitors on the formazan formation seem to coincide with those on cell wall-bound ATPases. That is, the cell wallbound ATPase was inhibited by orthovanadate but not by neomycin (Kiba et al., 1996a; Table IV), whereas the plasma membrane ATPases were inhibited by both inhibitors (Yoshioka et af., 1990; T. Shiraishi, T. Yamada, Y. Ichinose, A. Kiba, and K. Toyoda, unpublished results). These results suggest the possibility that the 02-generation in the cell wall fractions of pea and cowpea might be regulated together with cell wall-bound ATPases. However, because it was reported that vanadate acted as an inhibitor of peroxidase (Serra et al., 1990), it is yet unknown whether the inhibition of 02-generation by orthovanadate is a result of the inhibition of.cel1 wallbound peroxidase or ATPase. As shown in Fig. 5 , the elicitor from M . pinodes significantly enhanced the formazan formation in the fractions solubilized from cell walls of pea and cowpea in a nonspecific manner as well as the cell wall-bound ATPase. On the other hand, the suppressor inhibited the formazan formation only in pea fraction. Even the concomitant presence of the suppressor with the elicitor decreased the formazan formation in pea fraction to the level of water control. However, in cowpea fraction, the formation was not inhibited by the concomitant presence of the suppressor and was inversely enhanced even by the suppressor alone. These results showed that the activity of 02generation in the cell wall fraction is also regulated by both fungal signals and that the suppressor acts on the activity in a species-specific manner. Thus, the NADH-dependent O f generating system in cell wall seems to be tightly correlated with cell wall-bound ATPase. Our recent experiments showed that the cell wall-bound ATPases of pea and cowpea were copurified with the activity of peroxidase(s) by an affinity chromatography (T. Shiraishi, T. Yamada, Y. Ichinose, A. Kiba, and K. Toyoda, unpublished results). These results support the idea that the plant cell walls may recognize fungal signals and play a key role in determination of plant-microbe specificity. Further investigations are needed to elucidate not only the relationship between the receptors for these fungal signals and the regulatory systems of 02-generation in cell walls but also the role of 02-in signal transduction cascade leading to the early defense responses of plants.
VII. Concluding Remarks A pathogen must possess the capacities (i) to penetrate plant tissues, (ii) to overcome the host’s resistance, and (iii) to evoke disease (Gaumann
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TOMONORI SHlRAlSHl ET AL.
c
30
. I
3)
g 25
1 Pea
I
P
40
35 30
? 20
25 20 15
10
5 3)
2
e9
0
0 0
60
120
0
60
120
Time [sl FIG. 5 Effect of the elicitor and suppressor from Mycosphaerella pinodes on superoxidc generation in the fraction solubilized from cell walls that were isolated from pea and cowpea seedlings. The assay was carricd out i n 30 mM Tris-MES (PIT. 6.5) containing 20 m M MnCI2, 2.5 pLg/ml of nitroblue tetrazolium, 0.5 rnM p-coumaric acid in the absence ( X ) or presence of 100 units of superoxide dismutasc (A), I00 p g h l of the elicitor alone (0),100 pglml of the suppressor alone (A), and the elicitor plus suppressor (B) by the method of Nathan et 01. (J. Clin. Invest. 48, 1895-1904, 1969). Each value represents the mean with standard deviation from triplicate experiments. Notc that the elicitor enhanced superoxide generation in both fractions and that the suppressor inhibited the generation in pea fraction but enhanced that in cowpea fraction.
1951; Oku, 1980). From the accumulated evidence (Oku cf al., 1977, 1980; Shiraishi et a/., 197%;Yamamoto et al., 1986; Yamada et af.,1989; Yoshioka ct al., 1990, 1992b), the suppressor of M . pinodes is thought to be a key factor in overcoming the general resistance of its host and in determining host specificity. Currently, we are most interested in how the suppressor overcomes the host defense responses induced by the nonspecific elicitor and establishes the specific accessibility. A model for the fungal signal transduction cascade in pca plants is summarized in Fig. 6. After the elicitor of M. pinodes has been recognized by a putative receptor on cell walls (the elicitor-binding molecule on pea plasma membranes is presented as RE2 in Fig. 6), it activates the ATPase and 0 -generation system in cell wall and plasma membranc PI metabolism, which involves Ptdlns kinase, PtdInsP kinasc, PLC, and PLA, with resultant production of second messengers such as DAG, IP3, and fatty acids. It is possible that certain protein kinases, present in plasma membranes, cytosol, and nuclei, also participate in such a process, but thcse have been omitted from Fig. 6. Although the second messengers
ROLE OF SUPPRESSORS IN HOST-PARASITE SPECIFICITIES 0 2
a3
02-
FIG. 6 Proposed rnodcl o f pea signal transduction cascades for the elicitor and suppressor from a pathogenic fungus. The enzymes and cascade that are presented by solid lincs a r e regulated by tlic suppressor and elicitor from Mycovphaerella pinodes. The broken lincs indicate putatiLc enzyme\ or cascades that might be affected by both fungal signals. CIIS. chalcone synthase; CW. cell wall; DAG. diacylgl~czrol:ER. endoplasmic reticulum; IP3, inositol 1,3,S~trisphosphate:LK. phospholipid kinases such as PtdIns kinase and 1'tdlnrP kinase: I.OX, lipoxygcnase; PA. phosphatidic acid: PAL. phenylalanine ammonia-lyase; PI. phospliatidylinositol: PIPz, phosphatidylinositol 1.5-bisphosphate: PK. protein kinasc: PKC, protein kinase C ; PLA,, phospholipase Xz: PLC, phospholipase C; PM, plasma membrane; POX. peroxidase; PK protein, pathogenesis-related protein; RE. receptor for elicitor; KS. receptor (or suppressor.
that act to induce the transcriptional activation of defense genes have not been elucidated yet, it is hypothesized that IP7 evokes the release of CaZ+ from the endoplasmic reticulum or tonoplast into cytosol and that the released <'a2' stimulates the activation of target proteins, including protein kinases, as described by Bush (1993). In this connection, we also found that thc exogenous DAG induced the pisatin synthesis and transcriptional activation of PAL and CHS genes (T. Shiraishi, T. Yamada, Y. Ichinose, A. Kiba, and K. Toyoda, unpublished results). On the other hand, fatty acids might be released from phospholipids by PLA and might be converted by lipoxygenases to yield some messenger molecules, such as jasmonate as
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described by Farmer and Ryan (l992), finally inducing the establishment of chemical andlor physical barriers. In addition, the efflux of several ions, such as K' and Nat, was induced in pea epicotyl tissues within 30 s by the M. pinodes elicitor, whereas the elicitor-induced efflux was severely inhibited by the suppressor (Amano et al., 1996). In this cascade, the possible modes of action of suppressors are considcred to be the following: (i) they interfere with recognition of elicitor molecules at the receptor site on the host; (ii) they insulate the signal transduction system; (iii) they block the transcriptional activation of defense genes; (iv) thcy suppress the biosynthctic pathways that lead t o the establishment of physical and chemical barriers against the pathogens; and (v) they inhibit fundamental functions, such as ion transport and generation of energy, that sustain a series of defense responses. In this review, we demonstrated that the supprcssor of M . pinodes seems to possess at least the properties ii and v because the suppressor inhibits the ATPase and the signal transduction cascade dependent on PI metabolism in pea plasma membranes. The next question is where and when is the host-parasite specificity determined? We emphasize here that the isolated plant cell wall responded to the elicitor from M . pinodes nonspecifically but responded to the suppressor in a species-specific manner. This finding indicates that the system switching on or off thc plant defense responses exists in the cell walls and also that the specific suppression of defense responses, at least in this case, determined the specificity. Our current working hypothesis for the periodical mechanism on specific suppression of defense responses is as Follows:
1. The suppressor and elicitor are initially bound to respective putative receptors on the cell wall (in the case of pea- M. pinodes system, the suppressor must be recognized earlier than the elicitor as shown in Fig. 1). 2. On the cells or cell walls of nonhost plants, the suppressor of the pathogen is recognized as an elicitor. 3. Despite the presene or absence of the elicitor, the suppressor crucially inhibits the host cell wall functions, including ATPase activity and 0 2 generation, in a strictly species-specific manner. 4. Such an anesthetized state of the host cell wall systcm reflects the function of plasma membranes, including ATPase activity and PI metabolism, via an as yet unidentified signal transduction cascade, and defense responses with the defense gene expression are suppressed (delayed) for at least 3-6 h. 5. The anesthetized state of cell wall may also reflect another defense system in uninjured host tissues including the production of the infection inhibitor induced within 1 h after the elicitor treatment. That is, the primary process of determination of specificity scems to exist in and start from the cell wall when contacted with the suppressor molecule from the pathogen.
ROLE OF SUPPRESSORS IN HOST-PARASITE SPECIFICITIES
05
The suppressor is considered to be the substance determining at least the basic compatibility. In this chapter, it has been demonstrated that the suppressor overcomes the action of the nonspecific elicitor on host plants but inversely acts as a specific elicitor in a sense on nonhost plants. Resistance is the rule, therefore we believe that the mechanism for determining specificity exists in the phenomenon of specifically overcoming the resistance. In other words, the infection establishment on a given plant species (or cultivar) by a pathogen is dependent on the specific secretion of the substance disturbing fundamental functions, such as signal transduction, ion transport, energy generation, and so on, in cell wall and membrane systems that sustain the defense responses in the host cells as described previously (Shiraishi et al., 1991b). If the compatible plant species (or cultivar) recognizes the suppressor of an original pathogen as an elicitor, it must establish resistance to the producer of the suppressor. Regarding this concept, details will be presented elsewhere. We believe that further investigation on the suppressor, especially on the molecular approach to the receptor, will elucidate the principle of the specificity.
Acknowledgments We are grateful to Professor H. Shibaoka, Faculty of Science, Osaka University, Osaka, Japan, and Professor K. W. Jeon, Department of Zoology, University of Tennessee, Knoxville, Tennessee, for their kind invitation and advice to contribute this chapter. This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. Financial support from Sankyo Co. Ltd., Tokyo, Japan, and Institute for Life Science, Okayama, Japan, is also acknowledged.
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Yamamoto, Y., Oku, H., Shiraishi, T., Ouchi, S., and Koshizawa, K. (1986). Non-specific induction of pisatin and local resistance in pea leaves by elicitors from Mycosphaerello pinodes. M. tnelonis and M. ligulicolu and effect of suppressor from M . pinodrs. J . Phytopathol. 117, 136-143. Yarwood, C. E. (1959). Predisposition. /ti "Plant Pathology. Vol. I . The Diseased Plants'' ( J . G . Horsfall and A. E. Diniond, Eds.), pp. 521-562. Academic Press, New York. Yoder, 0. C.. and Schcffer, R. P. (1969). Role of toxin in early interactions of Hrluninihosporitrnr victoritre with susceptible and resistant oat tissue. Phytoptihulogy 59, 1954-1959. Yoshikawa, M., Yamauchi, K., and Masago, H. (1978). Glyceollin. its role in restricting fungal growth in resistant soybean hypocotyls infected with Phyioph/hortr megcrsprrnza vat-. sojur. Phy.yiol. Pl[irli P(1thol. 12, 73-82. Yoshikawa. M., Keen, N. T., and Wang, M. C. (1983). A reccptor on soybean membranes for a fungal elicitor o f phytoalexin accumulation. Plant Physinl. 73, 497-506. Yoshikawa M., Yamaoka, N., andTakeuchi, Y. (1993). Elicitors. their significance and primary niodcs of action in the induction of plant del'ense reactions Plant Cell Physiol. 34, I 163-1173. Yoshioka.H..Sliiraishi,T.,Yamada,T.,Ichinosc,Y..andOku,H. (1990). Supprcssionolpisatin production and ATPase activity in pea plasma mernbrancs by orthovanadate. verapamil and a suppressor from Mycosphaerellci pinotlrs. Pltrnt Cell Physiol. 31, 1139-1 146. Yoshioka, H., Shiraishi, T., Kawamata, S., Nasu, K., Yamada, T., Ichinose. Y., and Oku, H. (1992a). Orthovanadate suppresses accumulation of phenylalanine ammonia-lyase mRNA and chalcone synthase mRNA in pea cpicotyls induced by elicitor from Mycusphirerella pinodes. Plant Cell Physiol. 33, 201-204.
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Distribution and Functions of PlateletDerived Growth Factors and Their Receptors during Embryogenesis Paris Ataliotis and Mark Mercola Department of Cell Biology, Harvard Medical School, Boston Massachusetts 02115
~~
Platelet-derivedgrowth factors (PDGFs) are soluble proteins that mediate intercellular signaling via receptor tyrosine kinases. The patterns of PDGF and PDGF receptor expression during embryogenesis are complex and dynamic and suggest that signaling can be autocrine or paracrine, depending on the particular tissue and the stage of development. Mesenchymal cells throughout the embryo and within some developing organs produce PDGF receptors, whereas their ligands are often produced by adjacent epithelial or endothelial cells. Disruption of PDGF signaling in the embryo leads to morphogenetic defects and embryonic or perinatal lethality. Tissues that are particularly susceptible to the absence of PDGF signaling are migrating mesoderm cells during gastrulation, nonneuronal neural crest cell derivatives, and kidney mesangial cells. These tissues share the common feature of undergoing epithelial-mesenchymal transitions. We review current knowledge of the distribution of PDGF ligands and receptors and discuss how this distribution may relate to several roles for PDGF during embryogenesis, particularly the regulation of mesenchymal cell behavior. KEY WORDS: Platelet-derivedgrowth factor (PDGF), Embryogenesis, Morphogenesis, Mesenchymal cell migration, Neural crest, Epithelial-mesenchymal interaction.
1. Introduction Platelet-derived growth factor (PDGF) was first characterized as the major mitogenic component of serum (Kohler and Lipton, 1974; Ross et al., 1974) and is known to initiate DNA synthesis in a wide variety of mesenchymal Intrmanonol Review OO74-769hi97 $25.00
of Cytology,
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Copyright 9 1997 by Academic Press All rights of reproduction in any form reserved
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cells grown in culture (Raines et al., 1991). Subsequent studies have shown that PDGF is, in addition, capable of modifying cell behavior in a variety of ways. For instance, it can initiate gene transcription (Cochran et uL, 19x3; Kelly et af., 1983),promote membraiic ruffling (Ridley and Hall, 3 992; Ridley et al., 1992) and cell migration (Noble el al., 1988; Stoker and Gherardi, 1991), act as a neurotrophin (Smits et al., 1991), cause calcium fluxes (Cassel et nl., 1983), modulate differentiation (Noble et al., 1988; Raff et al., 1988), induce synthesis of collagen (Canalis, 1981; Owen et nl., 1984), act as a survival factor (Barres et al., 1992), and induce chemotaxis (Grotendorst et ul., 1982; Seppa et al., 1982; Kundra et ul., 1994). The varied activities of PDGF can be related to its physiological roles in the adult, which probably include wound healing in blood vessels, the nervous system, and cutaneous tissue. Clearly, the cellular processes affected by PDGF in the adult animal and in cultured cells can play equally vital roles during the normal development of the embryo and in the progress of disease states. Many studies of PDGF function have concentrated on its role in proliferative diseases such as cancer, atherosclerosis, inflammatory joint disease, and fibrosis (Raines et al., 1991). Molecular and genetic approaches have demonstrated an absolute requirement for PDGF signaling in the vertebrate embryo (Smith et af.,1991; Stephenson et ul., 1991; Levken et al., 1994;Soriano, 1994;Ataliotis eta]., 1995;Bostrom et al., 1996). Defects in animals deprived of PDGF signaling suggest that a major role for PDGF in the embryo is to influence the behavior of mesenchymal cells during their migration or their transition to or from an epithelium.
II. Biochemistry of PDGFs and Their Receptors
A. PDGF Ligands PDGF was first purified from human platelets using its mitogenic activity on cultured fibroblasts as the basis for a biological assay (Antoniades et ul., 1979;Heldin el al., 1979).The 28- to 35-kDa complex of heterogeneously glycosylated proteins obtained was composed of two similar but distinct polypeptides joined by disulfide bonds (Johnsson et uf., 1982; Waterfield et ul., 1983). Treatment with reducing agents destroyed the mitogenic activity of PDGF but allowed the further separation of PDGF-A and -B chains by high-performance liquid chromatography (HPLC) (Johnsson et al., 1982). PDGF-A and -B are the products of separatc genes (Chiu et al., 1984; Collins et al., 1985; Betsholtz et al., 1986; Rao et al., 1986) that, when expressed in the same cell, can dimerise with themselves or with each other to produce PDGF-AA, -AB, or -BB depending on the species or cell type
DISTRIBUTION AND FUNCTIONS OF PDGFs
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(Raines et al., 1991).The developmental significance of these various PDGF isoforms still remains unclear, but differences in their biochemical properties and the cellular responses they evoke in cultured cells suggest that they may play markedly different roles. PDGF-A and -B are hydrophilic, soluble proteins that share approximately 51% amino acid identity in the mature human protein and 35% similarity between Xenopus PDGF-A and mouse PDGF-B. PDGF isoforms are more highly conserved between species, with Xenopus and mouse PDGF-A sharing 71% similarity and mouse and human PDGF-B 89%. In addition, gene structure and organization are remarkably similar in both PDGF-A and -B (Raines et al., 1991), including 5' untranslated sequences that are known to inhibit translation (Ratner et al., 1987; Rao et al., 1988; Wang and Stiles, 1993). These findings suggest a chromosomal duplication of an ancestral gene and that PDGF molecules have retained distinct and important functions during evolution. Eight conserved cysteine residues in each polypeptide link PDGF dimers by disulfide bridges to form a cysteine knot motif (Murray-Rust etal., 1993). Of these conserved cysteines, four are essential for the transforming activity of v-sb (Giese et al., 1987; Sauer and Donoghue, 1988) (and by analogy, for the activity of PDGF-A and -B ). v-sis is the transforming gene of the simian sarcoma virus and shares 9 3 4 5 % identity with PDGF-B (Antoniades and Hunkapiller, 1983; Devare et al., 1983; Doolittle et al., 1983; Waterfield et al., 1983). It is clear that mutation of particular cysteines to serine or deletion of cysteine residues at t h e N terminus can affect dimer formation, which may account for some of the observed loss of PDGF activity in these cases (Sauer et al., 1986; Sauer and Donoghue, 1988), whereas at least one other cysteine to serine mutation affects the stability of the newly synthesized protein (Mercola et al., 1990a). PDGF-A and -B are both synthesized as longer precursor molecules that are extensively processed. Cleavage of N-terminal proregions and dimerization occur shortly after synthesis of PDGF-A or -B, whereas PDGF-B undergoes additional cleavage of a C-terminal proregion (Robbins et al., 1983, 1985; Raines et al., 1991). PDGF-A is probably glycosylated on both O-linked and N-linked sites, whereas PDGF-B is glycosylated only on the former (Raines et al., 1991). Glycosylation does not appear to be required for PDGF activity because recombinant material from bacteria can bind to PDGF receptors (Fretto et al., 1992), but there are conflicting reports regarding the activity of proteolytically processed and dimerized forms of PDGF. The major form of v-sis with transforming activity contains both N- and C-terminal proregions (Leal et al., 1985), whereas in contrast, blocking transport of v-sis into the endoplasmic reticulum prevents its dimerization, secretion, and biological activity (Lee et al., 1987). However, it has been shown that PDGF-B monomers also have mitogenic activity
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(Stevens et al., 1988). Also, coexpression of a mutant form of PDGF-A that blocks cleavage of the N-terminal propeptide prevents the processing of both endogenous PDGF-A and -B but abolishes the secretion and activity not AInutmt B dimers (Mercola et al., 1990a). It is not of only AAmUtSn', known which PDGF receptor combination(s) binds to and transduces the signal from AmutdntB dimers. Two splice variants of the PDCF-A chain have been found and are termed AL (long-form) or As (short form) (Betsholtz el uE., 1986; Collins et al., 1987; Tong et al., 1987). The long form of PDGF-A contains a long basic stretch at the carboxy terminus that is similar to a basic region at the carboxy terminus of PDGF-B. In addition, PDGF-AL and -B contain a nuclear transportation signal in this carboxy-terminal region of the mature protein (Lee et al., 1987; Maher et al., 1989). If import into the endoplasmic reticulum is blocked, this sequence is sufficient to mediate rapid translocation of PDGF (or other cytoplasmic proteins to which it is attached) to the nucleus (Lee et al., 1987; Maher et al., 1989). The biological relevance of this nuclear transportation signal remains unclear. However, the basic regions of both PDGF-B and PDGF-AL have been suggested to mediate association of secreted PDGF to the cytoplasmic membrane, although the data present an incomplete picture. Secreted PDGF-B is found predominantly associated with the cytoplasmic membrane of fibroblasts within 3 h of its synthesis and is only detectable in the tissue culture medium at low levels 4 h after synthesis (Robbins et al., 1985). Membrane association of PDGF-B is caused by a retention signal at the carboxy terminus of the protein that is not present in PDGF-A (LaRochelle et aZ., 1990, 1991). However, several cell lines and transfected cells are known to secrete soluble and readily recoverable PDGF-B into the medium (Johnson et al., 1985; Raines et al., 1991), suggesting that some of the observed differences may be related to the kinetics of secretion or may be cell line dependent. Although membrane association was not initially observed for PDGF-AL or PDGF-As under conditions in which PDGF-B was membrane associated (Beckmann et al., 1988; LaRochelle et al., 1990, 1991), subsequent studies have found that PDCF-AL can, like PDGF-B, remain cell associated in COS (Ostman et al., 1991) or Chinese hamster ovary (CHO, Kelly et al., 1993) cells. This PDGF-AL retention signal has been suggested to reside in the carboxyterminal basic region (Ostman et ul., 1991). Synthetic peptides corresponding to the basic region of PDGF-AL have also been shown to prevent the mitogenic activity of PDGF and other growth factors, including PDGF-As (Khachigian and Chesterman, 1992, 1994; Khachigian et a/., 1992). There is evidence that this region mediates association of PDGF with the extracellular matrix via glycosaminoglycans (Kelly et af.,1993; Khachigian and Chesterman, 1994). These data further
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imply that the retention of PDGFs at the extracellular cytoplasmic surface is probably related to association with matrix components rather than the cytoplasmic membrane itself. Because no compelling differences have been ascribed to the mitogenic or other signaling properties of these splice variants, for the purposes of this review they will be regarded as equivalent, particularly because no information exists on whether they are differentially expressed in vivo. In summary, although we know much about the biosynthesis and processing of PDGFs in cultured cells, we have yet to understand how subcellular localization, glycosylation, proteolytic cleavage, and the biochemical properties of the various PDGF homo- and heterodimers relate to the physiological roles of PDGF in the adult or during development. However, we should bear in mind that all these modifications are likely to have profound consequences for the availability and activity of PDGFs in vivo, whether by influencing binding to the extracellular matrix, their stability, their rate of diffusion, or their ability to interact with specific PDGF receptors. 6. PDGF Receptors PDGF signaling is mediated by two distinct, high-affinity receptors termed PDGFR-a and -/3 (Yarden et al., 1986; Claesson-Welsh et al., 1989; Matsui et al., 1989). PDGFR-a and PDGFR-/3 are transmembrane proteins with intrinsic tyrosine kinase activity. The extracellular portion comprises five immunoglobulin-like domains and the intracellular portion contains the catalytically active kinase that is interrupted by a hydrophobic kinase insert region. This latter feature is characteristic of members of the class 111 (Ullrich and Schlessinger, 1990) or PDGFR family of receptor tyrosine kinases, which also includes c-kit and c-fms. PDGFR-a and c-kit are clustered together on chromosome 5 of the mouse and chromosome 4 of humans, whereas PDGFR-/3 and c-fms are clustered on chromosome 18 of mouse and chromosome 5 of humans (Rosnet et al., 1993; Nagle et al., 1994; Dietrich et al., 1996). A third receptor tyrosine kinase, JEk-1, maps to the same region of mouse chromosome 5 and human chromosome 4 as PDGFR-a and c-kit. JEk-1 is related to the PDGFR family, containing a kinase insert region, but has seven extracellular immunoglobulin-like domains (de Vries et al., 1992), thus making it a class V receptor tyrosine kinase (Rosnet et al., 1993). In humans, a third class V receptor tyrosine kinase, flt-4, maps to the PDGFR-Plc-fms locus, but this is not the case for mouse, in whichfit-4 is located on chromosome 11 (Galland et al., 1992). However, an as yet unidentifiedflk gene is present on chromosome 18, adjacent to PDGFR-P and c-fms (M. BuCan, personal communication). Overall, the similar chromosomal organization and se-
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PARIS ATALlOTlS AND MARK MERCOLA
quence similarities between these genes suggests that they arose from duplication events of an ancestral gene followed by chromosome duplication 1993; Nagle et al., 1994). (Ciebel rr ul., 1992; Rosnet et d., PDGFK-a genes from rat, mouse, and human are 90-95% similar, and they share 73% similarity with Xenupus PDGFR-a. PDGFR-P from human and mouse are 85% similar, whereas human PDGFR-a and -p share 47% similarity overall but differ most in their extracellular regions. This degree of conservation indicates strong selective pressure for the individual features of each receptor. I n addition, there is evidence that PDGF-like molecules may function even in invertebrates such as Hydra and sea urchins (Hanai et ul., 1987; Ramachandran et ul., 1993, 1995; Govindarajan ef al., 1995). PDGFR-a binds both PDGF-A and -B chains with high affinity, whereas PDGFR-P binds only PDGF-B with high affinity (Seifert et al., 1989). Our current understanding of ligand-receptor interaction is that dimeric PDGF molecules initiate or stabilize receptor dimer formation (Bishayee et al., 1989; Heldin et al., 1989), which then triggers an intracellular signaling cascade (see below). PDGF receptors can form both homo- and heterodimers that bind with high affinity to the ligand combinations shown in Table I. Dimerization of PDGF receptors is accompanied by transphosphorylation between dimer partners of specific tyrosine residues within the intracellular domain (Heldin et al., 1989; Kelly et ul., 1991). These phosphorylated tyrosines serve as high-affinity binding sites for a variety of mcmbraneassociated and cytoplasmic proteins that contain src homology 2 (SH2) domains (Claesson-Welsh, 1994; Kazlauskas, 1994). It is largely these SH2 domain-containing proteins that mediate transmission of the PDGF signal inside the cell. Binding of parlicular SH-2 domain-containing proteins to phosphotyrosines is regulated by flanking amino acid sequences (Songyang et al., 1993) that determine whether a given SH2 domain-containing protein can interact with the phosphorylated growth factor receptor. This specificity may impart crucial differences in the signaling activities of PDGFR-a and -P.
C. Downstream Signaling Molecules The recruitment of intracellular signaling molecules to the phosphorylated PDGF-P receptor has been intensely studied over the past few years. A number of molecules have been identified and, in some cases, their binding sites determined (reviewed in Claesson- Welsh, 1994; Kazlauskas, 1994). Although much of this work relates to PDGFR-P, sequence similarities and direct comparison of PDGFR-a binding suggest that most of these SH2 domain-containing proteins can bind to both receptors (Eriksson et
101
DISTRIBUTION AND FUNCTIONS OF PDGFs TABLE I High-Affinity PDGF Ligand-Receptor Interactions
PDGF-AA ~~~~
PDGF-AB
PDGF-BB
PDGFR-aa
PDGFR-~CI
~
PDGFR-aa
~~~~~~~
PDGFR-@
PDGFR-aP
PDGFR-Pp"
PDGFR-fiP
~
The high-affinity binding of PDGF-AB to the -Pfi receptor homodimer has been the subject of some debate. Interaction has been shown following downregulation of cell surface PDGFR-a by PDGF-AA (Drozdoff and Pledger, 1991; Grotendorst et al., 1991; Olashaw eta[., Welsh e t a / . , 1991). Also, activation and dimerization of PDGF-Pfi receptors has been demonstrated in smooth muscle cells that had no detectable PDGFR-a protein and on which PDGF-AA had no effect (Inui e f al., 1993). Furthermore, 3T3 cell lines derived from mice in which the gene for PDGFR-a has been deleted can still respond to PDGF-AB, although higher concentrations of PDGF-AB than PDGF-BB were required (Seifert eta/., 1993). These authors concluded that the concentrations of PDGF-AB required (100-200 ng/ml) were unlikely to be reached in v i v a Although serum concentrations of PDGF-AB are about 20 ngiml in the adult, local concentrations around cells or within tissues of developing embryos might be much greater. In addition, PDGF binding molecules that are present in the extracellular matrix (Kelly et ul., 1993; Khachigian and Chesterman, 1994) or the cytoplasmic association of PDGF-B may act to concentrate or present the ligand to responding cells. "
al., 1992; Songyang et al,, 1993). However, it is interesting that within the same cell type, -aa and -&3 receptor dimers can activate different responses and associate with unique substrates (Eriksson et al., 1992). Furthermore, -ap heterodimeric receptors contain a unique, phosphorylated tyrosine that is not seen in -aa homodimers (Rupp et al., 1994). The significance of these differences is not entirely understood in vitro nor in vivo, but it is highly likely to be relevant to the observed differences in the sites and timing of PDGF receptor production in the developing embryo. Cellular responses to PDGF, including mitogenesis, cell migration, chemotaxis, survival, and differentiation, are all likely to play vital roles in the developing embryo. SH2 domain-containing proteins have been implicated in the first three of these processes. Much of our current understanding of these protein-protein interactions is based on the use of specific tyrosine to phenylalanine mutations. In many cases, a single tyrosine residue is both necessary and sufficient to mediate the binding of one SH2 domain protein
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PARIS ATALlOTlS AND MARK MERCOLA
without affecting others (e.g., a Tyr716 to Phe mutation in human PDGFRabolishes grb-2 binding; Arvidsson et a]., 1994). A consensus has thus emerged for the functions of specific intracellular effector molecules using cell lines, and these are summarized in Table 11. Data summarized in this table are taken from the reviews of Claesson-Welsh (1994) and Kazlauskas (1994) and references therein. For instance, CHO cells transfected with wild-type PDGFR-/3 show a chemotactic response to PDGF-B. By transfecting these cells with specific Tyr to Phe point mutants, this chemotaxis has been shown Lo be positively regulated by phospholipase C-y and phosphatidylinositol-3 kinase (PI-3K), and negatively regulated by GTPase activating protein (Kundra et al., 1994). In BALB/c 3T3 cells, PDGF-Bstimulated chemotaxis is mediated by a ras-dependent pathway (Kundra et af., 1995). Similarly, PDGF-induced membrane ruffling and chemotaxis have been shown to require PI-3K in porcine aortic endothelial cells (Wennstrom et al., 1994b). The challenge now is to extend these findings to the embryo to determine how the activation of PDGF receptors can exert such widely differing effects during different stages of development. One approach to this problem, which is in use in our laboratory as well as in others (Wennstrom el al., 1994a), is to construct chimeric receptors that TABLE II Possible Embryological Roles of SH2 Domain-ContainingProteins
Molecule
Cellular response
Possible embryological relevance
Receptor association
~~
PI3 kinese
PI turnovcr
Mi togenesis Chemotaxis Cell adhesion
Grb2
ras activation
Mitogenesis Cytoskeletal changes
SH-PIP2
ras activation?
PLC-y
Calcium release
Cell adhesion? Migration? Chemotaxis
src family
Cytoskeletal reorganization
Motility Mitogenesis?
Ras-GAP
Negative regulation of ras
Inhibits migration?
)
nck
?
shb
Mediates SH3 association?
?
B P P
shc
Mediates Grh2 association?
?
a?lP
n') I
fos induction'?
>
B
Mitogenesis?
DISTRIBUTION AND FUNCTIONS OF PDGFS
103
contain the intracellular portion of PDGFR-fl fused to the extracellular domain of another growth factor receptor. In our laboratory, the extracellular domain of c-fnzs has been fused to wild-type or mutated forms of the intracellular domain of PDGFR-fl that have then been introduced into embryonic mesoderm cells. Such cells spread onto a fibronectin substratum in response to colony stimulating factor-1, the ligand for c-fms, but only if the binding sites for PI-3K are present (Symes and Mercola, 1996). Delivering and activating similar mutant receptors in the intact embryo should allow the identification of important signaling pathways for various developmental processes in vivo. Such experiments are currently under way.
D. Dominant Negative Mutants The dimeric nature of PDGF ligands and receptors provides a valuable opportunity to probe their function both in vitro and in vivo. Because, in general, two active partners are required to produce secreted ligand (see Section II,A) or to initiate receptor phosphorylation (see Section II,B), the coexpression of a mutated form of PDGF or its receptor can block such activity within the cell. A cysteine 129 to serine point mutation of mouse PDGF-A (called 1308) has been shown to act as a dominant-negative mutant when coexpressed in cells producing PDGF-A or -B (Mercola et al., 1990a). This protein dimerizes to form unstable AA or AB dimers that are rapidly degraded within the cell. A separate mutation of a proteolytic processing site (called 1317) prevents the proteolytic cleavage of N-terminal sequences in PDGFA. The 1317 mutant can dimerize with wild-type PDGF-A or -B, but inhibits the activity only of PDGF-A, because the 1317-PDGF-A dimer cannot bind to PDGFR-a (Mercola et al., 1990a; M. Mercola, unpublished data). These mutants have also proven effective in blocking transformation by PDGF-A or -B and in suppressing the growth of certain astrocytomas (Shamah et al., 1993). Dominant-negative mutations of PDGF receptors fall into two categories. First, the truncation of a large part of the intracellular domain of either PDGFR-a or -0 removes the kinase region and results in the loss of biological activity of the receptor (Ueno et al., 1991,1993). These truncated receptors can suppress PDGF signaling via wild-type receptors when overexpressed in large molar excess. One such truncated form of PDGFR-P has also been shown to block glioma cell growth (Strawn er al., 1994). Second, point mutations of conserved amino acid residues in PDGFR-a are potent inhibitors of PDGFR-a and $3 function both in v i m and in vivo (see below; Ataliotis et al., 1995).
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111. Distribution of PDGFs during Embryogenesis PDGFs are sccreted, soluble proteins that might be expected to signal over long distances in the embryo. However, the analysis of ligand and receptor distribution in v i m suggests that both paracrine and autocrine signaling can occur, depending on the developmental stagc or tissue. Furthermore, the patterns of ligand and receptor expression suggest that switching between paracrine and autocrine signaling occurs within particular tissues. PDGF-A and PDGFR-a are produced at earlier stages and more widely than PDGF-B and PDGFR-P. In many instances PDGF receptors are found in mesenchymal tissues, whereas their ligands are present in adjacent epithelial layers. Representative examples of these epithelial/mesenchymal production patterns are given below, along with some exceptions to this rule. Furthcr examples of our current knowledge of PDGF ligand and receptor distribution during development are given in Table 111. There is some dcgree of overlap between ligand and receptor subtype production that may have profound implications for PDGF action in the embryo, but this has yet to be systematically investigated.
A. PDGF-A and PDGFR-(Y 1. Early Production
PDGF-A and PDGFR-a are synthesized as maternal transcripts in mouse and Xenupus embryos (Mercola et ul., 1988, 1990b; Rappolee et al., 1988; Palmieri et al., 1992; Jones et al., 1903). In Xenopus embryos, RNA levels of ligand and receptor decline during the initial stages of development, then increase shortly after the onset of zygotic transcription but in a spatially restricted pattern. At early gastrula stages, PDGFR-a mRNA is distributed evenly throughout mesodermal cells of the marginal zone, whereas PDGFA mRNA is present throughout the inner (sensorial) layer of the animal pole ectoderm (Jones et al., 1993; Ataliotis et al., 1995). As gastrulation proceeds, PDGFR-a production is maintained in the mesoderm, whereas PDGF-A is in the adjacent ectoderm. At early ncurula stages, PDGFR-a is widely distributed throughout the mesoderm, except for the notochord, whereas PDCF-A is present in the neural and epidermal ectoderm (Ho et al., 1994). A similar pattern is seen in the mouse embryo, in which ligand and receptor proteins are evenly distributed in two-cell- and blastocyst-stage embryos (Palmieri et al., 1992). At Embryonic Day 7.5, PDGFR-a is produced in the mesoderm, with the exception of the primitive streak (OrrUrtreger and Lonai, 1992), whereas PDGF-A mRNA is present in adjacent
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ectodermal and endodermal layers (Orr-Urtreger et al., 1992; Orr-Urtreger and Lonai, 1992; Palmieri et al., 1993). The patterns of production described previously suggest that at early stages of development, PDGF-A may be acting in an autocrine manner to stimulate cell proliferation. During the early periods of morphogenesis, it seems more likely that PDGF-A acts in a paracrine manner to influence the migration of mesenchymal cells. Experimental evidence supporting these models is discussed under Section IV.
2. Neural Crest After somitogenesis begins, PDGFR-a production is no longer confined to mesodermal derivatives but is also in the neural crest, which is ectodermally derived (Morrison-Graham et al., 1992; Orr-Urtreger et al., 1992; OrrUrtreger and Lonai, 1992; Palmieri et al., 1992; Schatteman et al., 1992; Ho et af., 1994). In Xenopus, PDGFR-a is produced in all migrating cephalic neural crest cells and PDGF-A is produced in a complementary pattern along the pathway of their migration in the neural ectoderm, epidermal ectoderm, otic vesicle, and pharyngeal endoderm (Ho et al., 1994). As the cephalic neural crest migrates through the branchial arches, it continues to produce PDGFR-a, whereas the adjacent pharyngeal endoderm produces PDGF-A (Ho et al., 1994). Trunk neural crest produces PDGFR-a at lower levels (Ho et al., 1994). In the mouse, PDGFR-a is also produced widely throughout the cranial neural crest and, as in Xenopus, is present in most, if not all, neural crest cells in the branchial arches (Morrison-Graham et al., 1992). However, neuronal derivatives of the neural crest do not produce PDGFR-a prior to E l 6 (Morrison-Graham et al., 1992; Orr-Urtreger et al., 1992; Orr-Urtreger and Lonai, 1992; Schatteman et al., 1992), although high levels of PDGFR-a and -p production are seen in neonatal rats (Eccleston et al., 1993). These production patterns suggest that in both mouse and frog the direct influence of PDGF is confined to only a subset of neural crest derivatives. 3. Somites
PDGFR-a mRNA is present throughout the presomitic mesoderm and the newly formed somite as epithelialization occurs, but as differentiation proceeds PDGFR-a production is maintained only in the sclerotome and downregulated in the dermamyotome (Morrison-Graham et al., 1992; OrrUrtreger and Lonai, 1992; Schatteman et al., 1992). Later still, the dermatome begins to produce PDGFR-a again, but the myotome does not and instead produces PDGF-A (Orr-Urtreger and Lonai, 1992). PDGF-A production is also widespread throughout the epidermal ectoderm of the
TABLE Ill
Localization of PDGF Ligands and Receptors during Embryogenesis Localization In the early embryo PDGF-A in oocyte, gastrula, neurula PDGF-A in oocyte. blastocyst PDGF-AIPDGFR-a in oocyte. blastocyst PDGF-NPDGFR-a in two-cell embryo. blastocyst PDGF-A in ectoderm PDGFR-a in mesoderm of early postimplantation embryo PDGF-A in inner layer of ectoderm, PDGFR-Q in mesoderm during gastrulation PDGF-B in placental cytotrophoblasts PDGF-A in smooth muscle cells. PDGFR-a in mesenchymal stroma of placental blood vessels PDGF-B and PDGFR-P in microcapillary endothelial cells PDGF-B in endothelium, PDGFR-/3 in smooth muscle cells and surrounding mesenchyme of macro blood vessels PDGF-A, -B. PDGFR-a and -P in placenta and decidua PDGF-A and PDGFR-a in E6.5 embryo PDGFR-P in E7.5 embryo, PDGF-B in E8.5 embryo PDGF-A in ectoderm and PDGFR-a in mesoderm of blastula stages
Species
Reference
Xenopus
Human Human Human Human
Mercola el a/. (1966) Rappolee el al. (1966) Watson er a/. (1992) Palmieri et al. (1992) Palmieri era!. (1992) Ataliotis et ul. (1995) Goustin et al. (1965) Holmgren er al. (1991) Holmgren ef a/. (1991) Holmgren e f ai. (1991)
Mouse Mouse Mouse Xenopus
Mercola er ai. (1990b) Mercola er ai. (1990b) Mercola et al. (1990b) Jones et al. (1993)
Mouse Mouse Mouse Mouse Mouse
Morrison-Graham er al. (1992) Schatteman et al. (1992) Orr-Urtreger and Lonai (1992) Orr-Urtreger and Lonai (1992) Soriano (1994)
Mouse
Mouse cow
Mouse Xenopus
Extensive surveys postgastrulation PDGFR-a in nonneuronal neural crest PDGFR-a in early mesoderm, heart. lens. neural crest-derived mesenchyme PDGF-A in early ectoderm, muscle, epithelial components of limb. lung, salivary gland, eye PDGFR-a in early mesoderm. somites, mesenchymal components of limb, lung, salivary gland, eye P-Galactosidase targeted to PDGFR-P locus produced in cephalic mesenchyme. heart. somites. limb bud mesenchyme, choroid plexus, vertebrae. notochord PDGFR-a in primitive endoderm, early mesoderm, somites. limb, mesenchymal tissue PDGF-A in neural ectoderm and pharyngeal endoderm PDGFR-a in cephalic and trunk neural crest
Xenopus Xenopus
Orr-Urtreger et a/. (1992) Ho er al. (1994) Ho et al. (1994)
During organogenesis PDGF-B throughout various regions of the brain and spinal cord
Monkey
Sasahara et al. (1991)
PDGF-B promoter driving chloramphenicol acetyltransferase shows production in brain. heart, lungs, placenta, liver, kidney, neurons of dorsal root ganglia PDGF-A, -B, PDGFR-a, -0 in cortex, brain stem, cerebellum, spinal cord PDGFR-0 in neurons throughout the brain PDGF-A in neurons of spinal cord and cortex PDGF-B in olfactory neurons, neurons of hippocampus, cerebellar cortex, optic chiasm PDGF-A in neurons of spinal cord, cortex, hippocampus PDGFR-a in glial cells? PDGF-A and -B in spinal cord, ventricular zone PDGFR-0 in spinal cord, dorsal root ganglia, although antibodies used d o not display correct specificity on Western blots PDGFR-a in lens, optic nerve, predominates in ventral E l 8 brain, white matter of cortex, hippocampus. cerebellum PDGF-A in lens, retinal neurons, optic nerve PDGFR-a in lens (adjacent to PDGF-A), retinal astrocytes, optic nerve PDGF-B and PDGFR-0 in lens, retinal microvasculature, optic nerve PDGF-B in radial glial cells of forebrain ventricular zone PDGFR-a in spinal cord PDGF-A and/or -B, PDGFR-a and PDGFR-0 in Schwann cells and neurons of dorsal root ganglia PDGFR-@in mesenchyme of developing tissues and organs, endothelium of small blood vessels and surrounding mesenchyme of larger blood vessels PDGFR-a in atrial and ventricular heart valves and cushions and adjacent myocardium PDGF-A in palatal epithelia, eye muscles, optic nerve, retina, tooth germs PDGFR-a in midline palatal epithelia, palatal mesenchyme, lens, optic nerve, retina, tooth germs PDGF-B and PDGFR-0 in retina, optic nerve PDGFR-a in mesodermal and ectodermal components of limb bud PDGF-B in glomerular epithelium, PDGFR-0 in metanephric blastema, interstitial cells, vasculature, then both PDGF-B and PDGFR-0 in mesangial cells PDGF-A and -B in lung epithelium and interstitial cells PDGFR-a and/or -p in lung epithelium and mesenchyme PDGF-B in lung epithelium PDGF-A in lung epithelium, PDGFR-a in mesenchyme PDGF-A in lung epithelium, PDGFR-a in mesenchyme
Mouse
Sasahara et al. (1991)
Mouse Rat Mouse Rat Mouse
Smits el al. (1991) Yeh et al. (1991) Sasahara et al. (1992) Yeh et al. (1993)
Mouse Mouse
Hutchins and Jefferson (1992) Hutchins and Jefferson
Rat
Pringle et ul. (1992)
Rat Rat Rat Rat Rat Rat Mouse
Mudhar et nl. (1993) Mudhar et al. (1993) Mudhar et al. (1993) Johnston and van der Kooy (1989) Pringle and Richardson (1993) Eccleston et ul. (1993) Shinbrot et nl. (1994)
Mouse Mouse Mouse
Schatteman et al. (1995) Qiu and Ferguson (1995) Qiu and Ferguson (1995)
Chicken Human
Potts and Camngton (1993) Alpers et al. (1992)
Rat Rat Rat Rat Mouse
Han et al. (1992) Han et al. (1993) Souza et al. (1994) Souza et al. (1995) Bostrom et al. (1996)
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mouse, thus leading to the apposition of receptor-producing cells in thc dermis and sclerotome with ligand-producing cells in the epidcrmis and myotome.
4. Central Nervous System A degree of ambiguity still exists regarding the distribution of PDGF-A and PDGFR-a in the embryonic central nervous system (CNS) partly because studies using in situ hybridization often lack sufficient detail to confidently assign labeling to specific cell types and also because of some discrepancies from different laboratories. The onset of production for PDGFR-a has been reported at El2 in the rat (Pringlc and Richardson, 1993) and at El5 (Yeh et al., 2993) or E13.5 (Schatteman et al., 1992) in the mouse. It appears that neurons, glia, and their precursors can all produce PDGFR-a depending on the region of the CNS and the time of development. For example, oligodendrocyte precursors probably produce PDGFR-a in vivo (Pringle and Richardson, 1993) as they are known to do in vitro (Hart et al., 1989), and retinal ganglion neurons also produce PDGFR-a (Mudhar et al., 1993). PDGF-A is abundantly produced in neurons in the late (EIX) mouse embryo (Yeh et aL, 1991) but is also produced in glial cells in regions such as the developing optic nerve (Mudhar ef al., 1993). 5. Limb PDGF-A is produced in the surface ectoderm and muscle masses of the mouse limb, whereas PDGFR-a is present throughout the limb bud mesenchyme and then in the perichondrium of the developing bone as production is lost from the condensing chondrogenic mesenchyme (Orr-Urtrcger et ul., 1992; Schatteman et al., 1992). PDGF may serve to prevent differentiation and/or promote the proliferation of chondrocytes in the limb because it can prevent chondrocyte differentiation in an in vitro system (Chen et ul., 1992). This inhibition may be mediated by the production of tumor growth factor$ (TGF-8) family members in the limb because TGF-P is known to stimulate production of PDGF-A and -B in microvascular endothelial cells (Kavanaugh et a[., 1988). Intriguingly, both PDGF-A and PDGFR-a are produced in the apical ectodermal ridge (AER) of the limb bud (Morrison-Graham ef al., 1992; Orr-Urtreger and Lonai, 1992; Potts and Carrington, 1993) along with many other growth factors. Thc role of PDGF here remains unclear but the AER is essential for normal limb development (Tickle and Eichele, 1994). However, defects of the limb have not been reported in Patch mouse mutant embryos that lack the PDGFR-a gene (see below). The only exception to
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this has been in mice of a particular genetic strain carrying the Patch mutation in which the overall phenotype was more severe than previously described and limbs appeared poorly formed (Orr-Urtreger et al., 1992). 6. Heart
PDGFR-a is first produced in cardiac mesenchyme cells in the heartforming region at E8 in the mouse. At E9 to E9.5, production is seen transiently in the endothelial lining of the heart (the endocardium), then in the pericardium and the endocardia1 cushions at E10.5 (Orr-Urtreger et ul., 1992; J. Payne and M. Mercola, unpublished observations). The latter give rise to the septa and valves between the atria and ventricles and between the ventricles and the outflow tract. At slightly later stages, PDGFR-a is present in the primitive trabeculae (Morrison-Graham et al., 1992; Schatteman et al., 1992, 1995) and production remains high in the forming atrial and ventricular valves and in the immediately abutting myocardium. Unfortunately, the production pattern of PDGF-A in the embryonic heart has not been well documented. A limited study in our laboratory shows low levels of production throughout the myocardium and in the endothelial cells of the aorta of an E11.5 mouse (J. Payne and M. Mercola, unpublished observations).
B. PDGF-B and PDGFR-P The patterns of production for PDGF-B and PDGFR-P have been less thoroughly described than those for PDGF-A and its receptor, particularly with respect to side-by-side comparisons of figand and receptor production in the early embryo. Given this qualification, the general pattern is that PDGF-B is produced in epithelia and endothelia, whereas PDGFR-fi is produced in adjacent mesenchymal tissue. PDGF-B mRNA is first detected in the mouse embryo at E8.5 by RNase protection assay and PDGFR-P at E7.5 (Mercola et al., 1990b). The spatial localization of PDGF-B has not been described before mid-gestation in the rodent embryo and in only a few locations thereafter. Briefly, PDGFB protein is found in the airway epithelial lining of the lung bud at E l 2 in the rat (Han et at., 1992) along with PDGFR-fi at E13. The receptor may also be present in the mesenchymal cells of the lung bud, mirroring the production pattern of PDGF-A and PDGFR-a in this organ (Han et al., 1992; Souza et al., 1995). Within the CNS, PDGF-B and PDGFR-0 are produced in neurons throughout the brain and spinal cord (Sasahara et al., 1991; Smits et al., 1991; Hutchins and Jefferson, 1992; Sasahara et al., 1992), but their relative cellular localizations have been poorly characterized.
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PDGFR-/3 production is seen in the periaortic, facial, and limb bud mesenchyme of E10.5 mouse embryos and at later stages in mesenchymal components of tissues derived from the primitive gut. Production is also seen in the pericardium and myocardium of the heart, in the endothelium of capillaries, and in the surrounding mesenchyme of larger blood vessels (Shinbrot et al., 1994). Similar patterns of production are observed for a /3-galactosidase gene targeted to the PDGFR-/3 locus of a transgenic mouse (Soriano, 1994). Within the kidney, PDCF-B is initially produced by the differentiating glomerular epithelium and PDGFR-P in the adjacent mesenchyme. As glomerulogenesis progresses, both PDGF-B and PDGFR-/3 are present in the mesangial cells of the glomerular tuft (Alpers et ul., 1992).
IV. Functional Studies on the Effects of PDGF PDGF is able to elicit a wide range of responses on cells grown in culture. We now understand some of the signaling pathways from PDGF receptors that mediate these responses, but we are only beginning to comprehend how and why a PDGF stimulus can elicit diverse reactions within and between individual cell types. Furthermore, we do not know the significance of differences between signaling from PDGFR-aa, -a& or $0 dimers nor where these differences might be relevant during embryogenesis. However, despite these limitations, a pattern is now emerging for PDGF function in a variety of tissues during development based on studies in vivo and in vitro.
A. Patch Mouse The earliest clear indication of the importance of PDGF during embryogenesis came from study of the Patch (Ph) mutant mouse. This mutation arose spontaneously and consists of a chromosomal deletion of between 45 and 550 kb (Nagle et al., 1994) that causes coat color abnormalities in heterozygote mice and leads to gross anatomical abnormalities and death at midgestation in homozygotes (Gruneberg and Truslove, 1960). It is now known that the gene for PDGFR-a is deleted in Ph mutants (Smith et al., 1991; Stephenson et al., 1991). However, the extent ol the deletion in Ph has not been fully mapped so it is unclear whether other genes have also been deleted (Nagle et al., 1994). The coding region of the adjacent c-kit gene is known to be intact (Nagle et al., 1994), and c-kit is ectopically expressed in Ph embryos in sites that normally produce PDGFR-a (Duttlinger et al., 1995; Wehrle-Haller et al., 1996). This probably occurs as upstream regulatory elements from the PDGFR-a gene are brought into proximity
DISTRIBUTION AND FUNCTIONS OF PDGFS
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with the c-kit coding sequence by the chromosomal deletion. It seems likely that the ectopic expression of c-kit causes the coat color defect in Phl+ mice as it does in certain Wmutants (Reith and Bernstein, 1991; Duttlinger et al., 1995; Wehrle-Haller et al., 1996), but it is probably not responsible for most of the defects seen in PhlPh embryos. PDGFR-a is normally not expressed in melanocytes but is present in many of the tissues that are affected in PhlPh embryos (Morrison-Graham et al., 1992; Orr-Urtreger et al., 1992; Schatteman et al., 1992). Furthermore, targeted disruption of the mouse PDGFR-a gene causes embryonic lethality and similar defects to Patch mutants in null mice but does not affect the coat color of heterozygotes (P. Soriano, personal communication). Thus, many of the defects seen in PhlPh mice can most likely be ascribed to the loss of the PDGFRa gene. Defects in Patch mutants first become obvious at E9 or E9.5 (Griineberg and Truslove, 1960;Morrison-Graham et aZ., 1992) when mice have a kinked neural tube and subepidermal blisters. Although the blisters in particular seem to improve with another day of gestation, by E l 2 embryos have a facial cleft caused by a failure of the mandibular processes to fuse (Griineberg and Truslove, 1960;Morrison-Graham et al., 1992). Most embryos on a C57BL6 background die at E9.5-10.5, although the cause is not clear. Histological examination reveals a variety of defects in mesenchymally derived tissues that normally express PDGFR-a, especially in the nonneuronal derivatives of the cranial neural crest such as craniofacial cartilage. Also affected are septa1 structures in the heart, including the aorticopulmonary septum and the atrioventricular valves (Morrison-Graham et al., 1992; Schatteman et aZ., 1995), both of which produce PDGFR-a and have a contribution of cells from the neural crest (Kirby et al., 1983). The severity of the embryonic phenotype for Ph varies with the strain of mouse used (Morrison-Graham et al., 1992; Orr-Urtreger et al., 1992). Homozygous PhlPh embryos from C57BL6 X BALB/c F2 mice survive until late gestation. These embryos reveal extreme skeletal abnormalities in addition to the craniofacial defects noted in earlier embryos. Defects include occult spina bifida and fusions of the ribs and cervical vertebrae (Morrison-Graham et al., 1992; Schatteman et al., 1992; J. Payne and M. Mercola, unpublished observations).
B. PDGF in the Early Embryo Maternal PDGF-A and PDGFR-a transcripts have been detected in frog, cow, and mouse embryos (Mercola et al., 1988,1990b; Rappolee et al., 1988; Palmieri et al., 1992;Watson et aL, 1992) and it is now apparent that PDGFA plays a vital role during early vertebrate development. It seems likely
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that its initial function is to promote proliferation of early blastomeres. PDGF-A and PDGFR-a are initially produced in the same cells, suggesting an autocrine stimulation of the receptor. Bovine embryos maintained in chemically defined medium in culture stop cleaving at the 8-cell stage. This block is overcome by addition of PDGF-B (which can stimulate PDGFRa ) to the culture medium (Larson et al., 1992). It is unclear whether this requirement for exogenous PDGF at the eight-cell stage reflects depletion or inaccessibility of maternal PDGF-A or results from the growth of embryos in a suboptimal medium in vifro. In support of the theory that PDGFA is required for early embryonic development, we have noticed that Xenopus embryos injected with a dominant-negative PDGFR-a mRNA (PDGFR-37) at high doses stop cleaving within a few hours and subsequently die (Ataliotis et al., 1995; P. Ataliotis, unpublished data). Furthermore, approximately SO% of embryos lacking the PDGF-A gene die before E10.5, although the reasons remain unclear (Bostrom et al., 1996). We have also used the dominant-negative PDGFR-37 construct to investigate PDGF function during gastrulation in the Xenopus embryo. An aspartate to lysine point mutation in the ATP-binding region of PDGFRu analogous to the W3’ point mutation of c-kit (Nocka et al., 1990; Reith et al., 1990) is sufficient to abolish the kinase activity of the receptor. PDGFR-37 is a potent transinhibitor of PDGFR-a and PDGFR-p in vitro and in vivo (Ataliotis et al., 1995). We have shown that PDGFR-a-expressing cells of the involuting marginal zone normally migrate over a substratum of ectodermal cells that produce PDGF-A. Blocking PDGF signaling in these migrating cells prevents them from adhering properly to their substratum and leads to their untimely death by apoptosis (Ataliotis rt al., 1995; P. Ataliotis and M. Mercola, unpublished observations). Thus, they do not migrate to their normal destinations within the embryo. Consequently, deficits of head mesenchyme and other anterior mesodermally derived structures are observed. We believe that the primary defect in these migrating cells is their ability to attach to the predominantly fibronectin-containing extracellular matrix of the inner surface of the blastocoel roof. The programmed cell death we observe is probably a secondary event caused by a process akin to anoikis, the programmed cell death observed when adherent cells are detached from their substratum (Ruoslahti and Reed, 1994). The gastrulation defects observed in Xenopus embryos injected with an mRNA encoding PDGFR-37 may appear initially to be at odds with the phenotype of PhlPh embryos, but it should be borne in mind that a proportion of Ph homozygotes do die at around the time OIgastrulation (Griineberg and Truslove, 1960; Graf et al., 1987; MorrisonGraham et NI., 1992) and that maternally derived PDGFR-a mRNA or protein may persist until this stage. The dominant-negative receptor will interfere with existing as well as newly synthesized protein. This view is supported by the finding
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that antisense oligonucleotides to PDGFR-a injected into cultured mouse embryos at E6.5 also lead to gastrulation defects in mesodermal derivatives (P. Lonai, personal communication). Furthermore, defects seen in mice with a targeted disruption of PDGFR-a are exacerbated by crossing to mice with a deletion of
[email protected] embryos die shortly after gastrulation, raising the possibility that signaling via PDGFR-@may be sufficient to compensate for the lack of PDGFR-a in the late gastrula stage mouse embryo (P. Soriano, personal communication). Mice with a deletion of PDGFR-P do not exhibit defects until the onset of organogenesis (Soriano, 1994). The requirement for PDGF during gastrulation may not be limited to vertebrate embryos because a dominant-negative form of PDGFR-/3 or blocking antibodies to PDGFR-@or PDGF-B are able to perturb gastrulation of the sea urchin Lytechinus (Govindarajan et al., 1995; Ramachandran et al., 1995; C. Tomlinson, personal communication).
C. PDGF in the CNS
PDGFs are widely produced in the developing CNS, but their functions remain largely unknown. Perhaps the best understood role for PDGF in the CNS involves oligodendrocyte precursor cells isolated from the optic nerve. This example illustrates the pleiotropic nature of PDGF action within a single cell type. Oligodendrocyte precursors produce PDGFR-a in v i m and, most likely, in vivo (Hart et al., 1989; Pringle et al., 1992; Mudhar et af., 1993; Pringle and Richardson, 1993). In v i m , PDGF acts as a mitogen for these cells and promotes their motility (Noble et al., 1988). Embryonic precursor cells grown in the absence of PDGF do not divide but instead differentiate prematurely into oligodendrocytes. Normally, oligodendrocytes do not appear in the rat optic nerve until after birth. Addition of PDGF to embryonic precursors in vitro delays their differentiation into oligodendrocytes until their counterparts have begun to appear in vivo (Noble et al., 1988). Finally, PDGF promotes the survival of oligodendrocytes and their precursors in vitro (Barres et al., 1992). Two pieces of evidence suggest that PDGF-A also performs at least some of these functions in vivo. First, PDGF-A is produced by both neurons and astrocytes in the developing optic nerve (Pringle et al., 1992; Mudhar et al., 1993; Qiu and Ferguson, 1995), suggesting that there is paracrine activation of PDGFR-a on oligodendrocyte precursors. Second, about 50% of oligodendrocytes in the rat optic nerve normally die by apoptosis, but this can be prevented by implanting COS cells producing PDGF-A, suggesting that amounts of this growth factor are limiting (Barres et al., 1992).
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The widespread production of PDGF-A and -B in neurons of the CNS suggests, at the very least, that these growth factors play a role in gliogenesis outside the optic nerve. Furthermore, the production of PDGF-A by astrocytes may indicate signaling to neurons or neuronal precursors. PDGF-A and -B are known to promote the differentiation of cortical neuroepithelial cells into neurons in vitro, and these cells produce both PDGFR-a and -/3 (B. Williams, personal communication). Neuronal cells from newborn rats also produce PDGFR-/3 in vitro and in vivo and show increased survival and neurite outgrowth in the presence of PDGF-B (Smits et al., 1991). However, no current studies have examined PDGF receptor distribution in the embryonic brain in sufficient detail to be sure of localization in viva A recent, detailed study of PDGF-A, -B, and PDGFR-/3 distribution in the mouse CNS is complicated by the finding that the antibody to PDGFR/3 used by these authors recognizes a 65-kDa protein on Western blots but not a 170- to 180-kDa protein, which is the expected size (Hutchins and Jefferson, 1992). No specific defects have been reported in the gliogenesis or neurogenesis of mice lacking either PDGFR-a (Griineberg and Truslove, 1960;MorrisonGraham et al., 1992; Schatteman et al., 1992; P. Soriano, personal communication) or PDGFR-/3 (Soriano, 1994), but in the former case these embryos usually die well before CNS development is complete. In both cases, deletion of either gene alone may not be sufficient to produce a defect in the CNS, but, unfortunately, deletion of both PDGFR-a and PDGFR-P leads to death shortly after gastrulation (P. Soriano, personal communication). Similarly, disruption of the genes for PDGF-A or PDGF-B has not been reported to adversely affect CNS development (Levben et ul., 1994;Bostrom et al., 1996). Interestingly, transgenic mice producing PDGF-B under the control of the myelin basic protein promoter exhibit retinal defects, although the cause remains unclear (K. Forsberg-Nilssen and M. NistCr, personal communication).
D. PDGF in the Heart Homozygous Patch embryos exhibit a range of severe cardiac abnormalities that include incomplete septation of the outflow tract, valvular defects, septa1 defects of the atria and ventricles, double outlet right ventricle, reduced myocardial tissue, and fewer vascular smooth muscle cells than wild-type mice (Griineberg and Truslove, 1960; Morrison-Graham et al., 1992; Orr-Urtreger and Lonai, 1992; Schatteman et al., 1995). The affected tissues are derived from both cranial neural crest and cardiac mesoderm, both of which produce PDGFR-a during development (Morrison-Graham et af., 1992; Orr-Urtreger et al., 1992; Schatteman et nl., 1992, 1995). The importance of PDGF-A in heart development is underscored by the finding
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that neutralizing antibodies to PDGF-A injected into the decidua of mouse embryos in utero cause defects similar to those seen in Patch embryos (Schatteman et af., 1996). However, the defects seem to be limited to the cardiac mesoderm-derived components of the heart and not to those derived from cranial neural crest. Interestingly, these authors did not report abnormalities in other regions of the embryo caused by injecting antibodies at E8.5, E9.5, or E l 0 5 This may indicate a particular susceptibility of the cardiac mesoderm to a loss of PDGF-A signaling or may be due to an inability of injected antibody to penetrate effectively, and at appropriate times, into all regions of the embryo. Heart defects are also seen in mice in which the PDGF-B gene has been deleted (LevCen el al., 1994), but these differ significantly from those of the Patch phenotype. The onset of defects is much later in the PDGF-B deletion-E16.5 compared to ElO-and is limited to an increased size and trabeculation of the ventricular myocardium and dilation of blood vessels. Furthermore, vascular smooth muscle cells and septa within the heart appear normal. It is difficult to ascribe the cardiac abnormalities in these mice directly to the loss of PDGF-B, particularly because the precise sites and timing of PDGF-B and PDGFR-P production in the developing heart remain unknown. Furthermore, these cardiac abnormalities do not occur in mice lacking PDGFR-P (Soriano, 1994), suggesting that PDGFR-a (which is widely produced in the heart) may compensate for the absence of PDGFR-P. It is possible that the cardiac defects seen in PDGF-B-deleted embryos are secondary to other defects such as the kidney abnormalities. However, similar kidney defects also occur when PDGFR-/3 is deleted. Therefore, the most likely explanation seems to be that signaling by PDGFBB via PDGFR-a is important for the late phases of cardiac development but not for its initial morphogenesis.
E. PDGF in the Lung The developing lung produces both PDGF ligand isoforms primarily in the epithelium, whereas both PDGF receptors are present at highest levels in the mesenchyme (Han et al., 1992, 1993; Orr-Urtreger and Lonai, 1992; Souza et af., 1994, 1995; Bostrom et af., 1996). I n vitro studies on lung bud explants have previously suggested that PDGF-A and PDGF-B serve distinct functions in the developing lung. Antisense oligonucleotides to PDGF-B inhibit DNA synthesis in these explants but do not affect branching of the lung buds. Addition of PDGF-BB, but not PDGF-AA, partially reverses the effects of the antisense oligonucleotide (Souza et al., 1994). This implies that DNA synthesis in the lung is dependent on signaling via PDGFR-P or by a PDGFR-a-mediated signal that can be stimulated
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only by PDGF-BB. However, addition of PDGF-AA alonc to lung explants can also stimulate DNA synthesis (Souza et al., 1995), which suggests that signaling via PDGFR-a is important for mitogenesis. Antisense oligonucleotides to PDGF-A decrease both DNA synthesis and branching in lung explants (Souza et al., 1995). PDGF-BB, however, is unable to reverse the effects of antisense PDGF-A oligonucleotides on branching or mitogenesis, even though PDGF-B can bind to PDGFR-a with high affinity. The failure of PDGF-BB to rescue branching morphogenesis in the lung could be ascribed to differences between PDCF-AA and PDGF-BB signaling via PDGFR-a. However, it is difficult to reconcile these findings with the inability of PDGF-BB to stimulate DNA synthesis in lung explants treated with PDGF-A antisense oligonucleotides. It should also be noted that the effect of antisense PDGF-A or PDGF-B oligonucleotides was most noticeable in the epithelial component of the lung explants (Souza et al., 1994,1995), even though this is primarily the site of ligand and not receptor expression (Han et al., 1992, 1993; Orr-Urtreger and Lonai, 1992; Souza et al., 1994, 1995). The previous findings seem to be at odds with the phenotype of a targeted disruption of the PDGF-A gene. Approximately half of the embryos lacking PDGF-A survive until birth but die shortly afterwards due to emphysema. This is caused by an absence of alveolar myofibroblasts, which are derived from the splanchnic mesenchyme and contribute to the formation of septa between individual alveoli (Bostrom et al., 1996). Development of these myofibroblasts is a much later event than the initial branching of the lung buds that is disrupted by antisense oligonucleotides in explant culture. One possible explanation for the discrepancy between the gene-targeting and antisense oligonucleotide studies is that the conditions of culture in the latter impose additional stress on lung bud explants that make them more dependent on the presence of PDGF.
F. Angiogenesis A role for PDGF-B and PDGFR-6 during angiogenesis has been proposed based on the production patterns of ligand and receptor during development and the effect of ectopically applied growth factor (Holrngren et a/., 1991; Risau rt al., 1992). At the initial stages of blood vessel formation and in microcapillaries, PDGF-B and PDGFR-/3 are coproduced by cndothelial cells, In contrast, cndothelial cells of larger blood vessels produce only PDGF-B, whereas PDGFR-6 production is detectable in surrounding smooth muscle and mescnchymal cells (Holmgren ~t al., 1991; Shinbrot et al., 1994). These production patterns suggest an initial phase of autocrine signaling within capillary endothelial cells, switching t o a paracrine signal
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from the endothelium to tissue surrounding the maturing blood vessel. It seems that PDGF-B signaling is not necessary for the initial formation or maturation of blood vessels because deletion of this gene, or the gene for PDGFR-P, does not prevent the formation of vascular endothelium (LevCen et al., 1994; Soriano, 1994). However, PDGF-B may be important for promoting the structural integrity of blood vessel walls because embryos lacking PDGF-B or PDGFR-fl begin bleeding late in development.
V. Concluding Remarks The production patterns of PDGF ligands and receptors in the vertebrate embryo have been widely examined during the past few years. In summary, PDGF-A and PDGFR-a are produced from the earliest stages of embryogenesis, initially in the same cells and then in an appostional pattern, where PDGF-A is produced primarily by epithelia and PDGFR-a by mesenchymal cells. The onset of production of PDGF-B and PDGFR-P in the mouse embryo does not occur until E7.SE8.5, and then in a far more restricted pattern than that of PDGF-A and PDGFR-a. PDGF-B is primarily produced in epithelial and endothelial tissues, whereas PDGFR-p is most often found in adjacent mesenchyme. Notable exceptions to these patterns are the production of PDGF-A by the myotome and forming limb musculature and of both PDGF-A and PDGF-B by neurons and glia of the central nervous system. In many instances, production data have been used to infer that the primary function of PDGFs during development is to influence the behavior of mesenchymal cells in a variety of tissues. However, because much of our interpretation of PDGF function is based on the production patterns of these ligands and receptors, we should bear in mind that posttranscriptional events may significantly affect ligand or receptor availability or activity. For instance, PDGF-A synthesis is known to be controlled at the level of translation (Ratner et al., 1987; Rao et al., 1988; Wang and Stiles, 1993). Also, a proteolytically processed form of PDGFR-a, consisting of only the soluble extracellular domain, has been found in human plasma and has been shown to compete for binding of PDGF-BB to cell surface receptors (Tiesman and Hart, 1992). Recently, targeted disruption of the genes for PDGF-A, PDGF-B, PDGFR-a, and PDGFR-P has revealed their importance for murine development. The loss of any one of these gene products results in embryonic or perinatal lethality. As predicted from expression studies, defects in these embryos occur in mesenchymal cells. Thus, the absence of PDGF-B or PDGFR-/3 prevents mesangial tuft formation in the kidney and when
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PDGF-A is disrupted alveoli do not septate normally, whereas the loss of PDGFR-a affects a variety of nonneuronal neural crest derivatives. A surprising finding from these gene-targeted embryos, particularly those lacking either of the PDGF ligands, is that they survive as long as they do and that defects are not more widespread. This is probably because in any one null embryo, signaling by the remaining ligand or receptor can still occur. For instance, in the PDGF-B-/- embryo, PDGF-A is still available to signal via PDGFR-a, whereas in the PDGF-A-I- embryo PDGF-B can signal via PDGFR-a and
[email protected] important step in the subsequent analysis of such embryos is to localize precisely the coproduction patterns of ligands and receptors in both affected and normal tissues. This should help determine where overlapping ligand or receptor production is likely to compensate for the loss of one gene or whether this compensation is provided by other growth factors. In particular, there are significant gaps in our knowledge of PDGFR-P and PDGF-B production in the early embryo and of PDGF-A and -B production in the heart, Similarly, interbreeding of mice to produce embryos lacking either both ligands or both receptors can further reveal the degree of compensation between these molecules. In the case of embryos lacking both PDGF receptors, lethality occurs at earlier stages (shortly after gastrulation) than it does for either receptornull embryo alone (P. Soriano, personal communication). Beyond the initial findings of the PDGF gene-targeted mice, further subtleties remain to be elucidated. To investigate the functions of PDGFs in specific tissues and organs without causing early embryonic lethality, more precise disruption of the PDGF signal must be achieved. In vivu, this will most likely involve the use of dominant-negative forms of the ligands or receptors under the control of tissue-specific promoters. Dominantnegative versions of PDGFR-a and PDGFR-@have been used to demonstrate a requirement for PDGF signaling during Xenupus (Ataliotis et al., 1995) and sea urchin (Ramachandran et al., 1995) gastrulation, respectively. Other blocking agents that are more suitable to embryos or tissues grown in culture are antisense oligonucleotides (Souza et al., 1994,1995; P. Lonai, personal communication) and antibodies (Schatteman el al., 1996). The availability of specific pharmacological inhibitors of PDGF receptor activity (Buchdunger et al., t99.5) may be immensely useful both therapeutically and in further defining growth factor function in v i v a In the years to come, many questions remain to be answered concerning PDGF [unction in the embryo. For instance, what is the developmental significance, if any, of the short and long splice variants of PDGF-A? What role do PDGF-AB and PDGFR-a@ heterodimers have in the embryo? Isolation of the promoter regions of PDGF ligand and receptor genes (Takimoto et al., 1991; Jin et ul., 1994; Wang and Stiles, 1994; Afink el al., 1995;Ballagi et al., 1995)should reveal how the complex production patterns
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seen in vivo are regulated. These promoters may provide the ideal means of targeting specific inhibitors of PDGF function both temporally and spatially within the embryo. Finally, probably the greatest challenge that we face is to discover the consequences of PDGF signaling at the cellular level in different tissues. How are these signals transmitted and how are they interpreted and acted upon in an appropriate manner?
Acknowledgments We thank Christer Betsholtz, Maja Bucan, Jon Epstein, Peter Lonai, Monica Nistir, Jennie Payne, Gina Schatteman, Phillipe Soriano, Craig Tomlinson, Bernhard Wehrle-Hallcr, and Brenda Williams for communicating results prior to publication, and we thank Jennie Payne for critical reading of the manuscript. Work in our laboratory is supported by NIH Grant HD28460 to M. M., American Heart Association Grant 94014850, March of Dimes Grant FY9S-0883, and The Council for Tobacco Research Grant 4211. P. A. was funded by a BBSRCiNATO postdoctoral fellowship.
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Adaptive Crystal Formation in Normal and Pathological Calcifications in Synthetic Calcium Phosphate and Related Biomaterials G. Daculsi,* J.-M. Bouler,* and R. Z. LeGerost *Centre de Recherche Interdisciplinaire sur les Tissus Calcifiis et les BiomatCriaux, Faculte dc Chirurgie Dentaire, 44042 Nantes Cedex 01, France; and ?New York University College of Dentistry, New York, New York 10010
Mineralization and crystal deposition are natural phenomena widely distributed in biological systems from protozoa to mammals. In mammals, normal and pathological calcifications are observed in bones, teeth, and soft tissues or cartilage. We review studies on the adaptive apatite crystal formation in enamel compared with those in other calcified tissues (e.g., dentin, bone, and fish enameloids) and in pathological calcifications, demonstrating the adaptation of these crystals (in terms of crystallinity and orientation) to specific tissues that vary in functions or vary in normal or diseased conditions. The roles of minor elements, such as carbonate, magnesium, fluoride, hydrogen phosphate, pyrophosphate,and strontium ions, on the formation and transformation of biologically relevant calcium phosphates are summarized. Another adaptative process of crystals in biology concerns the recent development of calcium phosphate ceramics and other related biomaterials for bone graft. Bone graft materials are available as alternatives to autogeneous bone for repair, substitution, or augmentation. This paper discusses the adaptive crystal formation in mineralized tissues induced by calcium phosphate and related bone graft biomaterials during bone repair. KEY WORDS: Enamel, Dentine, Bone, Calcified tissue, Apatite, Biomaterials, Calcium phosphates, Bone graft substitutes.
1. Introduction Mineralization and crystal deposition are natural phenomena widely distributed in biological systems from protozona to mammals. In mammals, normal lnrrrnarional Rcview vf Cyrology, Vol. 172
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and pathological calcifications are observed in bone, teeth, and soft tissues or cartilage. In other species, normal calcifications are observed in fish teeth, calcificd fish scales, and shells. In normal calcifications,crystals deposited are mainly apatites, first ideal(Beevers and McIntyre, izcd as calcium hydroxyapatite [Cal,,(P04)6(OH)2] 1946; DeJong, 1926). The biological apatites have been identified as nonstoichiornetric carbonate-substituted calcium hydroxyapatite (CHA) or carbonate-containing calcium fluoroapatite (CFA) (Aoba and Morcno, 1990; Daculsi, 1979; Daculsi and Kcrebel, 1980; Daculsi et ul., 1984; Elliott, 1974, 1994; Elliott et al., 1985; Glas, 1962; Kerebel and Daculsi, 1975, 1976; Lee and LeGeros, 1981;LeGeros, 1967,3981a, 1983a, 1991a, 1994;LeGeros and Suga, 1980; LeGeros et ul., 198S, 1995a, 1996; Lowenstam et al., 1992; Miake et al., 1991; Posner, 1985; Ripa et al., 1972; Rey et ul., 1991). In pathological calcifications (e.g., dental and urinary calculus and soft tissue calcifications) and discased states (e.g., dental caries, fluorosis, amelogencsis imperfecta, etc.) other calcium phosphate phases, e.g., amorphous calcium phosphate (ACP) dicalcium phosphate dihydrate (DCPD, CaHP04-2H20) octacalcium phosphate (OCP, Ca8H2(P04)6*5H2gO], magnesium-substituted tricalcium phosphate [P-TCMP, (Ca,Mg)3(P04)2], calcium pyrophosphatc dihydrate (CPPD, Ca2PZO7.2H20), and other inorganic crystals (e.g., calcium oxalates, calcium carbonate, and magnesium phosphatcs) and organic crystals (urates and uric acid) are observed with and without the simultaneous presence of biological apatite (Ali and Griffith, 1983; Backman et al., 1993; Bigi et al., 1980; Daculsi el al., 1979, 1987, 1992b;Jensen and Rowles, 1957;LeBouffant et al., 1973; Lee and LeGeros, 1981; LeGeros, 1974a, 1990, 1991a; LeGeros and LeGeros, 1984; LeGeros and Morales, 1973; LeCeros and Shannon, 1979; LeGeros et al., 1973,1975, 1988a, 1989a; Newesely, 1965; Sakac et al., 1981; Schroeder, 1969; Sutor and Schcidt, 1968; Vahl et al., 1964). In calcified tissue of invertebrates, the principal crystal is calcium carbonate (CaC03)in the structural form of calcite, aragonite, or, rarely, vaterite. However, in some species of shells (atremate brachiopods) apatites, CHA or CFA, instead of CaC03, constitute the mineral phase (LeGeros, 1994; LeGeros et al., 1980, 1985, 1995a; Lowenstam et al., 1992). The greatest number of studies have been made on enamel apatite crystals and their involvement in demineralization/remineralization (as in caries) and in biomincralization (Aoba and Moreno, 1990; Arends and Davidson, 1975; Arends et al., 1982; Bres et al., 1986; C . E. Brown et af., 1987; Daculsi and Kerebel, 1978;Daculsi et al., 1984,1987; Diekwiech et ul., 1995; Elliott, 1974; Elliott et al., 1985; Fearnhead, 1989; Featherstonc et al., 1979, 1981; Frank and Vocgel, 1980; Glas, 1962; Hallsworth et a1.,1972; Jensen and Moller, 1948; Johansen, 1965, 1965b; Jongbloed et al., 1975; Kambara and Norde, 1995; Kerebel et al., 1976, 1979; Lee and LeGeros, 1981; LeGeros,
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1967, 1974b, 1981a, 1983b, 1984, 1990, 1991a; LeGeros and Suga, 1980; LeGeros et al., 1967, 1971, 1978, 1983a; Menanteau et al., 1987; Michel et al., 1995; Nanci and Smith, 1992; Nelson and Williamson, 1984; Nylen, 1964; Nylen et al., 1963; Orams et al., 1976; Palamara et al., 1980; Robinson et al., 1992; Scott et al., 1974; Selvig and Halse, 1972; Silverstone, 1985; Siew et al., 1992; Simmelink and Nygaard, 1979, 1982; Simmer and Fincham, 1995; Spencer et al., 1989; Soremark and Gron, 1966; Terpstra and Driessens, 1986; Tohda et al., 1987; Tsuda and Arends, 1994; Voegel and Frank, 1974,1975; Voegel et al., 1981; Warshawsky, 1987; Warshawsky and Nanci, 1982; Weatherell et al., 1974; White et al., 1989; Woltgens et al., 1981; Young and Elliott, 1966). The enamel apatite crystals served as a model for other types of calcification such as those in dentin, cementum, and bone. However, the apatite crystals in enamel, compared to those in dentin, cementum, or bone or those in pathological calcifications, differ in size and shape (Table I). The enamel apatite crystals are much larger as evidenced by the higher crystallinity (reflecting greater crystal size and/or perfection) demonstrated in their X-ray diffraction (XRD) patterns. The XRD patterns of enamel apatites show narrower and sharper diffraction peaks compared to those in the XRD patterns of dentin or bone (Fig. 1).Morphologically, the enamel apatites appear as acicular crystals, whereas dentin and bone are much smaller and appear as plate-like crystals. In terms of composition (Table II), apatite crystals in enamel compared to those in either bone or dentin contain considerably lower concentrations of the two minor but important elements associated with biological apatites-carbonate (Fig. 2) and magnesium (LeGeros 1967, 1974a, 1974b, 1981a, 1984, 1990, 1991b, 1994; LeGeros et ul., 1995a)-and are significantly different in crystal properties from that of pure hydroxyapatite (HA) (Daculsi et al., 1976, 1989; LeGeros, 1981a, 1983a, 1991b, 1994; Young and Elliott, 1966).
TABLE I Crystallographic Properties of Adult Human Enamel, Dentin, and Bone"
Lattice parameters (?0.0003 nm) u
axis
Enamel 0.9441 nm
Dentin 0.9421 nm
Bone 0.941 nm
c axis
0.6880 nm
0.6887 nm
0.689 nm
Cristallinity indexh
70-75
33-37
33-37
Crystallite size (av. nm)
0.13 X 0.03 nm
0.02 X 0.004 nm
0.025
X
0.003 nm
'' From LeGeros (1981a) Cristallinity index is determined from the ratio of coherent to incoherent scattering ratio. In mineral OH-apatite (Holly Springs) crystallinity index is 100.
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ET AL.
20
22
I
I
I
I
I
I
I
I
I
24
26
28
30
32
34
36
38
40
Diffraction Angle (two-theta)
FIG. 1
X-ray diffraction pattcrns of the mineral phases of human enamel (a), dcntin (b), and
bonc ( c ) . All patterns indicate an apatite structure with enamel having much larger crystallites than either dentin o r hone apatitcs (LeGeros 1967, 1981a, 1991a).
This paper is a brief review of studies on the adaptive apatite crystal formation in enamcl compared with those in other calcified tissues (e.g., dentin, bone, and fish enameloids) and in pathological calcifications, demonstrating the adaptation of these crystals (in terms of crystallinity and orientation) to specific tissues that vary in functions or vary in normal or diseased conditions. The roles of minor elements, such as carbonate (CO,), magnesium (Mg), Auoridc (F), hydrogen phosphate (HP04), pyrophosphate (P207), and strontium (Sr) ions, on the formation and transformation of biologically relevant calcium phosphates are summarized. The use of a combination of analytical techniques allows the elucidation of the properties of biological crystals and the interaction between thc crystal (mineral phase) and the organic phase. The roles of specific proteins in the nucleation, maturation, and orientation of biological apatite crystals have been comprehensively covered in other reviews (Boskey, 1992; Bonucci, 1992; Diekwiech et al., 1995; Eastoe, 1960, 1963; Fearnhead, 1979; Fukae et al., 1993; Moradian-Oldak er ul., 1991; Robinson et al., 1979, 1992; Slavkin, 1990; Simmer and Fincham, 1995; Termine et al., 1979). The roles of nonapatitic precursor phases of biological apatites (e.g., initial formation of ACP, DCPD, or OCP and their subsequent transformation to CHA), covered in
133
ADAPTIVE CRYSTAL FORMATION TABLE I1 Composition of Apatites in Adult Human Enamel, Dentin, and Bone“ Composition
Weight % Enamel
Dentin
Bone
Calcium (Cali)
36.5
35.1
24.5
Phosphorus (P)
17.7
16.9
11.5
(Ca/P) molar ratio
1.63
1.61
1.65 0.7
Sodium ( Na’ )
0.5
0.6
Potassium (K’ )
0.08
0.05
0.03
Magnesium (Mg”)
0.44
1.23
0.55
Carbonate (CO; )
3.5
5.6
5.8
Fluoride (F )
0.01
0.06
0.02
0.01
0.10
0.10 70.0
0.07
Chloride (Cl )
0.3
Pyrophosphate (PzO,)
0.022
Ash (total inorganic)
97.0
Total organic
1.5
20.0
Adsorbed H20”
1.5
10.0
65.0 25.0 9.7
Trace elements: Sr”, Pb2‘, Fe”. Zn2’,Cu”, etc. Ignition products (SOOC) ~~~~~~~~~~~~~~~~
P-TCMP ~~~
+ HA
P-TCMP
+ HA
HA
+ CaO
~~
From LeGeros (1981a, 1991a). Ratio of coherent/incoherent scattering, with mineral HA value as 100. Weight analyses based on ashed samples except for CO?, which was determined on unashed samples using an IR method. f3-TCMP is Mg-substituted h-tricalcium phosphate, or whitlockite in biological systems [(Mg,Ca)@&)J. (I
other reviews (W. E. Brown et al., 1987; Eanes, 1979; LeGeros, 1991a; LeGeros et al., 1975; Simmer and Fincham, 1995), are not included in this paper. Another adaptative process of crystals in biology concerns the recent development of calcium phosphate ceramics and other related biomaterials for bone graft. Bone graft materials available as alternatives to autogeneous bone for repair, substitution, or augmentation include metals, resorbable and nonresorbable polymers, inert ceramics (e.g., alumina and zirconia), special glass ceramics described as bioactive glasses; calcium phosphates (calcium hydroxyapatite and HA), tricalcium phosphate (TCP), biphasic calcium phosphate (BCP), calcium carbonate (coral), and materials derived from natural sources (e.g., bovine bone or corals). These materials differ in composition and physical properties from each other and from bone (Aoki, 1991; Bonfield, 1988; De Groot, 1983; Hench, 1994; Hench and Wilson, 1984; Jarcho, 1981, 1992; LeGeros, 1983a, 1988, 1992; LeGeros et al., 1995b,c; Rey et al., 1991).
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G. DACULSI E T A .
FREQUENCY
FIG.2 Infrarcd spectra of the mineral phases of human enanici (a), dentln (h). and hone (c) showing the much lower COz intensity (and therefore concentration) in enamel compared to that in dentin or bone apatite (LeGeros 1967, 1981a. 1991a).
Based on their ability to induce bone formation, these matcrials are described as either osteoinductive [i.e., have osteogenic properties (Reddi, 198S)] or ostcoconductive (i.e., does not have osteogenic properties but serve as an efficient scaffold for the formation of new bone). Based on the nature of the interface formed by these biomaterials with bone, these materials are characterized (Osborn and Neweseley, 1980) as biotolerant, bioinert, or bioactivc (Table 11).Biotolerant materials include metals, bioinert materials include alumina or zirconia ceramics and perhaps titanium and titanium alloy, and bioactive biomaterials include calcium phosphate and bioactive glass ceramics. This paper discusses the adaptive crystal formation in mineralized tissues induced by calcium phosphate and related bone graft biomaterials during bone repair. Topics include (i) description of these materials in terms oE their physicochemical and crystal properties and their similarities and differences with thc bone mineral, (ii) characterization of the bone/material interface and the events occurring in the development of this dynamic interface such as cellular response, (iii) biodegradation or bioresorption of
ADAPTIVE CRYSTAL FORMATION
135
the materials and their transformation to C H A similar to biological apatites (specifically bone apatite), and (iv) the osteocoalescence process contributing to the formation of strong bone/material interface unique to these materials.
II. Analytical Techniques A wide range of analytical techniques are used in investigating crystal formation in calcified tissues and the physicochemical properties of these crystals. Each of these techniques provide specific information. However, each of these techniques also has limitations. Therefore, it is necessary to use a combination of analytical techniques to obtain more comprehensive and accurate information.
A. Determination of Mineral/Organic Distribution and Interaction. An X-ray microradiography is a picture on a photographic emulsion of a thin undecalcified section of calcified tissue taken with low-energy X ray. Such micrographs give information on the qualitative relationship between calcified and soft tissue and an assessment of the topographic distribution of the mineral (Boivin and Baud, 1984; Silverstone, 1985; White et af.,1989). B. Removal of Organic Phase To investigate the properties of only the mineral phases of calcified tissues, it is necessary to separate or remove the organic phase using different treatments. Treatment methods that have no significant effect on the crystal properties of the mineral phase include treatment with hydrazine (Termine ef al., 1973) or ethylenediamine (LeGeros, 1967,1991a) and sodium hypochlorite or low-temperature ashing (Daculsi et af., 1992b). Other methods, such as treatment with ethylene glycol or KOH and ashing at temperatures above 400"C, cause an increase in the crystallinity of the mineral phase (apatite) or transformation of other calcium phosphate phases (LeGeros, 1967; 1991a). C. Identification of Crystal Phases
1. X-Ray Diffraction Analyses XRD analyses are generally performed using Ni-filtered Cu radiation generated at 40-50 kV and 20-30 mA, with silicon or KCI as internal standards.
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G DACULSI ETAL.
A Debye-Scherrer or Guinier camera is used when only small amounts of samples are available. For larger amounts of samples, a goniometer stage may be used. XRD analyses provide accurate identification of phases present. In addition, XRD analyses provide information on the effects of substituents [e.g., F or CI for OH; CO?, HPO4, or SO4 for PO4 ; or Sr, Mg, and other cations for Ca in apatites (Baud and Very, 1975;Bonel, 1972; Elliott, 1974; LeGeros, 1967,1974a, 1981a,1983a, 1984,1991b; LeGeros and Suga, 1980; LeCeros et al., 1967,1978,1980;Montel et al., 1981); and Mg for Ca in 0-tricalcium phosphate (P-TCP (Ca,Mg)3(P04)2),also known as whitlockite (LeGeros, 1967, 1974b; Rowles, 1968; Jensen and Rowles, 1957)]. XRD analysis is also useful in determining factors affecting the crystallinity (reflecting crystal size and/or strain) of apatite (LeGeros, 1967, 1981~1,1983a, lY84, 1991b; LeGeros et al., 1968, 1995a), approximating the crystal shape (e.g., acicular, rod-like, or equiaxed) (LeGeros et al., 1967, 1971), and in discriminating between the effects on crystal size andlor strain (Klug and Alexander, 1Y74). 2. Infrared Absorption Analyses
fnfrared ( I R ) absorption analyses are usually performed on pellets prepared by mixing about 1 mg sample and 300 mg KBr (IR grade) pressed using 12,000 psi. Assignments of absorption bands of functional groups are based on earlier studies on apatites and related calcium phosphates (Berry and Badiel, 1967; Bonel and Montel, 1964; Dykes and Elliott, 1971; Elliott, 1974, 1994; Elliott er a[., 1985; Fowler et al., 1966, 1974; LeGeros, 1967, 1974a, 1991a; LeGeros et al., 1967, 1970,1971, 1975). The environment of the functional groups can be positively identified [e.g., OH from HA or OH from adsorbcd H20 or H20 of crystallization; CO? from CHA and CFA or CO3 from simple carbonate compounds (e.g., CaCO?); H P 0 4 in calcium-deficient apatites (CDA) or H P 0 4 in other calcium phosphate phases (e.g., DCPD and OCP) and other nonapatitic calcium phosphate phases; and PO4 in HA or in PO4 in P-TCP]. Such IR analyses allowed the distinction between C 0 3 from simple carbonates (e.g., CaC03 and NaHCO?) and between two types of C 0 3 substitution in the apatite: type A, COr for OH in carbonate apatite [CA, Ca10(P04)6(C03)](Bonel, 1972; Bond and Montel, 1964; Elliott, 1974; Elliott et LEI., 1985); and type B, C 0 3 for PO, coupled with Na for Ca C 0 3in CHA [(Ca,Na)lu(P04,C03)2(OH)2] (LeGeros, 1967; LeGeros et al., 1969, 1970, 1971). IR methods have also been used for the quantitative determination of C03, HP04, and OH in biological and synthetic CHA (Arends and Davidson, 1975; Dykes and Elliott, 1971; Elliott ef al., 1985; LeGeros, 1967; LeGeros and LeGeros, 1993; Featherstone et al., 1984) and the qualitative detection of nonapatitic component (Rey et al., 1991). The resolution of the absorption bands can
ADAPTIVE CRYSTAL FORMATION
137
give an indication of the crystallinity of the apatite: The greater the resolution of the absorption bands, the higher the crystallinity. Synthetic and biological ACPs are characterized by the absence of diffraction peaks in their XRD patterns and the loss of resolution in the IR absorption bands (LeGeros, 1967; LeGeros et al., 1975).
3. Raman Spectroscopy Raman spectroscopy (Nelson and Williamson, 1984; De Mu1 et al., 1986; Leach, 1990; Sauer et af., 1994; Sowa and Mantsch, 1994; Walters ei al., 1990) on large amounts of powder samples is achieved using an Argon laser, which provides a high spatial resolution when focused on a small area (
4. Nuclear Magnetic Resonance Nuclear magnetic resonance ( N M R ) analysis is noninvasive and does not use any ionizing radiation. It can give an absolute quantification of OH and absorbed H20. The use of a less common atom gives more precise information (Roufosse et al., 1984). For example, Yesinowski and Molley (1983) and White et al. (1989) demonstrated the ability of 19FNMR, coupled with MAS, to differentiate the F i n fluorapatite [FA, CaIO(P04)6F2], fluorohydroxyapatite [Cal,~(P04)6(OH,F)2], and calcium fluoride (CaF2). 31P NMR of solid calcium phosphate offers a possibility of detecting the different phosphate compounds and studying their structure. Rothwell et al,, (1 980) used precise Hartmann-Hahn sequencing, coupled with MAS and proton enhancing, with a H3P04reference. Tropp et al. (1983) characterized the ACPs by their spinning sideband patterns, comparing with and without polarization.
D. Determination of Crystal Morphology (Size and Shape] 1. Scanning Electron Microscopy Samples (blocks or isolated crystals) are cemented on stubs and coated with gold palladium or carbon. Sample examinations are made under 1025 kV with a resolution of 2.5-5 nm. Scanning electron microscopy (SEM) analyses are useful in surface characterization and elucidating the features of enamel prisms and in detecting large calcium phosphate crystals (e.g., P-TCMP and OCP) in dental calculus (Daculsi et al,, 1992b; LeGeros et al., 1988c) or in other pathological calcifications (Ali and Griffith, 1983;
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G. DACULSI ET AL.
LeGeros and Morales, 1973) but are not useful in characterizing the morphology of the much smaller biological apatite microcrystals of enamel and especially of dentin or bone. For the investigation of the crystal properties of biological apatites: transmission electron microscopy (TEM) is the more appropriate technique.
2. Transmission Electron Microscopy Thc following necessary steps in sample preparation for TEM analysis were recommended by Meek (1976): 1. Fixation: Glutaraldehyde, paraformaldehyde or osmium tetraoxyde are used alone or in a double fixation process: 2. Dehydration, infiltration, and polymerization; 3. Sectioning: A diamond knife is required. The best results arc obtained using a manual ultramicrotome (Sorvall Porter); 4. Mounting: For conventional electron microscopy, formvar-carbonated coppcr grids are used. For high-resolution electron microscopy, hole formvar-carbonated coated grids are used; 5. Section staining: Heavy metal solutions are used (lead and uranium). However, for calcifications it is necessary to use unstained sections and to compare observations with stained sections. The sections are observed in TEM under 60-100 kV for crystal shape and size. For high-resolution TEM (HR TEM) 200-300 kV is used. Because biological calcium phosphate crystals are very sensitive to electron irradiation, various devices can be used to reduce the damage. One of the devices suggested is the video camera with a low irradiation level. The advantage of this device is that it can be connected to a computer for image analysis. In the case of enamel, an original method was developed for preparing isolated enamel crystals to study the length and shape of the crystals during the growing process (Daculsi et al., 1984). Frozen powders were separated into different fractions in aqueous medium according to their respective densities. The crystals are laid down under water on Cu grids covered with carbonated formvar and examined with TEM. Combined with clcctron diffraction, TEM can provide positive identification of the crystals (Little, 1959; Nylen, 1964;Nelson etal., 1986;Landis and Glimcher, 1978). However, it cannot detect substitutions in the apatite, unless such substitutions cause changes in the crystal morphology (LeGeros, 1967,1981a, 1991a; LeGeros et al., 1967, 1971).
E. Determination of Crystal Composition Elemental Analysis from Solutions Quantitative analyses of cations (Ca, Mg, Sr, Na, K, etc) are possible using atomic absorption spectroscopy (Rubeska and Moldan, 1971). Specific ion
ADAPTIVE CRYSTAL FORMATION
139
electrodes can be used for the analysis of some ions (Ca and F). Colorimetric methods are used for calcium (Charlot, 1966) and most frequently for phosphorus (Chen et al., 1953). Flame spectroscopy and X-ray fluorescence techniques require large samples (1.5-2 g).
1. Electron Microprobe Analysis Two kinds of electron microprobe analysis (Castaing, 1975) can be used: electron dispersive spectrometry and wavelength dispersive spectrometry on bulk samples (SEM) or on ultrathin sections (TEM). Samples are carbon coated. Quantitative analyses can be performed using standards and ZAF (atomic number, absorption, and fluorescence). 2. Surface Analysis
Electron spectroscopy for chemical analysis, Auger electron spectroscopy, and secondary ions mass spectroscopy can be used (Hercules and Craig, 1976). However, these very sophisticated physical techniques are rarely used in biological analysis due to numerous technical limitations.
111. Crystal Formation, Composition, and Properties in Normal Calcifications The calcified tissues of enamel, dentin, and bone are composites of organic and inorganic phases. Enamel differs from bone and dentin in the organic/ inorganic ratios, type of the principal organic phases, and the crystallinity and composition of the mineral phases (Table 11).The mineral or inorganic phases of calcified tissues consists of nonstoichiometric CHA differing in crystallinity ,reflecting size, and perfection (Fig. 1) and in the concentration of C 0 3 and other minor elements (Fig. 2; Table 11) (LeGeros, l967,1981a, 1991b, 1994; Monte1 et al., 1981; Posner, 1985). The difference in their sizes and composition is reflected in the difference in their dissolution properties, with enamel apatite (more crystalline and containing less carbonate and magnesium) being least soluble (LeGeros et al., 199%; Moreno et al., 1977).
A. Enamel Early studies (Daculsi and Kerebel, 1978; Kerebel et al., 1976,1979) demonstrated that enamel mineralization (Figs. 3a-3c) is not due to an increase in the number but rather in the size of enamel crystals. In a study of
FIG. 3 Embryonic human enamel. TEM section showing the younger crystals formed near the Tomes processes (a), the older crystals in the core of the forming enamel (b), and the first crystals formed at the initial stcp of the amelogenesis (c). 'I'he size of the crystal increases during the maturation, and the number of crystals decreases. FIG. 4 Embryonic human enamel. TEM of the first crystal observed in the enamel near the Tomes processes is one unit cell in thickness (a). Crystal grows by increasing in thickness by
ADAPTIVE CRYSTAL FORMATION
141
embryonic enamel (Daculsi and Kerebel, 1978) crystals growth was shown to be a logarithmic increase in width with respect to thickness (Figs. 4a-4c). Because the enamel volume does not increase during crystal thickening in the course of enamel mineralization, two possibilities may be considered: Several crystals disappear and/or fuse together during the mineralization process. The fusion first occurs in depth, and later, in the course of the maturation process, when crystals get thicker, the fusion extends to the surface. At their mature stage, the crystals are no longer pyramidal but assume a regular rod-like shape. H R TEM allowed the determination of the section plane of a crystal and provided accurate measurements of both width and thickness of human enamel crystals to within a few nanometers (Daculsi et al., 1979) as shown in Fig. 5. However, this method did not permit accurate length determination. Estimations of the length of human enamel crystals were reported as 150 nm (Nylen, 1964,1979; Selvig and Halse, 1972) and 160 nm (Ronholm, 1962). To obtain accurate length measurements, it was necessary to develop a method of preparing isolated crystallites (Daculsi et al., 1984). Although the grinding process induced breaking of the longest crystals, length values beyond 100 F ) were obtained (Fig. 6). Considering the limits of the observation field in TEM, it is most likely that the real size of these crystals is larger, in good agreement with the hypothesis developed by Warshawsky and Nanci (1982) based on stereological models. Length must be considered as infinite with respect to section thickness. Thus, even if crystallites have infinite true ends, these would probably never be recognized in TEM sections. The enamel apatite crystals could be continuous from the dentinoenamel junction to surface enamel; however, this does not explain the observed irregular course of the enamel prisms. Such phenomenon could be demonstrated by the long and flexuous crystal ribbons classically described within the early mineral deposits during enamel formation (Fig. 6). Crystal defects (dislocations) inducing crystal curvatures have been previously observed with H R TEM (Daculsi and Kerebel, 1978; Daculsi et al., 1991) and the incidence of these defects on the curvature radius has been calculated
apposition of unit cell (b and c) until it reaches the final crystal size for one tissue and one species. FIG. 5 Human enamel crystal observed in HR TEM. The 001 section shows the three equivalent lattice fringes (100) with an angle of 120" between them. The identification of the lattice plane provides information on the real shape and size of the crystals. FIG. 6 Isolated human enamel crystals observed in TEM showing long and flexuous ribbon.
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(Voegel et al., 1981). T o our knowledge the smallest value observed for crystal curvature (0.1 p ) represents the maximal strain that can be supported by the crystals before splitting occurs. The mature human crystal are flattened hexagons of 68 nm in width and 26 nm in thickness (Nylen et al., 1963). The orientation of the crystals within the prisms seems related to the presence of specific crystal proteins (Bonnucci, 1992; Daculsi and Kerebel, 1978; Daculsi et al., 1984; Fincham et al., 1981; Hohling et al., 1982; Simmer and Fincham, 1995; Warshawsky, 1987). The size and shape of the crystals may be largely attributed to such interactions, although the concentration of some important ions (e.g., Coltand Mg) has been shown to affect the crystal size and shape of synthetic apatites (LeGeros, 1967, 1981a, 1984,1991b; LeGeros et al., 1967,1968,1971). We have demonstrated that the sheath at the surface of the crystals is not an artifact but results from specific proteidcrystal interactions. This sheath, observed in highresolution TEM (Fig. 4), is only a part of a protein complex transformed by fixation techniques and electronic bombardment. It is likely that the crystal surface is coated with a product of interactions betwee; proteins and inorganic clcments at the surface of the growing apatite. This structure, which constitutes the crystal surface, is part of the crystal. After decalcification, the surface layer of the crystal constitutes the “crystal ghost.” The observed sheath is the consequence of a highly significant protein/crystal interactions. lnvestigations on the exact nature and properties of this sheath continue. However, the molecular size of the species isolated from the youngest crystals suggests that the cnamelins play an important role (Fincham et d., 1981; Slavkin, 1990; Simmer and Fincham, 1995). A protective role of thc organic phase intimately associated with biological apatite crystals against susceptibility to acid dissolution has also been suggested (LeGcros et al., 1995a). In carious or sound enamel, the concentrations of C03, Mg, and F in enamel vary from the surface to the dentin-enamel junction (Hallsworth et d.,1Y72; LeGeros et al., 1996; Weatherell et al., 1983). A recent study showed that the observed compositional variation is reflected in the crystallinity and the dissolution properties: Higher Mg and CO1 concentrations are related to lower crystallinity (implying smaller and/or more strained or less perfect apatite crystals) and a higher extent of dissolution in acidic buffer of apatite in enamel regions closer to the enamel/dentin junction compared to those closer to the enamel surfacc (LeGeros et ul., 1996).
6. Dentin The studies on early studies on dentin are much more limited compared to those on enamel (Arsenault, 1989; Arsenault and Robinson, 1989; Bradford,
ADAPTIVE CRYSTAL FORMATION
143
1960; Jensen and Moller, 1948; Johansen, 1965; LeGeros, 1967; LeGeros et al., 1973; Lester and Boyde, 1968; Newesely, 1965; Takuma et al., 1975). The apatite crystals in dentin are similar to those in enamel and bone with respect to chemical and crystallographic structure. However, the apatite crystals of dentin or bone are much smaller than those of enamel as shown in their XRD patterns (Fig. 1) and differ in the amount and type of organic matrices and in the concentration of minor elements such as carbonate (Fig. 2) and magnesium (LeGeros, 1967, 1981a, 1983b, 1984, 1991a; LeGeros et al., 1995a). Higher concentrations of the organic phase [20% organic content essentially consisting of type 1 collagen and higher concentrations of magnesium and carbonate in the mineral phase (LeGeros 1981a, 1991a)l may contribute to the growth of smaller dentin apatite crystals distributed along the collagen fibers (Fig. 7) compared to the much larger crystals of enamel. The mineralization process described for dentin was different from that of enamel, consisting of a linear mineralization, a globular process, and a combination of the two. HR TEM analysis of the dentin crystals was difficult to obtain with a high resolution due to the small size of the crystals and their interaction with organic matrix (Figs. 8a and 8b). The size and shape of the crystals obtained by our methods of lattice plane identification (Daculsi et al., 1978) was more precise than the indirect method using X-ray diffraction (Jensen and Moller, 1948) and revealed the parrallepiped shape. The 001 cross section showed a needle-like crystal and a 100-section rectangle. The size of the sound dentin crystal was 3 nm in width, 20 nm in thickness, and 20 nm in length (Daculsi, 1979). Compared to the enamel apatite crystal consisting of 30-40 units of cell thickness, the dentine apatite crystal consisting of 3-5 unit cells has a very large surface area, amounting 10-200 m2/g, thus exposing an enormous area for chemical reactivity involving substitutions, exchanges, and absorption.
C. Bone Bone crystals are very similar to dentin crystals. X-ray diffraction analyses [DeJong, 1926; LeGeros, 1967,1981; Posner, 1985) and HR TEM demonstrated the apatitic nature of the mineral phase (Glimcher, 1984; Handschin and Stern, 1992). IR analyses identify bone apatite as CHA (LeGeros, 1967, 1981a; Rey ef al., 1991). The bone apatite crystals are rectangular and platelet shaped, distributed along the collagen fibers (Figs. 9a and 9b). The width is 2-4 nm, the thickness 5-10 nm, and the length 40-50 nm (Robinson and Watson, 1952; Steve Bocciarelli, 1970; Landis and Glimcher, 1978; Moradian-Oldak ef al., 1991; Weiner and Price, 1986). Using darkfield
FIG. 7 The dentin scction observed in TEM revealed smaller dentin apatite crystals distributed along the collagcn fibers. FIG. 8 The HR 'I'EM or the dcnlin crystal shows thickness of only 3-S unit cells (a and b).
FIG. 9 kluman bone crystals are reclangular and platelet shaped distributed along thc collagen liher (a and b).
ADAPTIVE CRYSTAL FORMATION
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electron microscopy, Arsenault (1989) considered that the length of the crystals was 17 nm longer or 5 nm shorter than the dentin apatite crystals (11 t 3 nm). Changes in bone crystal composition with maturation have been reported (LeGeros, 1991b; Rey et ul., 1991).
D.Fish Enameloids Selachian enameloid can be compared to enamel of mammal (Garant, 1970; Glas, 1962; Kerebel and Daculsi, 1975; Lee and LeGeros, 1981; LeGeros et ul., 1983b;Miake eta]., 1991). The inorganic or mineral phase is essentially composed of FA (Daculsi and Kerebel, 1980; LeGeros, 1991a; LeGeros and Suga, 1980; LeGeros et af., 1983b). The observation that the high concentrations of fluoride F in the enameloids of some species of fish and not in others regardless of the F concentrations in their environment (Kerebel and Daculsi, 1975; LeGeros and Suga, 1980; Suga et al., 1978) led to the speculation that an F-concentrating mechanism must be operating during the formation of the FA in the enameloid (LeGeros and Suga, 1980). The observed lower caries progression in shark enameloid compared to human enamel is related to the lower solubility of the shark enameloid FA compared to human enamel CHA, which is in turn attributed to the higher F and low C03F concentrations in shark enameloid apatite (LeGeros et a/., 1983b). The crystals of shark enameloid are a more regular hexagonal shape compared to human enamel as shown in Figs. 10a and 10b (Daculsi and Kerebel, 1980; Lee and LeGeros, 1981; LeGeros et af., 1983b). The (width to thickness) (W/T) ratio in enameloid apatite crystals was about 1.1 compared to 2 or 3 for human enamel. The crystal size, determined by HR TEM, was 52 nm in width and 47 nm in thickness (Daculsi et al., 1979; Kerebel and Daculsi, 1975).However, the abundance of lattice defects in shark enameloid apatite (which is CFA) is not significantly different from that of human enamel (which is CHA) (Lee and LeGeros, 1981).
IV. Crystal Formation, Composition, and Properties in Pathological Calcifications In contrast to the mineral phases of normal calcifications (enamel, dentin, cementum, and bone) that consist principally of “apatitic” calcium phosphate or CHA (LeGeros, 1967, 1981a, 1991b; LeGeros et af., 1970, 1971; Posner, 1985; Rey et af., 1991), the mineral phases of pathological calcifications usually consist of other types of calcium phosphates (e.g., ACP,
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FIG. 10 Selachian enameloid is made up of fluorapatite crystals. These crystals are shown with more perfect hexagonal profile (a) and larger crystal size (b) compared to human enamel apatite crystals. FIG. 11 TEM shows the articular and periarticular calcification appcaring as a glohular structure of various diameters (4-7 pin) within a stroma of scattered crystallites.
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DCPD, OCP, and P-TCMP) and/or other phosphatic and nonphosphatic compounds (e.g., magnesium phosphates, oxalates, etc) in addition to or in place of the CHA (Gatter and McCarthy, 1967; Daculsi et al., 1992a; LeGeros, 1974a, 1991a;LeGeros and LeGeros, 1984;LeGeros and Morales, 1973; LeGeros and Shannon, 1979; LeGeros et at., 1973, 1988a, 1988d; Rowles, 1968; Schroeder, 1969; Sutor and Scheidt, 1968).
A. Soft-Tissue Calcifications The articular and periarticular calcifications showed globular structure of various diameters (4-7 um) within a stroma of scattered crystallites (Daculsi et al., 1992b). The crystal density was about 97% of the surface within the globules and 35% between them. At higher magnifications, the globules appeared as a network of numerous crystallites within a dense matrix of underlined structure. Variations in the ratio of globules/isolated crystals could be different; the structure could also be different, sometimes with concentric lamellar organization (Fig. 11). However, common feature were observed: (i) Different crystal sizes existed between intraglobular and interglobular crystals, and (ii) the different crystal size precipitations were independent of collagen fibers. Identification of the section plane in HR TEM allowed measurement of the thickness and width (37 t 11 and 90 t 28nm, respectively). Their approximate length was estimated to be 320 nm (Daculsi et d., 1983, 1992b). X-ray diffraction analyses identified the mineral phases in these calcification to be apatitic (Paegle, 1966, LeGeros et al., 1973). However, investigations on tendon calcifications(Sael-Clavere et al., 1980) suggested that this material might be a nonstoichiometric carbonated calcium-deficient apatitic solid phase. Bigi et al., (1980), studying human subcutaneous ectopic calcifications using XRD and IR spectroscopy, demonstrated the presence of two different types of calcium phosphates: The main crystalline phase was CHA, with small amounts of P-TCMP (i.e., Mg-substituted P-TCP). Pathological calcifications associated with uremia showed the presence of ACP (containing Mg and pyrophosphate but no C03) and CHA (LeGeros et al., 1973).
B. Dental and Urinary Calculus Several calcium phosphate phases (DCPD, OCP, P-TCMP, and CHA) coexist in human dental calculus, whereas principally calcium carbonate (calcite form) mixed with small amounts of CHA is observed in animal calculus (LeGeros, 1974b; LeGeros and Shannon, 1979; LeGeros et al., 1988c; Schroeder, 1969). The difference in composition between human
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and animal dcntal calculi reflects the difference in the average pH and composition between human and dog saliva (for example, pH 7.4 in human and pH 8.5 in dog; much higher C03/P04 or HCO3/PO4 molar ratios in dog saliva compared to those in human) (LeGeros and Shannon, 1979). The presence of various calcium phosphate phases in human dental calculus may be attributed to the changing pH and composition o f the oral environment because each of these calcium phosphate phases forms and transforms from one type to another under different conditions of pH and composition (Daculsi et ~ l . ,1987; Johnsson and Nancollas, 1992; LeGeros, 1967, 1974a, 1984; 1991a; LeCeros et al., 1973, 1975, 1983, 19894. The common crystals found in urinary calculus are calcium oxalales, magnesium ammonium phosphate, and magncsium phosphates. CHAs are sometimes found in the nidus of some types of urinary calculus (LeGeros and Morales, 1973; Sutor and Scheidt, 1968).
C. Diseased Conditions 1. Enamel Caries
Dissolution of biological apatites occurs during natural processes such as dental caries (Daculsi and Kerebel, 1977; Frank and Voegel, 1980; Voegel and Frank, 1974, 1975) and bone remodeling (Posner, 1985). Dissolution of human enamel apatites occurs preferentially at the crystal core (Daculsi and Kerebel, 1977; Kerebcl et al., 1976; Little, 1959; Nylen, 1964; Frazier, 1968; Jongbloed et al., 1975; Lee and LeGeros, 1985; Scott et al., 1974; Tohda et a!., 1987; Voegel and Frank, 1974,1975). Dissolution of biological apatite (cnamel or enameloid) was observed to consistently extend along the crystal c axis according to the largest lattice plane (100). The initial sites of dissolution were generally the top and bottom of the crystal along their c axis (Fig. 12a), although crystal dissolution starting from the latcral surface has also been observed. The dissolution features of biological apatite in vitro were similar to those in vivo (e.g., dental caries), i.e., characteristic preferential dissolution of the crystal cores (Fig. 12b). The dissolution was shown to progress along linear defects (edge dislocations), as shown by the distortion of the latticc planes (Fig. 12c).The preferential dissolution of the crystal core of biological apatites is well documented (Daculsi and Kerebel, 1977; Daculsi et al., 1989a,b; Lee and LeGeros, 1985; Scott et al., 1974; Vocgel and Frank, 1975;Tohda et al., 1987). The theoretical considerations (Welch, 1968; Arends and Jongbloed, 1977; Jongbloed et al., 1975; Lovell, 1958) of the role of dislocations in crystal dissolution have been corroborated by SEM observations on synthetic apatites (Arends and Jongbloed,
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1977; Jongbloed et al., 1975) and by H R TEM observations on biological (Daculsi and Kerebel, 1977; Voegel and Frank, 1974, 1975) and synthetic apatites (Nelson et al., 1986; Bres et al., 1986). Dislocations are crystal lattice defects, and it has been established that acid dissolution starts or occurs at dislocation sites (Lovell, 1958; Lee and LeGeros, 1985). Crystal populations of different sizes were observed in the different zones of enamel caries, with the largest crystals being observed at the surface (Silverstone, 1985). Changes in the chemistry of the enamel apatite are also involved in caries progression and remineralization (Featherstone et al., 1979; Ingram and Silverstone, 1981; LeGeros, 1990). It was suggested that dissolution of C03, Mg-rich, F-poor enamel apatite and precipitation of C 0 3 , Mg-poor, F-rich apatite may occur, making the remineralized enamel less susceptible to acid dissolution (LeGeros, 1990). 2. Dentin Caries
Ultrastructural study of dentin demonstrated that the dentinal tubules in the area between the sound and carious zones were obstructed with large crystals (Bradford, 1960; Lester and Boyde, 1968; Hawkinson and Eisenmann, 1983). The intertubular dentin of this area also appeared to have greater mineral density. Ultrastructural studies and electron diffraction demonstrated the whitlockite nature (i.e., P-TCMP) of these large rectangular crystals, which completely occluded the tubule lumen (Daculsi et ai., 1979,1987; Frank and Voegel, 1980; Neweseley, 1965; Takuma et al., 1975; Vahl et al., 1964) as shown in Fig. 13a. However, it has been shown that pure P-TCP does not form in aqueous solution and that all biological whitlockites are Mg substituted [Ca,Mg)3(P04)2] (LeGeros, 1967, 1974; LeGeros and LeGeros, 1984; Jensen and Rowles, 1957; Rowles, 1968); thus, these large crystals associated with arrested dentin caries were identified as P-TCMP (Daculsi et al., 1987). The region of carious dentin that has undergone a dissolution process revealed a fine granular more or less amorphous calcium phosphate inside the tubule, a highly mineralized peritubular dentine, and an intertubular dentine composed of larger crystal than sound dentin (Fig.13b). The region that has been partially demineralized by the caries process contained smaller number of crystals but the crystals were larger than those observed in the sound dentin region (Fig. 13c). An early study using H R TEM on arrested dentin caries led to the suggestion that different events occur during this process (Daculsi et al., 1987). Four events, not necessarily in sequence, were described as follows: (i) occlusion of tubules by fine, granular, amorphous materials; (ii) narrowing of the tubule lumen concomitant with a thickening of peritubular dentin; (iii) large crystals of whitlockite completely occluding the tubule lumen;
FIG. 12 TEM of shark enameloid apatite (exposed to acid in uitro) showing inilial dissolution (arrow) from thc top and the hottoni of crystals (a). The dissolution features of biological apatite revealed ch;iracteristic preferential dissolution of the crystal cores both in vitro and in uivo like in human enamel caries (h). The dissolution was shown to progress along linear dclccts (edge dislocations), demonstrated by the distortion of the lattice planes (c).
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and (iv) association of large intratubular crystals with thinner needle-shaped crystals in the lumen. A possible chronological sequence of physicochemical events occurring at the crystal level during the progress and arrest of caries in human dentin can be described as follows: (i) Dissolution of the dentin mineral (a C03and Mg-rich apatite) caused by the acid produced by bacteria increases the Mg/Ca ratio in this environment; (2) this condition (i.e., high Mg/Ca) in solution could promote the precipitation of ACP containing high levels of Mg, ACP-Mg, or of tricalcium phosphate containing high levels of Mg or P-TCMP (LeGeros, l967,1981a, 1991b;LeGeros et aL, 1973); (iii) transformation of the initially formed ACP-Mg to DCPD or P-TCMP under acid conditions (LeGeros, 1991); (iv) DCPD could initially form under acid conditions and then transform to P-TCMP and/or CHA which is C 0 3 and Mg poor compared to the original dentin apatite (LeGeros et al., 1983b); and (v) direct formation of P-TCMP and CHA under basic conditions and increasing HC03/P04ratio in the microenvironment (Daculsi et al., 1986; LeGeros, 1990,1991b).This sequence of events could explain the difference in size of CHA and the presence of P-TCMP in the dentin tubules in arrested dentin caries. The higher Mg/Ca ratio in dentin compared to that in enamel could allow the formation of P-TCMP in dentin caries but not in enamel caries (LeGeros, 1991b).
3. Fluorosis Fluorotic enamel is not only hypomineralized but also microhypoplastic. Histological features of fluorosed human dental enamel have been well described in the studies of Fejerskov et al. (1975). Mineral deposition can be effected by exposure during either the secretory or the maturation phase and is possibly due to interference with matrix mineral interactions or matrix withdrawal (Robinson et al., 1992). An increase of crystal size, alterations in their morphology, and lattice dislocations were observed in fluorotic enamel (Kerebel and Daculsi, 1976). The size of these crystal is more irregular and 20-30% larger. The hexagonal shape of the crystals from fluorotic enamel showed higher symmetry, the crystal angles observed
FIG. 13 Dentine tubules in the area between the sound and carious zones were shown to be obstructed with large crystals (a). The carious dentin area caries (which underwent a dissolution process) revealed the presence of fine granular material inside the tubules, a highly mineralized peritubular dentine, and an intertubular dentine containing larger crystals than those observed in sound dentin (b). The dentine area partly demineralized by the caries process consisted of less numbers of crystals that are larger in size (c).
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were often 60-120" and the W/T ratio was shorter than that of sound enamel (Figs. 14a and 14b). Albumin was shown to affect crystal growth and morphology (Robinson et al., 1992).
4. Amelogenesis Irnperfecta Two types of amelogenesis imperfecta have been suggested according to stages of enamel development. However, no correlations between classification and ultrastructural observations have been made (Backman et al., 1993). Hypoplasia can be related to defective matrix mineral intcractions (Shafer et al., 1974). Kerebel and Daculsi (1976, 1977) had shown two crystal morphologies: acicular and rectangular (Figs. 1Sa-1Sd). Studies of the different lesions in amelogenesis imperfecta have not been done. Extracellular matrix interactions with crystal development may occur in the initially formed apatite or later in thc maturation stage. This supports a relationship between an insufficient amount of extracellular proteins and physical stability of enamel apatite crystals similar to that speculated for the role of collagen in osteogenesis imperfecta (Kerebel and Daculsi, 1977). Crystal defects similar to those observed in CHA and apatitic calcium phosphates (Daculsi and LeGeros, 1986) were observed in apatite of enamel with amelogenesis imperfeta (Daculsi and Kerebel. 1977; Arends et nl., 1982).
5. Osteoporosis The well-known phenomena associated with osteoporosis is the thinning of the cortical bone and the increase in macroporosity of trabecular bone. The bone mineral of osteoporotic bone was reported to contain less Mg and lcss COT (Cohen et al., 1987). This may be due to the dissolution of the C03-and Mg-rich bone apatite and precipitation of C 0 3 -and Mg-poor apatite. Administration of sodium fluoride (NaF) is one of the recommended regimen. However, although ingested fluoride was shown to cause the formation ol bone apatite that have better crystallinity and less suscepti-
FIG. 14 The shapc of the crystals from fluorotic enamel showcd hexagonal symmetry (a). The WIT ratio was shorter than sound enamel traducing the more regular hexagonal shape (b). FIG. 15 TEM sections of teeth with amelogenesis imperfecta revealed two kinds o f crystallites. one of about 25 nm i n diameter with an acicular shape and the other of SO nni and more rectangular in shape (a). The crystals not presenting a hexagonal shapc generally had a rectangular shape (h) with very irregular sidc (c). These crystals were very sensitive to beam darnage, traducing crystal sensitivity (d).
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G. DACULSI E T A L .
bility to acid dissolution than the F-free bone apatite (LeGeros, 1981a, 1981b, 1983a, 1991a), the total effect of F on the density and strength of bone is still controversial.
V. Factors Affecting Crystal Formation and Properties of Biologically Relevant Calcium Phosphates Minor elements (occurring in concentrations of 0.01-7.0 wt%) and trace elements (occurring in ppm concentrations) are associated with biological apatites. Some of these elements have been shown to promote or inhibit crystal growth of synthetic and biological apatites and related calcium phosphates and/or facilitate or inhibit the transformation of one calcium phosphate to another (Boskey and Posner, 1974; Fleisch, 1981; LeGeros, 1967, 1981a, 1984, 1991a; LeGeros et ul., 1968, 1973, 1980, 1983b; 1988b, 1995; Posner, 1985). The more important minor elements are carbonate, magnesium, fluoride, chloride, HP04, pyrophosphate, and strontium ions. Some proteins and macromolecules have also been demonstrated to affect crystal growth of biologically relevant calcium phosphates (Boskey, 1992; Hay et al., 1984). For example, some salivary proteins prevent calcium phosphates from precipitating out from saliva (Hay et al., 1984), which is normally supersaturated with respect to several calcium phosphate phases, namely, DCPD, OCP, and HA. On the other hand, in v i m elevation of calcium ions resulted in the precipitation of CHA from human saliva and calcium carbonate from dog saliva (LeGeros and Shannon, 1979), indicating that the inhibitory effect of these proteins can be nullified by increasing the supersaturation of the synthetic or biological fluids. Formation of acid calcium phosphates (DCPD and OCP) is facilitated at low pH but inhibited by the presence of F- even at low pH, promoting instead the formation of apatite (LeGeros et al., 1984,1988b). Transformation of acid phosphates (DCPD, DCPA, and OCP) to CHA is facilitated in the presence of C 0 3or HC03and inhibited by the presence of Mg or P207 or citrate ions (Johnson and Nancollas, 1992; LeGeros, 1991b; LeGeros et ul., 1984, 1989b). ACP formation is facilitated by the presence of CO3, Mg, and/or P207 and stabilized by these ions (Boskey and Posner, 1974; LeGeros, 1967, 1981a, 1991b; LeGeros et al., 1975).
A. Carbonate The presence of C032-ions in solution causes its incorporation in synthetic apatites. The mechanism of C 0 3incorporation is by a coupled substitution of Na for Ca and C 0 3 for PO4 and causes a contraction in the n- and
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expansion in the c-axis dimensions compared to C03-free apatite (LeGeros, 1967, 1981a, 1991b; LeGeros et al., 1968, 1971; Zapanta-LeGeros, 1965). Incorporation of C 0 3 causes a reduction in the crystallinity of the apatite, eventually causing the formation of amorphous calcium phosphate containing carbonate (LeGeros, 1967; LeGeros et al., 1973). Incorporation of CO3 causes changes in the morphology of apatite-changing from acicular to rod like to equiaxed crystals-with increasing C 0 3incorporation (LeGeros, 1967,1981a,1991b;LeGeros etal., 1968,1971),causes crystal defects (Featherstone et al., 1984; Daculsi and LeGeros, 1986), increases the solubility of the apatites (LeGeros and Tung, 1983; Nelson, 1981; Okazaki et al., 1983), and causes very irregular size and shape (Fig. 16). The effect of C 0 3 on the crystallinity and dissolution properties of apatites is synergistic to that of magnesium (LeGeros, 1984; LeGeros et al., 1995) and antagonistic to that of F (LeGeros and Tung, 1983; LeGeros et al., 1983a). Another mode of C 0 3substitution in apatite is by C 0 3 for OH, observed in apatites prepared at 1000°C under very dry conditions (Bonel, 1972; Elliott, 1974). This C 0 3 for OH substitution was referred to as type A, whereas the C 0 3for PO4substitution first reported by LeGeros in carbonate apatite prepared from aqueous systems at low temperatures, e.g., at 37, 60, 80, and 100°C (LeGeros, 1967, 1981a, 1991b, 1994; LeGeros et al., 1968, 1971; Zapanta-LeGeros, 1965), was described as type B. The a-axis dimension of human enamel apatite is larger than that of pure HA (0.9441 vs 0.9422 -+ 0.0003 nm). This observation initially appeared to support the C03-for-OH (type A) substitution that causes an a-axis expansion (Elliott, 1974) and not the C03-for-P04substitution (type B), which causes an a-axis contraction (LeGeros, 1967; Zapanta-LeGeros, 1965). However, a combination of results from XRD (for lattice parameter measurements), chemical analyses (which demonstrated concomitant decrease in PO4 with increasing C 0 3concentrations in the apatite), and IR analyses [which showed that the spectral properties of C 0 3 in enamel apatite are more similar to those of the C 0 3 in CHA than in CA (LeGeros, 1967; LeGeros et al., 1969, 1971)] led to the conclusion that the C 0 3 in enamel and other biological apatites, e.g., dentin or bone (Fig. 2), are principally incorporated as a substituent for PO4 coupled with an Na-for-Ca substitution (LeGeros, l967,1981a, 1991b, 1994;LeGeros et al., 1971).This observation has been confirmed by subsequent studies (Elliott, 1974; Nelson et al., 1986). Thus, the expanded a axis of enamel compared to HA could be explained by additive effects of C1-for-OH, Sr-for-Ca, and HP04-for-P04 substitutions (LeGeros, 1967, 1974b, 1981a, 1991b). 6. Fluoride When present in solution fluoride ions become readily incorporated in the apatite even under acid conditions (LeGeros, 1981b, 1991a; LeGeros et al.,
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FIG. 16 The carbonate content (8.3%)involved irregular crystal size and shape, these crystals with irregular parallelepipedic shape look iike amelogenesis imperfecta enamel crystals
FIG. 17 Thc incorporation of fluoride (2.3%) in the crystals involved a larger sizc of crystals and more regular crystals with hexagonal symmetry FIG. 18 SEM of synthetic magnesium-substituted tricalcium phosphate, 8-TCMP, o r whitlockite crystals (large cubes) observed with acicular apatite crystals.
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1984,1988a). The F-for-OH substitution in synthetic and biological apatites causes a contraction in the a-axis dimension, promoting the formation of larger, more stoichiometric and less soluble crystals (Baud and Very, 1975; Larsen and Jensen, 1989; LeGeros, 1967, 1981a, 1983b, 1988, 1991a; LeGeros and Tung, 1983; LeGeros et al., 1973, 1977; Moreno et al., 1977). Biological apatites containing F (e.g., from shark enameloid) show larger crystal size and more regular hexagonal symmetry (Fig. 17) compared to F-free apatite (e.g., from human enamel) (Daculsi et al., 1976, 1978, 1989). Comparative caries progression between human enamel and shark enameloid shows a much lower progression in shark enameloid demonstrating the effect of F in facilitating apatite formation and in inhibiting apatite dissolution (LeGeros et al., 1983b). When F is simultaneously present with other ions (e.g., C03,Mg, or Sr) that induce negative effects on the crystallinity and dissolution properties of apatites, the positive effect of F is the dominant effect (LeGeros et al., 1988~). Topical fluoride treatment of either enamel or dentin results in the formation of (F or OH) apatite and/or CaF2, depending on the F concentration (LeGeros, 1991d; Ogaard et al., 1983). The morphology of the CaF2crystals is influenced by the presence of phosphate ions that cause the formation of small aggregated crystals that appear to be cemented by ACP (LeGeros, 1991a), causing the appearance of globules.
C. Magnesium
Biological apatites (inorganic phase of enamel, dentin, cementum, bone, and some pathological calcifications) and biological whitlockite are always associated with Mg (LeGeros, 1967, 1974b, 1581a, 1984, 1991b; LeGeros and LeGeros, 1984). Studies on synthetic systems showed that Mg can be incorporated in the synthetic apatites to a very limited extent, up to 0.3 to 0.6 wt% (LeGeros, 1984; Terpstra and Driessens, 1986). The incorporation of Mg was shown to cause the following effects on some of the properties of the synthetic apatites: slight contraction in the unit-cell parameters, reduction in “crystallinity” (indicating reduction in crystallite size and/or increase in crystal strain), and increase in solubility (LeGeros, 1984; LeGeros et al., 1995a; Okazaki and LeGeros, 1992; Spencer et al., 1989). In vivu, whitlockites have often been identified associated with apatites and/or other calcium phosphates in pathological calcifications, e.g., soft tissue calcifications in lungs and cartilage (Ali and Griffiths, 1983, LeBouffant et al., 1973; Rowles, 1968; Gatter and McCarthy, 1967), in human dental calculi (Rowles, 1968; Jensen and Rowles, 1957; Schroeder, 1969; LeGeros, 1974; LeGeros and Shannon, 1979; LeGeros et al., 1988d), and occasionally in human urinary calculi (Sutor and Sheidt, 1968).Whitlockites
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have also been identified in carious human dentin (Daculsi et al., 1979, 1987; Takuma et al., 1975; Vahl et al., 1964). These bioloqical whitlockites havc been described as “cuboidal” crystals in human dental calculi and in calcified arthritic cartilage (Ali and Griffith, 1983) or “rhombohedral” or “larger’ crystals in human carious dentin (Takuma et al., 1975; Vahl et al., 1964; Daculsi et al., 1979, 1987). X-ray diffraction analyses showed differences in lattice parameters between pure whitlockites, (P-TCP), and biological whitlockites, (P-TCMP) (LeGeros, 1967, 1974, 1984; LeGeros and LeGeros, 1984; Rowles, 1968; Jensen and Rowles, 1957). The observed decrease in lattice parameters of P-TCMP whitlockites is caused by partial Mg-for-Ca substitution. P-TCMP has also been observed when some biological apatites, e.g., enamel, dentin, and bone (depending on age and species), are ignited at 800°C (LeGeros, 1967, 1984, 1991a), or when ACP deposit from pathological calcifications associated with uremic patients was ignited at 600°C (LeGeros et al., 1973). In synthetic systems, rhombohedral crystals of P-TCMP associated with acicular apatite crystals (Fig. 18) are obtained from solutions with Mg/Ca 1 0.05 either by precipitation or by hydrolysis methods (LeGeros, 1984,1991a). SEM samples of human calculus showed the presence of cubic or rhombohedral crystals of assorted sizes. TEM of undecalcified ultrathin sections of samples of human dental calculus and of arrested dentin caries in human teeth showed the presence of large cubic crystals associated with smaller needle-like crystals. Electron microdiffraction of the area of needlelike crystals gave 0.34- and 0.28-nm rings corresponding to the 002 and 300 reflections of apatite; electron nanodiffraction allowed the diffraction from single crystals of apatite. The electron diffraction of the area consisting of cubic crystals gave the dominant ring corresponding to the 0210 spacing of P-TCP, whereas electron nanodiffraction of single cubic crystals gave rings of 0.52 and 0.25 nm corresponding to the 120 and 0114 spacings of P-TCP with an angle of 107.5’. Microanalyses of cubic and needle-like crystals in carious dentin gave Ca/P weight ratio of 1.6 % 0.1 and 2.0 ? 0.1, respectively (Daculsi et al., 1987).
D. HPO, Calcium-deficient synthetic biological apatites contain HP04, as seen in the IR and as evidenced by the formation of P-TCP (or P-TCMP in the case of biological apatites) after ignition of these apatites (LeGeros, 1967; 1991; Rey et al., 1991). The incorporation of H P 0 4 ions in synthetic apatites is facilitated when the solution pH is lower than 9 (LeGeros, 1991a).
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P207
The presence of P207ions during the formation of calcium phosphates promote the formation of ACP (LeGeros, 1967,1981a, 1991b; LeGeros et al., 1975). These actions act synergisticallywith Mg or C 0 3 in the formation and stability of ACP and may explain the occurrence and stability of ACP in pathological calcifications (LeGeros et al., 1973). These ions were also reported to inhibit calcification (Fleish et al., 1968).
F. Sr and CI Substitution of Sr for Ca in synthetic apatite causes expansion of the lattice parameters and an increase in solubility (LeGeros, 1991a; LeGeros et al., 1977, 1988a). C1 incorporation in apatite prepared in aqueous systems requires a very high C1 concentration in the solution (LeGeros, 1974b), explaining the low C1 content associated with biological apatites (Dykes and Elliott, 1971) despite the large C1- concentrations in the biological fluid. The effect of C1 substitution is an expansion on the a axis and contraction in the c axis compared to C1-free apatite (Dykes and Elliott, 1971; LeGeros, 1974b). Strontium incorporation in synthetic and biological apatite causes the apatite to be more soluble (LeGeros, 1991a; LeGeros et al., 1989b; Sillen and LeGeros, 1991).
VI. Calcium Phosphates and Related Bone Graft Biomaterials Between 1920 and 1975,a very limited number of scientific articles reported that the use of calcium phosphate materials, described as “tricalcium phosphate,” to repair bone defects successfully promoted bone formation (Albee, 1920; Bhaskar et al., 1971; Ray and Ward, 1951) or periodontal defects (Nery et al., 1975). The tricalcium phosphate material used by Nery was subsequently identified by LeGeros in 1985 (reported in LeGeros, 1988) as consisting of a mixture of 20% P-TCP and 80% HA. This material and other mixtures of P-TCP and HA were later described (first by Nery) as a BCP. In the past decade, through the pioneering efforts of Jarcho (1981), De Groot (1983), and Aoki (1991), synthetic calcium phosphate materials, principally HA ceramics, were commercially introduced as alternatives to autogenous bone or allografts for bone repair, substitution, or augmen-
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tation. Other calcium phosphate materials from natural sources, such as bovine bone-derived and coral (calcium carbonate) and coral-derived biomaterials, were later introduced. These materials have gained acceptance for several dental and medical applications that include fillers for periodontal defects, alveolar ridge augmentation, maxillofacial reconstruction, ear implants, spine fusion, and as adjuncts to uncoated implants or as extenders of autogenous bone or demineralized bone matrix (Aoki, 1991; Barney et al., 1982; Basle et al., 1993; Chiroff et al., 1977; Cranin et al., 1987; Daculsi ef al., 1990a, 1992a; Daculsi and Dard, 1994; Damien et al., 1994; Dard et al., 1994; De Groot, 1983; Denissen, 1979; Ellinger et al., 1986; Frank et al., 1987; Galgut et al., 1990; Froum et al., 1982; Guillemin et al., 1989; Jarcho, 1981; Han et al., 1984; Holmes, 1979; LeGeros, 1988; Moskow and Lubarr, 1983; Nery et al., 1975, 1992; Niwa et al., 1980; Passuti et al., 1989, 1991, 1992; Shors and Holmes, 1993; Troster, 1993; Van Blitterswijk, 1985; Wagner, 1989; Williams, 1982; Wilson and Menvin, 1988; Yukna et al., 1984). Other suggested applications include pulp capping materials (Frank et ul., 1991; Jean et ul., 1988), bioreactors (Blottiere et al., 1995; Cheung and Haak, 1988; Frayssinet et al., 1991; Gregoire et al., 1992; Ohgushi et al., 1992, 1993; Soueidan et al., 1995), and for drug delivery systems (De Groot, 1987). Comparative properties of calcium phosphate materials are summarized in Tables I11 and IV. Calcium phosphate (Cap) materials are also used as components or fillers in polymeric composites (Bonfield, 1988; Ducheyne et al., 1993; LeGeros and Penugonda, 1984; Rawls er al., 1985) and in cements (Brown and Chow, 1995; Constanz et al., 1995; LeGeros et al., 1982; Mirtchi et al., 1989) or largcly as the source material for coating dental and orthopedic implants (De Groot, 1987;Ducheyne et al., 1986;Jarcho, 1992;Shirkahanzadeh, 1994). Despite the desirable bioactive properties of CaP materials, their low fracture strength makes them unsuitable for use in load-bearing areas (De Groot, 1983) in which metals have been the material of choice because of their strength (Branemark, 1985). The development of coated dental and orthopedic implants was based on the documented bioactivity of CaP mate-
TABLE Ill Classificationof Bone Substitutes Based on the Nature of the Interface Formed with Bone
Biomaterial/bone interactions
Biornaterialhne interface
~~
Blotolerant
Distant osteogenesis
Fibrous tissue
Bioinert Bioactive
Contact osteogenesis Osteocoalescence (chemical bond)
Direct contact bone-biomat Diffused interface
161
ADAPTIVE CRYSTAL FORMATION TABLE IV Comparative Crystal Characteristics Concerning CaP of Various Origins“
Coralline HA Crystal size ( p m ) Specific surface area (rn’
.g
’)
Ionic and inorganic impurities
Bone-derived HA
Synthetic HA, TCP, and BCP
0.02 x 0.1
0.1-2
1-5
50-100 Mg”. C012-
10-50
2-7
Mg”, C032-
CO,’., CdO, other CaP
“From Le<;eros (1967, 1981a, 1991a)
rials combined with the strength of metals. In this regard, one of the important uses of CaP materials, principally H A ceramic, is as a source material for plasma-spraying bioactive coatings on commercial dental and orthopedic metal implants. Such coated implants, compared to uncoated ones, were reported to allow accelerated skeletal fixation and maximum bone c ont xt without fibrous encapsulations and exhibit high interfacial strength (Cook et al., 1987, 1992; Daculsi et al., 1990a; De Groot, 1987; Jarcho, 1992; Klein et al., 1993;LeGeros er al., 1995d). The coatings were also reported to minimize the leakage of metal ions (Ducheyne and Healy, 1988).
A. Coral and Derived Biomaterials Natural coral (Porites) consists of mineral phase (principally calcium carbonate in the structural form of aragonite with impurities such as Sr, Mg, and F ions) and organic matrix. Commercially available coral (Biocoral) is used as bone graft material and reported to be biocompatible and resorbable (Guillemin et al., 1989; Richard et al., 1996). Coral-derived material described as coralline H A is commercially available (Interpore) as bone graft material. It is prepared by the hydrothermal conversion of the original calcium carbonate of the coral (Porites) in the presence of ammonium phosphate (Roy and Linnehan, 1974). This process maintains the original interconnecting macroporosity of the coral (Fig. 19a). The change in size and shape of the crystals in coralline H A (Fig. 19b) reflects the change in composition from CaC 0 3 (Fig 19c) in the original coral to apatite. Coralline H A was identified to be a C H A and differs from synthetic HA in composition and crystallinity (reflecting crystal size and/ o r strain or perfection), resulting in differences in dissolution properties and reactivity (LeGeros et al., 1988c, 1991).
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FIG. 19 (a) SEM of coralline HA (Interpore) showing the interconnecting pore; (b) higher magnification showing a group of apatitc crystals and microporosity; (c) aragonitc (CaC03) crystals in coral (Biocorail).
6 . Bone-Derived Biomaterials Materials from bovine bone are either unsintered (e.g., BioOss and Pyrost) or sintered (BonAP, Endobon, and Osteograf). The unsintered materials are usually processed to remove the organic phases providing the bone mineral that is principally a calcium CHA containing a small amount of magnesium, sodium, and other trace elements. Depending on the sintering conditions, the porosity (micro/macroporosity) of the original bone is essentially conserved (Fig. 20a). The process of sintering or hydrothermal treatment of bone retains some of the minor (e.g., magnesium and sodium) and trace elements present in the unsintered bone mineral but loses most of the carbonate of the original bone mineral (LeGeros, 1991a; LeGeros et al., 1988c, 1992) and results in an increase in crystal size and change in crystal morphology as previously reported for enamel (LeGeros, 1991a) shown in Fig. 20b. Changes in crystal size and composition have been reported resulting from sintering of enamel or dentin or bone (Holcomb and Young, 1980; LeGeros, 1991a). The loss of carbonate and increase in crystal size are expected to make the sintered bone less soluble than the original bone mineral. Bovine-derived materials have been successfully
FIG. 20 (a) SEM of sintered bovine bone. (h) Higher magnification showing the crystal size and shape. FIG. 21
SEM of P-TCMP (Mg-substituted p-TCP) crystals.
FIG. 22 TEM of ceramic HA crystal (Calcitite) prepared at high temperature. FIG. 23 TEM of ceramic HA crystal prepared at 900°C showing three-dimensional lattice defect (intrinsic porosity). FIG. 24 TEM of HA and P-TCP crystals in BCP (Triosite).
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used in bone rcpair (Basle et al., 1993; Daculsi and Dard, 1994; Dard et al., 1994; Triister, 1993).
C. Synthetic Calcium Phosphate Bone Graft Biomaterials
Based on composition, synthetic calcium phosphate currently used as biomaterials is classified as HA, P-TCP, BCP for mixtures of HA and 0-TCP, and unsintered apatite or calcium-deficientapatite. HA and P-TCP ceramics are usually prcpared by precipitation and subsequent sintering at about 1100°C (Hayek and Newesely, 1968; Jarcho, 1981; LeGeros, 1983a, 1988, 1994; LeGeros and LeGeros, 1993). The sintering process involved the formation of larger crystal (Fig. 21), grain and twin boundaries as previously described by Daculsi et al. (1991) (Fig. 22), and specific three-dimensional crystal defects (Fig. 23) (Daculsi and Legeros, 1996). BCP, with varying PTCP/ HA ratios, can be prepared by sintering precipitated CDAs of varying Ca/P ratio (LeGeros, 1991b; LeGeros and Daculsi, 1990; R. Z. LcGeros et ul., 1988a; 1994). The unsintered or calcium-deficient apatite can be prcpared by precipitation or hydrolysis of DCPD, dicalcium phosphate anhydrous (DCPA, CaHP04), or OCP (LeGeros, 1991b; LeGeros et al., 1971, 1984, 1989a; Tiselius et ul., 1956). CaP biomaterials are available in various physical forms (particles or blocks; dense or porous). Macroporosity (pore size >50 pm) in the material is intentionally introduced by the addition of volatile substances (e.g., naphthalene) before sintering at high temperatures (Hubbard, 1974). Microporosity (pore size <10 pm) results from the sintering process and the size of microporosity (Fig. 24) depends on the sintering period and temperature. The specific surface area, associated with the crystal size, ionic impurities shown in Table 11, and the macroporosity and microporosity have an influence on the physicochemical properties (Daculsi and LeGeros, 1996). All these parameters have a specific influence on bioceramic final mechanical properties (Bouler et af., 1996). Calcium phosphate biomaterials differ in their solubility or extent of dissolution in acidic buffer, which may reflect the comparative dissolution or degradation in vivo (LeGeros, 1991a, 1993;LeGeros et al., 1995b,c). The comparative extent of dissolution is: ACP >> a-TCP >> P-TCP > CdA >> HA For BCP, extent of dissolution depends on the P-TCPIHA ratio, the higher the ratio, the higher the extent of dissolution (LeGeros et id., 1988d; LeGeros and Daculsi, 1990; Daculsi et al., 1989a).
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D. Calcium Phosphate Coatings on Dental and Orthopedic implants The plasma-spraying process causes changes in the composition of the source material (HA ceramic) resulting in the formation of other calcium CaP phosphate phases besides HA-principally ACP, small amounts of a- and P-TCP, tetracalcium phosphate, and, sometimes, calcium oxide (LeGeros and LeGeros, 1991; LeGeros et al,, 1993, 199.5~;Klein et al., 1993). Variations in composition, principally in the ACP/HA ratios, were observed between the outer and inner layers of the coatings and among the coatings on implants from different manufacturers (LeGeros, 1991b; LeGeros and LeGeros, 1991; J. P. LeGeros et al., 1994; LeGeros et al., 1995d). Such differences in the composition of the coatings on dental and orthopedic implants are expected to affect the biodegradation of these coatings (LeGeros, 1993; LeGeros et al., 1993, 1995a).
VII. Comparative Properties of Bone and Calcium Phosphate Materials
Bone is an integrated composite of an organic phase (principally collagen) and an inorganic or mineral phase, with an inorganiciorganic ratio of approximately 75/25 by weight and 65/35 by volume. The inorganic or mineral phases of enamel, dentin, and bone were initially identified as an apatite, idealized as HA, with the structural formula, Calo(P04)6(OH)2(De Jong, 1926; Beevers and Mclntyre, 1942). Actually, the biological apatites (i.e., mineral phases of enamel, dentine, cementum, and bone) are associated with minor (e.g., carbonate, magnesium, sodium, potassium, chloride, fluoride, and HP04) and trace elements (e.g., strontium). Although carbonate substitution in biological apatites has been proposed by McConnel (1952), experimental evidence for such substitution was originally presented by LeGeros (1967; LeGeros et al., 1967, 1971) and later confirmed by other studies (LeGeros, 1981; Elliott, 1974; Monte1 et al., 1981; Nelson and Featherstone, 1982; Rey et al., 1991). Biological apatites are more accurately described as CHA, approximated by the formula, (Ca,Mg,Na,X)lo. (P04,C03,HP0&(OH,CI,F);?,where X may be other substituents for calcium (e.g., strontium, etc.). Magnesium is incorporated in synthetic and biological apatites to a very limited extent (LeGeros, 1984,1991b; Okazaki and LeGeros, 1992). The concentrations of carbonate and magnesium are higher in both dentin and bone apatite compared to that in enamel apatite (LeGeros, 1967, 1981, 1991b; LeGeros et al., 1995d). Biological apatites,
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unlike HA (Table V), are nonstoichiometric with Ca/P molar ratio <1.67 (calcium deficient) for enamel and dentin and >1.67< for bone apatite, depending on age and specie of the animal (LeGeros, 1981a, 1991b, 1994; Rey et al., 1991). Biological apatites can more appropriately be represented as (Ca,X)lo(P04,Y)6(OH,Z)2where X is possible substituents for calcium (e.g., Mg,Na,K,Sr, etc.), Y is possible substituents for PO4 (e.g., C 0 3 ,HP04, and SO4), and Z is possible substituents for OH (e.g., C1 or F) (LeGeros, 1967,1981a, 1991b; Monte1 et al., 1981; Young and Elliott, 1966). The bone apatite microcrystals have rod-like or plate-like morphology, with average dimensions of 25 X 3 nm, compared with about 200 nm for HA ceramic.
TABLE V Comparative Composition and Crystallographic and Mechanical Properties of Human Bone and Synthetic Hydroxyapatite Ceramic"
Weight %
Human bone
HA
24.5 11.5 1.65 0.1 0.03 0.55 5.8 0.02 0.1 65.0 25.0 9.7
39.6 18.5 1.67 Trace Trace Trace
Crystallographic propertics Lattice parameters (t0.003 11 axis b axis Crystallinity indcy" Crystallite sizc (A)
9.419 6.880 33-31 250 X 25-SO
9.422 6.880 100 2000
Products after sintering (950°C)
HA
Mechanical properties Elastic modulus (10' MPa) Tensile strcngth (MPa)
Cortical bone 0.020 150
Constituents Calcium (Ca2') Phosphorus (P) (CalP) molar ratio Sodium (Na') Potassium ( K ' ) Magnesium (Mg") Carbonate (CO? ) Fluoride (F ) Chloride (CI-) Ash (total inorganic) Total organic Adsorbed H20" Trace elcments: Sr2', Pb", Ba2+,Fe", ZnZ', Cu" etc.
-
I00
A)
" From LeGeros (1967, 1981a, 1991a).
+ CaO
HA HA 0.01 100
* Ratio of coherenthcoherent scattering in mineral HA crystal index value is 100.
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Sintered bone-derived apatite is less soluble than unsintered bone apatite but more soluble than synthetic ceramic HA (LeGeros et al., 1995b,c,). Another unique property of bone apatite crystals is their association with an organic matrix that plays an important role in the formation, growth, orientation, and size of the biological apatite crystals (Boskey, 1992;Daculsi and Kerebel, 1978; Daculsi et al., 1989a;Wright et al., 1992). Collagen fibrils, for example, give the direction of epitaxies (Boskey, 1992). Moreover, biological crystals are covered by a proteic sheath. This sheath may influence their reactivity (Daculsi et al., 1989a) and their dissolution properties (LeGeros et al., 1995d). The protective effect of the organic phase is suggested from the observation that bone or dentin is less soluble before the removal of the organic phase (LeGeros et al., 1995d). Cellular activity causes biodegradation and dissolution of calcium phosphate materials, eventually contributing to the formation of carbonate apatite on the surfaces of these materials as described in Section VIII. Moreover, numerous lattice defects appear in biological apatite crystals, such as dislocations that disturb the normal arrangement of the lattice planes (Jongbloed et al., 1974; Daculsi and Kerebel, 1977, 1978; Kerebel et al., 1976; Lee and LeGeros, 1981; Voegel and Frank, 1975). Crystal defects have also been observed in HA ceramics that appeared to depend on sintering temperature (Daculsi and LeGeros, 1996; Daculsi et al., 1989b). These ultrastructural differences may explain the difference in mode of dissolution between the biological and ceramic apatite crystals, i.e, preferential dissolution of the crystal core in biological apatites compared to nonspecific dissolution in ceramic HA crystals, and the difference in bioactivity related to the sintering temperature of the implanted HA (Nagai and LeGeros, 1993).
VIII. Bone/Biomaterial Interface The main attractive feature of bioactive bone graft materials, such as CaP ceramic and special glass ceramics (bioactive glass), is their ability to form a strong direct bond with the host bone resulting in a uniquely strong interface compared to bioinert or biotolerant materials that form a fibrous interface (Daculsi et al., 1990b; De Groot, 1983; Hench et al., 1971; Hench, 1994; Osborn and Newesely, 1980). The strong bond associated with HA ceramics has been attributed to a “bonding zone,” also described as “electron dense” or “amorphous” zone (Jarcho et aL, 1976; Ganeles et al., 1986; Gross et al., 1983). The formation of this dynamic interface is believed to result from a sequence of events involving interaction with cells; formation of carbonate hydroxyapatite CHA (similar to bone mineral) by dissolution/
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precipitation processes, and mineralization (Hench, 1991; LeGeros et af., 1991, 1992).
A. Cellular Events The CaP materials elicit responses from bone cells and related cells in vitro and in vivo that are similar to those elicited by bone. These materials allow cell attachment, proliferation, and expression (Bagambisa et al., 1990; Blottier et al., 1995; Cheung and Haak, 1988; Davies, 1990; Frayssinet et al., 1991;Galgut etal., 1990; Gregoire et al., 1992; Gross et al., 1991;LeGeros et al., 1991;Nagai and LeGeros 1993; Niwa et al., 1980; Ohgushi et al., 1993; Soueidan et al., 1995). The first biological events after CaP ceramics implantation are biological fluid diffusion, followed by cells colonization. These cells include monocytes macrophages (Fig. 25), giant cells (Fig. 26), osteoclasts for resorption, and fibroblast and osteogenic cells for tissue repair (Fig. 27). The nature of the multinucleated cells involved in the resorption processes occurring inside macroporous calcium phosphate biomaterials grafted into rabbit bone was demonstrated using light microscopy, histomorphometric analysis, enzymatic detection of tartrate-resistant acid phosphatase (TRAP) activity, and SEM and TEM microscopy (Bade et al., 1993). The combination of these analytical techniques allowed the observation that osteogenesis and resorption occur at the surface of the biomaterials and inside the macropores as early as Day 7. Resorption of both newly formed bone and CaP materials was associated with two types of multinucleated cells. Giant cells were found only at the surface of biomaterials, showed a large number of nuclei, were TRAP negative, developed no ruffled border, and contained numerous vacuoles with large accumulation of ceramic crystals from the biomaterials. The number of these cells decreased with time. The other multinucleated cells observed were osteoclasts. These cells exhibited well-defined ruffed border and were TRAP positive. They were observed at the surface of the newly formed bone and on the surface and inside the macropores of the CaP biomaterials. The increase in microporosity of the CaP ceramics underneath this type of cell were greater than that observed underneath giant cells or in the depth of the biomaterials. Calcium phosphate ceramics elicit osteogenesis (bone ingrowth) and the recruitment of a double multinucleated cell population having resorbing activity, macrophages with monocytes (Blottiere et al., 1995; Benhamed et af., 1994, 1996; Soueidan et af., 1995), and multinucleated giant cells that resorb biomaterials and osteoclasts that resorb newly formed bone and biomaterials. These observations suggest that resorptionidissolution must occur before osteoblastic adhesion and
FIG. 25 TEM of monocytes macrophages in contact with calcium phosphate ceramics.
FIG.26 TEM of giant cells invading macropores of calcium phosphate ceramic. FIG. 27 Light microscopy of the interface of BCP ceramic (Triosite) ( C )and bone (B) showing osteoclasts and osteoblasts. FIG. 28 TEM of extracellular dissolution of HA single crystal (arrows) (a) and crystal core dissolution (b).
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phenotypic expression [similar to that described for bone remodeling to takc place (Daculsi and Dard, 1994)l.
B. Biodegradation and Biodissolution The degradation of the CaP ceramics after implantation has been described using light microscopy of decalcified sections (Drobeck et ul., 1984; Froum etal., 1982;Moskow and Lubarr, 1983;Osborn, 198.5) or TEM of decalcified and undecalcified sections (Daculsi et al., 1986, 1989a; Ganeles et al., 1986; Tracy and Dorcmus, 1984). The biodegradation of these ceramics included the dissolution of the individual HA or P-TCP crystals (Daculsi et al., 1989a; Daculsi and Delecrin, 1994; LeGeros and Daculsi, 1990; LeCeros et al., 1988a, 1991, 1992) as shown in Figs. 28a and 2%. The proportion of HA to 0-TCP crystals in BCP appeared greater after implantation (Richard et al., 1996), in agreement with previous reports (LeCeros et al., 1988a) and the known higher reactivity or solubility of P-TCP compared to HA (LeGeros, 1991b, 1993; Osborn, 1985). Dissolution of biological apatites occurs during natural processes such as dental caries and bone remodeling. Dissolution of human enamel apatites crystals was shown to be site specific, occurring preferentially at the crystal core (Bres et al., 1986; Daculsi et al., 1979; Daculsi and Kerebel, 1977; Daculsi and LeCeros, 1996; Jongbloed et al., 1974; Lee and LeGeros, 198.5; Scott et af., 1974; Voegel and Frank, 1975). The dissolution of some synthetic ceramic HA crystals, unlike biological apatite crystals, did not show site specificity (Daculsi et al., 1989b; Daculsi and Delccrin, 1994). Dislocations are crystal lattice defects, and it has been established that acid dissolution starts at the dislocation sites (Lee and LeGeros, 1985). Such phenomena were observed in the dissolution of both the ceramic HA and biological apatite crystals: dissolution on grain boundaries and crystal cores in ceramic HA and preferential dissolution in crystal cores in biological apatites (Daculsi et al., 198%). However, significant differences were also observed: (i) preferential core dissolution of biological apatites compared to the nonspecific crystal dissolution of ceramic apatite crystals and (ii) correlation with crystal c axis in biological apatite dissolution and noncorrelation observed with ceramic apatite. The observed differences in dissolution features, i,e., preferential dissolution at the crystal core (site specific) for biological apatite and nonspecific dissolution for ceramic HA (Fig. 28), may be attributed to the differences in the chemical and physical properties of the biological and ceramic apatite crystals resulting from the differences in the conditions of their formation: for example, (i) the presence of screw dislocation at the crystal base and along thc c axis in biological apatites (Jongbloed ef al., 1974): Thcse screw
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dislocations, responsible for the helicoidal crystal growth, could explain the elongated tunnel observed along the c axis of the biological apatite crystals subjected to in vivo and in vitro acid dissolution. This morphological aspect was not observed in ceramic HA crystals because, during the sintering process (at temperatures above 90OOC), the individual elongated crystals fuse into large equiaxed crystals; (ii) difference in the origin of crystal defects in biological and ceramic apatite crystals: Biological apatites form by nucleation and growth in aqueous systems of different chemical composition that may contribute to the observed preferential core dissolution. HA ceramic, on the other hand, is prepared by compacting and sintering apatites. These processes may induce different types of crystal defects causing the nonsite-specificdissolution process; (iii) intimate crystal/protein interaction associated with individual biological apatites (Bonucci, 1987; Daculsi and Kerebel, 1978; Daculsi et al., 1995) but not with ceramic apatites: Although serum-derived proteins can adsorb on ceramic HA implant, the interaction with individual HA crystals of the ceramic implant is purely physical. This crystal/protein interaction unique to biological apatites (Bonucci, 1987; Daculsi et al., 1984; Daculsi, 1995) appears to make the crystal surfaces less susceptible to acid dissolution (LeGeros et al., 1994~). The observed decrease in average size of crystals in BCP ceramics after implantation is associated with an increase in the size of microporosities in the surface and at the core of the ceramic indicating that in vivo dissolution has taken place (Daculsi, 1988; Daculsi and Passuti, 1990; Daculsi et al., 1990a; LeGeros and Daculsi, 1990; Passuti et al., 1989, 1991). The resorbability (reflecting in vivo dissolution) of BCP ceramics depends on their P-TCPIHA ratios; the higher the ratio, the greater the resorbability (Daculsi et al., 1989a, LeGeros et al., 1988a; LeGeros and Daculsi, 1990). Formation of microcrystals (which are able to diffract X rays) with Ca/P ratios similar to those of bone apatite crystals were also observed after implantation (Daculsi et al., 1988,1990a). The abundance of these crystals was directly related to the initial P-TCPIHA ratio in the BCP; the higher the ratio, the greater the abundance of the microcrystals associated with the BCP crystals (Daculsi et al., 1989a; LeGeros et al., 1988b; LeGeros and Daculsi, 1990). It was proposed that the formation of the bone apatite-like crystals may be due to the precipitation of calcium and phosphate ions released from the dissolving BCP crystals with the P-TCP component dissolving preferentially to the H A component (LeGeros et al., 1988b; LeGeros and Daculsi, 1990).
C. Bonding Zone
TEM of undecalcified sections from the bone/HA interface showed the presence of microcrystals, described as biological apatite, deposited perpen-
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dicular to the ceramic HA crystal surface and associatcd with collagen fibers (Heughebaert ef al., 1988; Jarcho, 1981; Tracy and Doremus, 1984) (Fig. 29). Using high-resolution TEM, Daculsi et a!. (1990a) demonstrated
FIG. 29 TEM of coalescing zone between CaP residual crystals (C) arid newly formed bone. FIG. 30 Biological apatite precipitation in microspores and at the surface of the implant. FIG. 31 Biological apatite precipitation in microspores and in close contact with CaP ceramic crystals. FIG. 32 High resolution TEM of biological apatite crystals growing heteroepitaxially on the surface of p-TCP ceramic crystal after implantation.
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for the first time that the formation of these microcrystals after implantation (Fig. 30) was nonspecific, i.e., not related to implantation site, subjects of implantation, and types of CaP ceramics. It was further observed that the new crystals were not necessarily deposited on collagenous fibers and demonstrated some specific crystallographic interactions with the HA or /3-TCP crystals of the ceramic (Fig. 30). The microcrystals associated with the CaP biomaterials were described as apatitic, similar to biological apatites based on TEM and electron diffraction studies (Tracy and Doremus, 1984), and were established as CHA, similar to biological apatite based on IR analyses (Daculsi et al., 1990a; Heughebaert et al., 1988; LeGeros et al., 1988a; LeGeros and Daculsi, 1990) (Fig. 31). These biological apatites associated with CaP ceramics were similar in spectral properties to those observed associated with HA ceramics implanted in nonosseous sites (Heughebaert et al., 1988) and those suspended in serum in vitro (Gregoire et al., 1987; LeGeros et al., 1987, 1991~).The observed formation of carbonate apatites in association with the CaP biomaterials, regardless of implant sites (osseous or nonosseous), indicated a process of calcification without cell differentiation. The presence of these microcrystals was interpreted by some investigators to show “osteogenic” properties of the HA ceramic (Frank et al., 1987). However, the histological properties of true bone formation were observed only in osseous sites, confirming the current general agreement regarding the nonosteogenic but osteoconductive properties of the CaP ceramics (LeGeros er al., 1988a; Amler, 1988). The CHA crystals associated with CaP ceramic materials form by the processes of dissolution and precipitation: (i) Partial dissolution of the HA or P-TCP crystals of the ceramic causes an increase in the supersaturation level of the immediate microenvironment of the CaP implant, subsequently leading to the precipitation of the new apatite crystals incorporating other ions (e.g., C 0 3 ,Mg, and H P 0 4 from the biological fluid) during its formation; and/or (ii) precipitation of the new apatite crystals with or without the dissolution of the ceramic crystals, the ceramic particles acting as nucleator or seeds, and epitaxic and heteroiepitaxic growing processes, was observed (Fig. 32).
D. Biological Significance of Carbonate Apatite Precipitation The coalescing interfacial zone of biological apatite and residual crystals (Fig. 31) provides a scaffold for boneicell adhesion and further bone ingrowth (Fig. 29) (Daculsi and Dard, 1994). The restoring process involves a dissolution of calcium phosphate crystals and then a precipitation of CHA needle-like crystallites in micropores close to the dissolving crystals. The formation of CHA may occur by secondary nucleation and an epitaxial
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growth process (Fig. 32). The dissolution of CaP ceramic or related materials (e.g., bioactive glass) and precipitation of CHA is one of the characteristics of bioactive materials (Hench, 1991; LeGeros et al., 1988a, 1991,1992). The coalescing zone constitutes the new biomaterial/bone interface that includes the participation of proteins and CHA crystals originating from the CaP materials but does not include the biomaterial surface. The following events of bone ingrowth and the newly formed bone progressively replaces the initially formed CHA from the CaP biomaterials. In the case of calcium carbonate biomaterial (coral), the dissolution/ precipitation process cannot be similar to that of the CaP biomaterials. The dissolving CaCO? crystals from the coral provide an environment enriched in calcium and bicarbonate ions but deficient in phosphate ions. Based on results of studies on the formation of carbonate apatite from synthetic systems, such an environment will permit the formation of highly carbonated apatite with very poor crystallinity and even amorphous calcium carbonate phosphate that will be readily soluble (LeGeros, 1967, 1981a, 1991b; LeGeros et al., 1973; LeGeros and Tung, 1983). The incorporation of large amounts of carbonate in apatite causes dramatic changes in morphology (size and shape) (LeGeros, 1967, 1981a, 1991b; LeGeros et al., 1967, 1971, 1986). This explains the observation that needle-like apatite crystals normally associated with crystals of CaP materials were not observed on the surfaces of the CaC03crystals of the coral implants (Richard et af., 1996).CaP layer bordering coral grains has been described by Damien et al. (1994). This layer may consist of highly carbonated apatite that are more sensitive to physiological dissolution during bone remodeling compared to those observed with implanted CaP materials. It is possible that, in the case of coral biomaterials, bone ingrowth takes place after a dissolution process has occurred. Further investigation on the new bone formation associated with CaC03 and CaP materials should clarify this issue.
E. Osseo-Coalescing interface: A Dynamic Process An apparent tight bonding of tissue to the calcium phosphate and related bioactive materials is the most important requirement for ceramic materials in order to be osteointegrated and described as bioactive (Hench, 1994; Hench and Wilson, 1984; Hench et al., 1971). Nevertheless, the exact nature of the bonekalcium phosphate interface is, at the present time, intensively discussed. Bone formation seems to start directly on the surface of bioactive materials, whereas it stays away from the surface of nonbioactive materials, resulting in fibrous tissue interposition. From studies in which the behavior of the blood clot and subsequent cellular reaction were studied by use of combined morphological methods it is known that the first cells colonizing
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the surface are macrophages (Van Blitterswijk et al., 1986; Muller-Mai et al., 1990). After the macrophages disappeared, the various steps of primary mineralization of bone begin, including bone cell proliferation and appearance of matrix vesicles, and then synthesis of extracellular matrix (ECM) consisting of collagen and glycoaminoglycans(Gross et al., 1991). The phenotypic expression of undifferentiated cells from the bone marrow on the surface of bioactive materials supports the osteogenic differentiation on the surface (Ohgushi et al., 1992). Interface reactions with marrow-derived cells, osteoblast-like cells, or bone tissue are dependent on the crystal structure (Daculsi et al., 1989a), degree of crystallinity (De Bruijn et al., 1992, 1993), and surface roughness and composition of the calcium phosphates (De Bruijn et al., 1992). According to Osborn and Neweseley (1980) interface processes are based on epitaxy, “apatite-protein affinity,” and structural osteotropism. Using HR TEM Daculsi et al. (1990~)have demonstrated epitaxial growing process and secondary nucleation of biological apatite on both H A and 0-TCP ceramic crystals (Fig. 32). LeGeros et al. (3988b, 1991, 1992; LeGeros and Daculsi, 1990) made a strong argument for the importance of the formation of carbonate apatite in reflecting the bioactivity of biomaterials and to the development of the biomaterial/ bone interface unique to bioactive materials. The following mechanism was proposed: (i) acidification of the microenvironment as a consequence of cellular interaction with the materials; (ii) dissolution of the CaP biomaterials followed by the formation of CHA associated with an organic matrix incorporating the carbonate (and Mg) ions from the biological fluid; (iii) production of extracellular matrix (collagenous and noncollagenous proteins); and (iv) simultaneous mineralization of the collagen fibrils and incorporation of the newly formed CHA crystals in the remodeling new bone. The relationship of CHA formation to bioactivity of materials (calcium phosphate materials and bioglass) has been supported in subsequent studies (Hench, 1994) and its importance in cellular activity has also been elucidated (Yamada et al., 1994). The process of cell colonization, adhesion, phagocytosis and osteoclastic resorption, ECM elaboration and mineralization, and bone ingrowth and bone remodeling associated with the biological apatite precipitation during CaP ceramics dissolution is continuously in progress. Consequently, the interface is not static but dynamic, in constant evolution, taking into account bone physiopathology, biomechanical factors, and bone maturation.
IX. Summary and Conclusion Dental and skeleton mineralization are natural phenomena involving complex interactions among cellular activities, extracellular components, and
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condition and composition of the biological environment. The type of calcium phosphate phase that forms depends on the conditions (e.g., pH) and composition (e.g., Ca/P, Mg/Ca, H C 0 3 / P 0 4 ,specific proteins, and macromolecules). The orientation and morphology of the apatite crystals in enamel is influenced by specific proteins and proteidmineral interactions. The stability of calcium phosphate phases in vitro and in vivo also depends on the presencc of some of the inorganic and organic molecules. In normal calcifications (enamel, dentin, and bone) the mineral phase consists principally of CHA, differing in crystal size and shape, orientation, and concentration o f minor but important elements, (C03and Mg). The crystal size and shape, and consequently the mechanical property of the tissue, and the physicochemical properties (specific surface area, ionic substitution, and dissolution properties) are adapted to the nature and function (mechanical or physiological) of the tissue. In pathological calcifications (e.g., dental and urinary calculi and soft-tissue calcifications) in human, several types of calcium phosphate phases (ACP, DCPD, OCP, j3-TCMP, and CHA) coexist, suggesting changing conditions of pH and composition of the biological environment. The incorporation of F, Mg, C 0 3 , H P 0 4 , Sr, or CI affects the crystal growth and crystal properties of synthetic apatites. The presence of some of these ions and of P 2 0 7in solution affects the formation of apatites and other biologically relevant calcium phosphates. The knowledge on biological crystals has involved the development of synthetic bone graft substitute. Commercial and laboratory prepared CaP materials (HA, P-TCP, BCP, and coralline HA) or calcium carbonate (natural coral) have gained acceptance as materials for bone repair, substitution, and augmentation. Results from studies demonstrated that all these materials are dissolved or biodegraded to a greater or less extent, depending on the physical (density, macro- and microporosity, and surface topography) and crystallographic (defects and crystallinity) properties and composition of the biomaterials and on the extracellular and intracellular activity (phagocytosis processes). The cells involved in the biological resorption processes are still unknown. However, fibroblasts, monocytes, and osteoclast-like and giant cells are observed. During these dissolution/transformation processes, bioactive ceramics have the same evolution and adaptation to the tissues: (i) Partial dissolution of the CaP ceramic macrocrystals causes an increase in the calcium and phosphate concentrations in the local microenvironrnent; (ii) formation of C H A (either by direct precipitation or by transformation from one CaP phase to another or by seeded growth) incorporating ions (principally, carbonate) from the biological fluid during its formation; (iii) association of the carbonate/apatite crystals with an organic matrix; and (iv) incorporation of these microcrystals with the collageneous matrix in the newly formed bone (in osseous sites). The events at the CaP biomaterial/bone interface represent a dynamic process, including
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physicochemical processes, CrystaUproteins interactions, cells and tissue colonization, and bone remodeling, finally contributing to the unique strength of such interfaces. Acknowledgment The individual and collaborative studies involving the authors were suppported by research Grant CJF 93-05 from the INSERM and Grant EP 59 from CNRS (Dr. G. Daculsi, Director) and Grants DE04123 and DE07223 from the National Institute for Dental Research of the National Institutes of Health and special Calcium Phosphate Research Funds (Dr. R. Z. LeGeros, Principal Investigator).
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Passuti, N., Daculsi, G., Martin, S., and Deudon, C. (1991). Macroporous polycristalline calcium phosphate implant for spinal fusion in man and dogs. In “Handbook of Bioactive Ceramics,” Vol. 2, pp. 345-354. CRC Press, Boca Raton, FL. Passuti, N., Delecrin, J., and Daculsi, G. (1992). Bone substitution for spine fusion. In “Implants in Orthopaedic Surgery” (D. F. Williams, and P. Christel, Eds.), Chap. 12A. Arnold, Kent, UK. Piecuch, J. J. (1992). Augmentation of the atrophic edentulous ridge with porous replaniform hydroxyapatite (Interpore-200). Dent. Clin. North Am., 291-305. Posner, A. S. (1985). The mineral of bone. Clin. Orthop. Rel. Res. 200, 87-99. Rawls, H. R., LeGeros, R. Z., and Zimmerman, B. F. (1985). A radiopaque composite restorative using an apatite filler. .I. Dent. Res. 64,209. [Abstract 3071 Ray, R. D., and Ward, A. A. (1951). A preliminary report on studies of basic calcium phosphate in bone replacement. Surg Form. 3, 429-439. Reddi, A. H. (1985). Implant-stimulated interface reactions during collagenous bone matrixinduced bone formation. J . Biorned. Muter. Res. 19, 233-239. Rey, C., Renugopalakrishnan, V., Collins, B., and Glimcher, M. J. (1991). Fourier transform infrared spcctroscopic study of the carbonate ions in bone minerai during aging. Culcif Tissue Int. 49, 251-258. Richard, M., Aguardo, E., Daculsi, G., and Cottrel, M. (1996). Ultrastructural and electron diffraction of the bone-ceramic interfacial zone in coral and biphasic calcium phosphate in press. implants. Calcif: Tissue h., Ripa, L. W., Gwinnett, A. J., Gusman, C., and Legler, D. (1972). Microstructural and microradiographic qualities of lemon shark enameloid. Arch. Oral Bid. 17, 165-173. Robinson, C . , Kirkham, J., Brookes, S. J.. and Shore, R. C. (1992). The role of albumin in developing rodent dental enamel: A possible explanation for white spot hypoplasia. J . Dent. R ~ s 71, . 1270-1274. Robinson, R. A,, and Watson. M. L. (1952). Collagen-crystal relationships in bone as seen in the electron microscope. Anar. Rec. 114, 383-410. Ronholm, E. (1962). The amelogenesis of human teeth as revealed by electron microscopy. 11: The development of the enamel crystallitcs. J. Ultrustruct. Res. 6, 249-303. Rothwell, W. P., Waugh, J. S., and Yesinowski, J. P. (1980). High resolution variable temperature 31P NMR of solid calcium phosphates. .I. Am. Chem. Soc. 102(8), 2637. Roufosse, A. H., Aue, W. P., Roberts, J. E. I., Glimcher, M. J., and Griffin, R. G. (1984). An investigation of the mineral phases of bone by solid state magic angle sample spinning nuclear magnctique resonance. Biochemistry 23, 61 15-61 28. Rowles, S. L. (1968). The precipitation of whitlockitc from aqueous solutions. Bull. Soc. Chinz. (France). 1797- 1802. Roy, D. M., and Linnehan, S. A. (1974). Hydroxyapatite formed from coral skeleton carbonate by hydrothermic exchange. Nature 247, 220-227. Sakae, T., Yamamoto, H.. and Hirai. G. (1981). Mode of occurrence of brushite and whitlockite in sialotih. J . Dent. Res. 60, 842-844. Sauer, G. R., Zunic. W. B., Durig, J. R., and Wuthier, R. E. (1994). Fourier transform raman spectroscopy of synthetic and biological calcium phosphates. Calcif: Tissue Int. 54,414-420. Schroeder, H. (1969). “Formation and Inhibition of Dental Calculus.” Han Hubert, Vienna. Scott, D. B., Simmelink, J. W., and Nygaard, V. (1974). Structural aspects of dental caries. J. Dent. Res. 53, 165. Sclvig, K. A,. and Hake, A. (1972). Crystal growth in rat incisor enamel. Anar. Res. 173, 453-468. Sharer, W. G., Hine, M. R., and Levy. B. M. (1974). A textbook of oral pathology. Saunders Press, Philadelphia. Shirkahanzadeh, M. (1994). Bioactive calcium phosphate coatings prepared by electrodeposition. .I. Muter. Sci. Lett. 10, 1415-1417.
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The Biogenesis, Traffic, and Function of the Cystic Fibrosis Transmembrane Conductance Regulator Tamas Jilling and Kevin 1. Kirk Gregory Fleming James Cystic Fibrosis Research Center and Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, Alabama 35294
The cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMPactivated chloride channel that is encoded by the gene that is defective in cystic fibrosis. This ion channel resides at the luminal surfaces and in endosomes of epithelial cells that line the airways, intestine, and a variety of exocrine glands. In this article we discuss current hypotheses regarding how CFTR functions as a regulated ion channel and how CF mutations lead to disease. We also evaluate the emerging notion that CFTR is a multifunctional protein that is capable of regulating epithelial physiology at several levels, including the modulation of other ion channels and the regulation of intracellular membrane traffic. Elucidating the various functions of CFTR should contribute to our understanding of the pathology in cystic fibrosis, the most common lethal genetic disorder among Caucasians. KEY WORDS: Cystic fibrosis, Genetic disease, Ion channels, Membrane traffic, Protein processing.
1. Introduction The cystic fibrosis (CF) gene encodes a CAMP-activated C1- channel (i.e., the cystic fibrosis transmembrane conductance regulator; CFTR) that resides at the apical membranes of a variety of epithelial cells. The goals of this article are to review our current understanding of the biogenesis, intracellular processing, and traffic of the CFTR and to summarize our knowledge regarding its participation in epithelial cell function. Under~nr~wnurronsi nrVIPIV of
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standing early events in CFTR biogenesis is becoming one of the prime interests of C F researchers because a defect in the posttranslational processing of CFTR is considered to be the predominant mechanism by which the most common mutation causes disease. The mechanism and regulation of CFTR traffic beyond the biosynthetic pathway are also of interest because of evidence that CFTR recycles between endosomes and the apical plasma membranes of epithelial cells. We discuss the emerging notion that CFTR is not a passive cargo molecule in intracellular organelles and transport vesicles but rather it possibly regulates the composition and traffic of these compartments. A more detailed understanding of such a putative intracellular function for this ion channel might provide insights into the pathogenesis of CF, which is a pleiotropic disease that affects many epithelial surfaces. Cystic fibrosis is the most common lethal, autosomal, and recessive hereditary disease among Caucasians. The morbidity is approximately 1: 2000 live births in the general Caucasian population and can be considerably higher among families of northern European origin and mormons in Utah. The disease is characterized by impaired mucociliary clearance in the lung, recurrent respiratory infections, intestinal obstruction, male infertility, and, in most cases, pancreatic insufficiency (Boat et al., 1989). The life-limiting aspect of CF is a loss of pulmonary function as a result of recurrent respiratory infections and inflammation. However, other aspects of the disease such as male infertility are becoming more relevant as improvements in clinical treatments have prolonged the mean life expectancy of CF patients from 10 years in the 1960s to 28 years in 1990 (Fitzsimmons, 1993). This improvement has been due primarily to the refinement of CF treatment methods that focus on alleviating symptoms such as pancreatic enzyme replacement therapy, treatment of recurrent infections with antibiotics, and reducing inflammation and airway obstruction with pharmacological and physical methods. Novel therapeutic approaches that attempt to correct or bypass the cellular defect caused by CFTR mutations including in vivo gene therapy are currently under development (Welsh, 1995). Streptococcus and hnemophilus bacteria are the predominant lung pathogens in the first 2 years of life. Subsequent colonization of airways by Pseudornonas ueruginosa provides the major clinical challenge (Rubio, 1986). Recent data indicate that, in addition to bacterial infections, an exaggerated inflammatory response also contributes to the deterioration of pulmonary tissue in CF. An elevated level of inflammatory mediators exists in the airways of CF infants prior to the development of bacterial infections (Khan et al., 1995). The finding that CF patients respond favorably to high doses of the anti-inflammatory drug ibuprofen indicate that inflammation plays an active role in CF pathogenesis. Maintenance of a plasma concentration of ibuprofen at 50-100 pg/ml €or 4 years resulted in a statistically significant reduction in the rate of lung function deterioration in CF patients (Konstan et al., 1995). The beneficial effect of ibuprofen was most
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evident in patients who were enrolled in the study at less than 13 years of age before or during the early stages of pulmonary infection. As discussed below, a defect in the apical membrane C1- channel function of CFTR plausibly explains many of the cellular abnormalities in CF such as impaired mucociliary clearance. The relationship between this functional attribute of CFTR and the exaggerated inflammatory state of the CF lung is less clear. Studies addressing novel cellular roles of CFTR in epithelial cells, including a possible role in regulating membrane traffic and epithelial secretory processes (see Section VIII), might help us understand the link between CFTR and inflammation.
II. Cystic Fibrosis Transmembrane Conductance Regulator
A. CFTR: The Protein The CF gene encodes a protein that is 1480 amino acids (aa) in length (Riordan et aZ., 1989). CFTR splice variants of differing lengths have been reported (Chu et aZ., 1992; Strong et al., 1993), although no functional significance of alternative splicing has yet been described. Five functional domains were predicted on the basis of the deduced amino acid sequence at the time of the discovery of the CFTR gene (Riordan et al., 1989). This five-domain model of CFTR protein is now generally well accepted (Fig. 1). Four of the five domains form two symmetrical halves of the CFTR molecule, each being composed of a set of six membrane-spanning a helices and a cytoplasmic nucleotide binding domain (NBD). The two halves are separated by a cytoplasmic regulatory (R) domain that includes multiple phosphorylation sites for a number of protein kinases including cyclic AMPdependent protein kinase (PKA) and protein kinase C. The characteristic symmetrical arrangement of the membrane-spanning domains and the two NBDs places CFTR in the family of ATP binding cassette (ABC) transporters (Hyde er al., 1990). The R domain is a feature of CFTR that distinguishes it from other members of this family. Interestingly, several other members of the ABC transporter family serve as active transport pumps that couple ATP hydrolysis at one or both NBDs to solute transport (e.g., the bacterial oligopeptide permease, OPP; Hiles et al., 1987). No such active transport function for CFTR has been described.
B. CFTR: The CI- Channel The most well-accepted function of CFTR is that of a CAMP-regulated chloride channel. Although it was known for more than 50 years that an
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OUT
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IN FIG. 1 A schematic diagram of the CFTR. The 12 putative membrane-spanning domains are indicated by stippled rectangles. NBD, nucleolide binding domain; R, R domain; AF508, approximate location of the AFS08 mutation. Asterisk denotes approximatc locations of two extraccllular glycosylation sites.
elevated sweat NaCl content is a hallmark of CF, it was Ouinton (1983) who first showed that a reduced C1- conductance in the sweat duct is responsible for the defect in NaCl reabsorption in the CF sweat duct. The laboratories of Frizzell (Schoumacher et al., 1987) and Welsh (Li et al., 19SS) subsequently used patch clamp techniques to establish that the regulation of C1- channels by PKA was defective in C F airway epithelial cells. After the gene was cloned in 1989 in a joint effort by the laboratories of Collins, Tsui, and Riordan (Kerem et al., 1989; Riordan et al., 1989; Rommens et al., 1989), several groups showed that the defective CI conductances of CF epithelial cells could be complemented by introducing the wild-type CFTR cDNA or gene into these cells (Rich et al., 1990; Drumm et al., 1990; Jilling et al., 1990). The most definitive evidence that CFTR is a CI- channel rather than a regulator of endogenous CI- channels is that purified CFTR protein, when reconstituted into liposomes and incorporated into artificial lipid bilayers, generates PKA-activatable CI- channel activity that is indistinguishable from that observed in patch clamp studies of CFTR-producing cells (Bear et ai., 1992). On the basis of the results of these patch clamp
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and bilayer experiments, the following biophysical and pharmacological “fingerprint” of CFTR has emerged: (i) 5-10-pS single channel conductance (Tabcharani et al., 1991), (ii) linear I/V relationship under symmetrical ionic conditions; (iii) permselectivity of Br- 2 C1- > 1- > F- (Cliff and Frizzell, 1990; Cliff et al., 1992); (iv) activation by PKA phosphorylation and dependence of channel activity on cytosolic ATP (Bear et al., 1992; Cheng et al., 1991); (v) insensitive to the disulfonic stilbene DIDS, which blocks other CI- channel types (Cliff and Frizzell, 1990; Cliff et al., 1992); and (vi) blocked by the broad-spectrum CI- channel blocker, diphenylamine carboxylate (Schwiebert et al., 1994a), and the sulfonylurea, glibenclamide (Schultz et al., 1996), the latter of which also blocks ATP-dependent potassium channels in pancreatic p cells (Aguilar-Bryan et aZ., 1995). Figure 2 shows a typical patch clamp record of CFTR C1- channel activity stimulated by PKA plus ATP. The results of a large number of structure-function studies indicate that the C1- channel activity of CFTR is coordinately regulated by the R domain and the two NBDs. Channel activation requires both phosphorylation of the R domain and ATP binding by the NBDs. In most tissues the physiologically relevant kinase for CFTR phosphorylation is CAMP-dependent protein kinase (Li et al., 1988; Cheng et al., 1991), although the type I1 cGMPdependent protein kinase may also play a role in CFTR activation within the intestine (French et al., 1995). At least 11 different serine residues are phosphorylated by PKA in vivo, 10 of which occur within the R domain. Site-directed mutagenesis of these residues has revealed that all 11 phosphorylation sites and possibly others as well contribute to channel activation (Cheng et a/., 1991; Seibert et al., 1995). On the basis of these results, it has been suggested that CFTR C1- channel activity can be elevated in a graded fashion rather than all or none in response to stimulation of the cAMP pathway (Chang et al., 1993). Interestingly, deletion of the R domain (aa 708-835) from CFTR results in a C1- channel that is constitutively active in the absence of cAMP or PKA (Rich et al., 1991). This observation implies that phosphorylation of the R domain reverses an otherwise negative modulation of CFTR C1- channel activity by this structural domain. Conceivably, the minimally phosphorylated R domain occludes or blocks the conducting pore in a manner crudely analogous to the “ball and chain” mechanism for K+ channel inactivation originally described for the Shaker K+ channel in Drosophila (Zagotta et al., 1990). Each of the NBDs contains consensus sequences for ATP binding and hydrolysis (i.e., Walker A and B sequences) that are characteristic of the ABC transporters (Hyde et al., 1990). Each CFTR NBD binds ATP in vitro (Hartman et al., 1992; Randak et al., 1995), although only NBDl has been shown directly to possess ATP hydrolytic activity (KO and Pedersen, 1995). Several groups have argued on the basis of the results of mutagenesis
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Cell-attached
Excised
+ PKA & MghTP
0.3
PAL Is
FIG. 2 Representative current tracings of CFTR C1- channel activity in transl'ected mouse I, fibroblasts. Thc records shown were obtaincd from a single membrane patch that contained two active CFTR channels. The dashed lines represent the baseline or zero currcnt Icvcl. The pipette contained only impermeant cations in addition to CI so that the currents shown can only be due to the flow of CI- into the pipette. Forskolin (10 p M )was added to increase cellular CAMP and activatc the CFTR channels in thc cell-attached configuration approximately 100 s prior to the lirst record. Both channels inactivated within seconds following patch excision. Addition of protein kinase A (PKA, 150 unitslml) and Mg-ATP (300 p M ) to the solution bathing the cytosolic side of the excised patch immediately restored the channel activity. The holding potential (-60 mV) and the single channel current amplitude in the excised configuration (0.48 PA) yield an estimated unitary conductance of 8 pS, which is the cxpcctcd conductance of CFTR CI- channels at room temperature (22-25°C). The cells and experimental conditions were identical to those described by Venglarik ef ul. (1994), except for the lower temperature. The recordings were filtered at 100 Hi! (-3 dB attenuation) and sampled at 500 Hz. ~
experiments that both NBDs hydrolyze ATP, but with distinct functional consequences (Carson et al., 1995; Gunderson and Kopito, 1995; Wilkinson et ul., 1996).In particular, mutating the strictly conserved lysine residue in the Walker A box in N B D l (K464) reduced the rate of channel activation.
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Conversely, the analogous mutation in NBD2 (K12.50) stabilized C1- channel activity. Neither mutation decreased azido-ATP binding to CFTR (Carson et d., 1995), whereas analogous mutations in other ATP-binding proteins decreased the rate of ATP hydrolysis (e.g., Tian et d., 1990). These results indicate that CFTR C1- channels are either activated or primed for activation by ATP hydrolysis at NBD1. The duration of channel activation is then controlled by ATP hydrolysis at NBD2. In this regard, Carson et al. (1995) have argued that the NBDs serve as timer switches to regulate the amount of time that CFTR C1- channels are open, by analogy to the function of GTPases to regulate the duration of activation of downstream effectors. According to this model, ATP hydrolysis at NBDl and at NBD2 opens and closes the channel, respectively. Gunderson and Kopito (1995) have proposed a somewhat different model in which ATP hydrolysis at NBDl primes CFTR for channel activation, whereas ATP hydrolysis at NBD2 provides the thermodynamic driving force for the cycling of CFTR between closed and open conformations. Despite these differences in interpretation, the available data clearly indicate that the NBDs are critical and functionally distinct modulators of CFTR C1- current activity. The membrane-spanning domains presumably play a role in forming the conducting pore and perhaps the selectivity filter for this pore. Anderson et al. (1991) have reported that the halide selectivity of CFTR is determined in part by positively charged residues in the first and sixth putative transmembrane domains. Namely, they observed that the mutants K95D, K335E, and R347E exhibited a reversed permeability ratio for 1- and C1- compared to wild-type CFTR ( P I > PClrather than Pa > P I )when analyzed by whole cell patch clamping in transfected HeLa cells. These results have since been called into question by Hipper et al. (199.5), who observed no such reversal of the permeability ratios for the same mutants produced in Xenopus oocytes. The reason for this discrepancy is unclear but probably relates to the different expression systems and electrophysiological techniques used by these two groups. Regarding the ion selectivity of CFTR, this channel also conducts bicarbonate (Paulsen et al., 1994) and has been reported to conduct ATP as well (Reisin et al., 1994). As a bicarbonate channel, CFTR could contribute to the regulation of luminal and perhaps intracellular pH. The notion that CFTR conducts ATP is more controversial; in particular, Reddy et al. (1996) failed to detect ATP currents through CFTR C1- channels in a variety of experimental systems. It is important to resolve this issue because several groups have argued that CFTR could regulate neighboring ATP-dependent ion channels by serving as a transmembrane conduit for ATP secretion into the luminal space (see Section V1,A). In summary, the C1- channel activity of CFTR is regulated by an interplay between phosphorylation of the R domain and ATP binding and/or hydroly-
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sis at each of the NBDs. The available data indicate that the NBDs are not functionally equivalent CFTR domains, with several groups having proposed that NBDl and NBD2 serve as positive and negative regulators of channel activity, respectively (Carson et al., 1995; Baukrowitz et ul., 1994; Wilkinson et al., 1996). A certain degree of functional redundancy between the two halves of this bipartite molecule may also exist, however, as indicated by the analysis of a CFTR truncation mutant (D836X) by Sheppard et ul. (1 994). This mutant, which contains the first six transmembrane domains, NBD1, and the R domain, is capable of forming regulated C1- channels when expressed in HeLa cells. In cell lysates D836X protein cosedimented with mature CFTR on a sucrose gradient implying that this truncation mutant forms homodimers. D836X generates CI- channels that can be activated by CAMP and PKA and that have the same halide selectivity as wild-type CFTR. In several other respects D836X and wild-type CFTR differ; in particular, the truncation mutant exhibited a modest level of Clchannel activity in the absence of PKA phosphorylation and a considerably greater sensitivity to stimulation by Mg-ATP. These differences may be attributable to the proposed negative modulatory role of NBD2, which is lacking in D836X. The fact that D836X can form regulated C1 channels that are otherwise very similar to the wild-type protein implies that the two halves of this symmetrical molecule share functional similarities.
111. CFTR Mutations That Cause Disease A. Classes of Mutations Over 550 disease-causing mutations in CFTR have been reported, although only a small fraction of these have been functionally characterized (Zielenski and Tsui, 1995). Welsh and Smith (1993) have categorized CFTR mutations into four groups based on their influence on CFTR processing and function (Fig, 3). Class I mutants constitute nonsense, splice, and frameshift mutants that encode truncated or aberrant forms of CFTR (e.g., G542X). Such mutants constitute about one-half of the 550 different CF mutations that have been reported. A number of the mutants in this class associate with greatly reduced mRNA and protein abundance due presumably to instability of the altered transcripts. Many of these mutants associate with severe pathology (e.g., G542X) including pancreatic insufficiency, the most reliable indicator of disease progression in CF (see Section I). Interestingly, Howard et al. (1996) have reported that two premature stop mutations in the CFTR coding region (G542X and R553X) can be suppressed by treating cclls with modest doses of the aminoglycoside antibiotics G418 and gentami-
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LUMEN
4 Class
2 (AF508)
Class 1 (G542X)
INTERSTITIUM FIG. 3 The four major classes of CFTR mutations. An example of each class of mutation i s shown in parentheses. MSD, membrane spanning domains. Adapted from Welsh and Smith (1991) with permission from Cell Press.
cin. HeLa cells that were transiently transfected with GS42X or R553X CFTR cDNAs produced full-length CFTR protein and exhibited CAMPactivated halide permeability when cultured in the presence of 0.1 mg/nil G418. These results are consistent with previous reports that aminoglycosides can suppress stop mutations in a wide variety of organisms ranging from bacteria to mammalian cells (Martin et al., 1989). If clinically appropriate aminoglycosides are capable of suppressing premature stop mutations in endogenous CFTR transcripts within airway epithelial cells, such drugs may be useful for treating CF patients who harbor such mutations, which account for approximately 5% of all CF alleles. Class 11 mutants are processing mutants that produce protein that is incompletely glycosylated because it is retained within the endoplasmic reticulum (ER). These mutants constitute the most prevalent diseasecausing alleles including the AF.508 mutation, which accounts for approxi-
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TAMAS JILLING AND KEVIN L KIRK
mately two-thirds of all CF alleles. In general, class I1 mutations represent single amino acid substitutions or deletions in one of the two NBDs, although this feature is not unique to mutations in this class (see below). As expected, disease severity within this class correlates with the amount of mutant protein that can be released from the ER. For example, the AF508 mutation results in the production of full-length protein, but virtually none of this protein escapes the endoplasmic reticulum (Cheng et a]., 1990; see below for discussion of mechanism of E R retention). Consequently, there is a nearly complete failure of CFTR AF508 to mature beyond the E R glycosylated form of this protein, as can be monitored by one-dimensional SDS-PAGE analysis of immunoprecipitated protein (see Fig. 4). Not surprisingly, the AF508 mutation associates with severe disease (Johansen et al., 1991). In contrast, A455E and P574H are two processing mutants that associate with milder disease (Kerem et al., 1990; Kristidis et al., 1992). For each of these mutants some protein escapes the E R and becomes fully glycosylated at amounts that are intermediate between wild-type CFTR and the AFS08 mutant. Interestingly, the latter of these two mutants (PS74H) exhibits a compensatory increase in channel open probability relative to wild-type CFTR when produced as recombinant protein in HeLa or Vero cells (Sheppard et af., 1995; Champigny et af., 1995). This result provides additional evidence for the involvement of the first NBD and proline 574, in particular, in CFTR channel gating. The increased C1 chan-
WTCFTR
AF508CFTR
BZ"++
I
+
I
+
FIG.4 Characteristic profile of WT CFTR and AFS08CFTR visualized by SDS-PAGE analysis. LLCPKI cells stably transduccd with WT CFTR or AF508CFTR cDNAs under the transcriptional regulation of the Zn2'-inducible metallothionein promoter were subjected to CFI'R immunoprccipitation followed by in v i m phosphorylation. The fully processed form (band C) of WT CJTR protein can be detected even in the absence of exogenous Zn2' (is., the serum in the tissue culture medium contains some heavy metal), whcreas AF508CFTR remains virtually undetcctable under these unstimulated conditions. Following 25 h treatment with 100 gM Zn", an increase in WT CFTR band C and the appearance of hand B (i.e., ER form) in both the WT CFTR and AFS08CFTR immnnoprecipitates is obscrved.
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nel activity exhibited by P574H presumably contributes to the milder clinical phenotype in patients with this mutation. Class I11 and class IV mutants encode proteins that are fully glycosylated and that are targeted to the plasma membrane but are defective either in channel regulation (class 111) or in ion conduction (class IV). The class I11 regulatory mutants generally exhibit single amino acid substitutions in one of the NBDs (usually NBD1) that lead to reduced channel activation by ATP. The loss of channel activation by ATP can be partial (e.g., G551S) or virtually complete (e.g., G551D), which correlates with mild or severe disease, respectively (Anderson and Welsh, 1992; Strong et al., 1991; Cutting eta/., 1990). The conduction mutants (class IV) represent a small group of rare mutations in the membrane-spanning domains (e.g., R117H and R347P; Sheppard et al., 1993). These mutant channels exhibit reduced single channel conductances; namely, when open they conduct fewer ions per second than wild-type CFTR at the same electrochemical driving force (ca. 20 and 70% fewer for R117H and R347P, respectively). Not surprisingly, these partial loss-of-function mutants associate with milder clinical phenotypes such as pancreatic sufficiency (Kristidis et al., 1992). This categorization of CF mutations into four classes has heuristic value; however, it should be noted that some mutations can be included within multiple categories. For example, the AF508 mutation, which leads to profound defects in CFTR processing, also apparently results in a partially reduced open probability at the single channel level relative to wild-type CFTR under conditions when the production of fully glycosylated AF508 protein is artificially induced (Dalemans eta/., 1991; Denning et aL, 1992b, see below). In addition, because many CF patients (ca. 50%) are compound heterozygotes, some patients will possess mutant alleles from different classes that may associate with differing clinical severity. Compound heterozygotes that carry alleles associated with both severe and mild phenotypes typically exhibit the milder phenotype, given the recessive nature of this genetic disorder. Finally, there are reports of complex alleles in which second site mutations may influence the primary mutation. The R553Q mutant is a particularly interesting example of a second site mutation that has been found on an allele carrying AF508. The milder clinical pathology that associates with this complex allele implies that the R553Q mutation alleviates, or reverts, the severe phenotype that normally associates with the AF508 mutation (Dork eta/., 1991). This notion is consistent with the observation that the R553Q mutant serves as an intragenic suppressor of the AF508 mutation in a hybrid molecule containing a portion of CFTR NBDl that replaces an analogous region of the corresponding NBD of the yeast ABC transporter, STE6 (Teem et al., 1993).
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L. KIRK
B. Defective Biosynthetic Processing of the Most Common Mutant, AF508
What is the mechanism by which the most common mutant is retained within the endoplasmic reticulum? Interest in this question has been fueled by the observation that AFS08 protein is capable of eliciting CI- current activity, although probably not at wild-type lcvels (see previous section). On this basis it has been proposed that pharmacologic maneuvers that release AF508 protein from the E R and allow it to be targeted to the apical plasma membrane could be therapeutically beneficial (Denning er al., 1992b). No such maneuvers that are clinically appropriate have yet been identified in large part because of our limited understanding of the fundamentals of protein folding, retention, and degradation within the ER. However, certain aspects of the basic biology of protein folding in general and CFTR processing in particular are becoming clearer as a result of the pioneering studies of several laboratories, as discussed below. One of the most striking features of CFTR biosynthesis is that wild-type CFTR protein is also inefficiently processed within the ER. Typically, less than 20-30% of newly synthesized wild-type CFTR molecules escape from the E R to become fully glycosylated within the Golgi complex (Ward and Kopito, 1994; Lukacs et al., 1994). Instead, the majority of wild-type CFTR protein and virtually all AF508 protein is degraded within the ER with rclatively rapid kinetics (Tli2of 15-40 min depending on cell type). This inefficient processing of wild-type CFTR occurs both for native protein in epithelial cclls and for recombinant CFTR protein when heterologously expressed in nonepithelial cells (Ward and Kopito, 1994). Thus, it is a feature of the protein itself and not the epithelial tissues that normally cxpress it. The degradation of immature CFTR protein within the E R appears to involve in part the proteasome, a 26s cytosolic complex of multiple peptidases. The proteasome degrades a variety of short-lived cytoplasmic proteins that have been “tagged” by polyubiquitination, the covalent addition of a chain of ubiquitin molecules catalyzed by a series of ubiquitinating enzymes (see review by Ciechanover, 1994). Until recently there had been no compelling evidencc for the involvement of this cytosolic complex in the dcgradation of integral membrane proteins within the ER. However, two groups have now shown that the E R degradation of immature wildtype and AFS08 CFTR protein is slowed substantially by cell permeant inhibitors of the proteasome such as lactacystin (Jensen et nl., 1995; Ward et al., 1995). Polyubiquitinated forms of CFTR accumulate coincident with the lactocystin-induced inhibition of degradation within the ER. Moreover, the degradation of AFS08 CFTR within the E R is inhibited when this protein is coexpressed with a dominant-negative ubiquitin mutant (K48R)
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in HEK cells or when wild-type protein or AF508 is expressed in a cell line that contains a temperature-sensitive mutant of the ubiquitin-activating enzyme E l (Ward et al., 199.5). These results provide strong evidence for an important role for ubiquitination in the degradation of CFTR within the ER. This covalent modification presumably targets CFTR for degradation by the proteasome, although the involvement of other proteolytic pathways cannot be ruled out on the basis of the protease inhibitor studies performed to date. It is perhaps not too surprising that this cytosolic protease complex could participate in CFTR degradation because approximately 80% of the mass of CFTR is predicted to be cytosolic (Fig. 1). Establishing that ubiquitination and the proteasome participate in CFTR degradation represents an important advance in our understanding of the cell biology of this protein. Unfortunately, neither blocking this degradative pathway at the level of CFTR ubiquitination nor inhibiting the proteasome itself using protease inhibitors releases AF.508 CFTR protein from the ER. For example. when AF508 CFTR is coexpressed with the dominant-negative ubiquitin mutant K48R, AF508 protein that escapes short-term degradation gradually accumulates as a pool of Triton-X 100 insoluble material rather than being converted to soluble, mature CFTR (Ward et a/., 1995). Thus, ubiquitination appears not to be the only factor that prevents AF508 protein from proceeding along the biosynthetic pathway. Other possibilities include interactions between AF.508 protein and two putative molecular chaperones-cytosolic hsp70 and the E R resident membrane protein, calnexin. Both are candidate facilitators of protein folding; the latter of which binds to the carbohydrate moieties of partially folded glycoproteins within the E R (Ora and Helenius, 1995; Ware et al., 1995). The results of pulsechase, immunoprecipitation, and cosedimentation experiments indicate that immature wild-type CFTR and AF.508 CFTR each associate with hsp70 (Yang et al., 1993) and calnexin (Pind et al., 1994) within the ER. Wildtype CFTR molecules that have progressed to the Golgi, which occurs within 30-45 min of synthesis, do not bind either chaperone. Conversely, the interactions between AF508 protein, calnexin, and hsp70 are long lived (>1..5-3 h). It has not been formally demonstrated that hsp70 and calnexin bind to those immature wild-type molecules that ultimately escape the E R and progress to the Golgi; for example, it could be that these chaperones bind only to that population of wild-type molecules that is targeted for degradation within the ER. However, if calnexin and hsp70 do indeed transiently bind to wild-type CFTR molecules that are destined to be completely processed, then the release of CFTR protein from hsp70 and calnexin would be an important step in the commitment of newly synthesized protein to further biosynthetic processing in the Golgi. The stable association of AF508 CFTR protein with calnexin and/or hsp70 is conceivably due to incorrect folding of the mutant protein. Indeed, one
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of the proposed functions of calnexin is to retain partially folded proteins within the ER (Hammond and Helenius, 1994). These considerations lead to an important question: To what extent is AF.508 protein in the E R misfolded? Simply deleting phenylalanine 508 from bacterially produced NBDl has no obvious effect on nucleotide binding or on the solution structure of this recombinant polypeptide as determined by circular dichroism spectra (Hartman et af., 1992). Of course, this deletion could have more profound structural consequences in the context of the folding of full-length CFTR protein. In this regard, however, Pasyk and Foskett (1995) have shown quite elegantly that there exists AF508 protein within the ER that can fold sufficiently well to function as a CAMP-regulatable CI- channel. These investigators patch clamped the outer membranes of nuclei with attached E R that were isolated from Chinese hamster ovary (CHO) cells producing recombinant wild-type or AF508 CFTR. In either case single C1 channels were observed with properties identical to cell surface CFTR such as PKA dependence, DIDS insensitivity, and a single-channel conductance of about 8-10 ps. No such channels were observed for nuclei that were isolated from mock-transfected CHO cells. One cannot ascertain from these single-channel measurements the relative amount of AFS08 protein that is functionally viable, although CFTR CI- channels were observed with the same approximate frequency for nuclei that were isolated from AF508 CFTR-expressing cells compared to wild-type CFTR-expressing cells. The simplest interpretation of these results is that some AF508 protein can fold sufficiently well within the E R to perform the function of a regulatable ion channel. How can we incorporate these observations into a model that accounts for the inefficient processing of wild-type CFTR and the virtually complete retention of AF508 CFTR in the ER? Several groups have proposed such a model that is based on the relative kinetics of CFTR folding and degradation within the E R (Ward and Kopito, 1994; Lukacs el al., 1995). According to this model CFTR folding occurs with relatively slow kinetics due to the complicated and interrupted topology of this polytopic protein, which includes 12 membrane-spanning domains that are interrupted by two NBDs and by a unique R domain. Consequently, the kinetics of CFTR folding overlap with the kinetics of sorting incompletely folded proteins to the E R degradative pathway. As a result, a large fraction of newly synthesized CFTR becomes targeted for degradation before it can fold completely (Fig. 5). The AF508 mutation likely further retards the kinetics of CFTR folding without inducing gross misfolding, such that virtually all AF50X molecules are sorted to the degradative pathway before they can fold completely. The sorting of AF508 protein between the biosynthetic pathway and the degradative pathway presumably occurs upstream of ubiquitination; otherwise, blocking this covalent modification would lead to maturation of AF508 protein. The nature of this putative sorting event is currently unknown,
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WTCFTR
AF508CFI’R
Time Deadline for folding
FIG. 5 Putative timeline for the processing and degradation of wild-type (WT) and AF508 CFTR. Shaded regions represent populations of newly synthesized WT CFTR or AF508CFTR molecules. The “threshold for transit to Golgi” represents an arbitrary state of folding that permits newly synthesized molecules to proceed from the E R to the Golgi for further processing. The “deadline for folding” represents an arbitrary time window beyond which molecules that have not reached the aforcrnentioned threshold are targeted for degradation. Maneuvers that allow some AF508 protein to escape the ER, such as reduced temperature and glycerol, could extend the deadline for folding, increase the rate of folding within the ER, and/or reduce the threshold for transit to the Golgi.
although the release of calnexin and hsp70 from CFTR protein may be a corollary to this event (see above). Jensen et al. (1995) have also provided evidence for an ATP-dependent step in the maturation of wild-type CFTR within the E R than can be blocked by the peptide aldehyde, MG-132. The relationships between this step and the interactions between CFTR, calnexin, and hsp70 are currently unclear. One of the major challenges t o workers in this field will be to define at a molecular level the nature of the event or events that sort newly synthesized CFTR protein between the biosynthetic and degradative pathways in the ER. Are there any circumstances in which AF.508 protein can mature into fully glycosylated protein? According to the model described previously, if the kinetics of CFTR folding could be accelerated relative to the kinetics
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of protein sorting to the degradative pathway, some AF508 protein might fold completely before being committed to degradation and thereby escape to the Golgi. Hope for this scenario comes from two observations. First, the AF508 mutant is a temperature-sensitive mutant. Denning et al. (1992b), following up the observation that CAMP-activated C1- channels appeared on the surfaces of AF508 CFTR-expressing SF9 insect cells and Xenopus oocytes cultured at reduced temperature, showed that a moderate amount of AFS08 protein escapes the ER when mammalian fibroblasts are cultured at 23-26°C for 2 or 3 days. The maturation of AF508 protein at reduced tcmpcraturc was evidenced both as the appearance of the fully glycosylated form of CFTR by SDS-PAGE analysis and by the appearance of PKAactivated CI- channels at the surfaces of AFS08-expressing fibroblasts. Presumably, by reducing temperature the kinetics of CFTR folding are accclcrated relative to the kinetics of protein sorting to the degradative pathway with the result that some AF508 protein can “beat the clock” and fold completely before being targeted for degradation. Second, Sat0 et al. (1996) have shown that a substantial amount of AFSOX protein can mature, be delivered to the cell surface, and generate CAMP-activated C1- currents when AF.508-expressingcells are grown in 10%glycerol. Glycerol treatment retarded the degradation of both immature AF508 and wild-type CFTR protein. Unlike the situation in which ubiquitination is blocked, a significant fraction of AFS08 protein that escaped short-term degradation progressed to the Golgi and becamc fully glycosylated. This polyol likely functions as a chemical chaperone that facilitates AF508 protein folding by analogy to its well-known effects on protein folding and stability in vitro. By facilitating AF.508 protein folding, glycerol would be expected to shift the balance between maturation and degradation in favor of the former, as predicted by the kinetic model outlined previously. The utility of reducing temperature or of glycerol treatment as therapeutic strategies for releasing AFS08 protein from the ER is obviously limited. Indeed, neither maneuver has been rigorously shown to augment AFS08 protein maturation and function in polarized epithelial tissues, the target tissues of interest. Nonetheless, these observations raise the possibility that clinically appropriate maneuvers to release AFS08 protein from the E R that are based on facilitating protein folding may be feasible.
IV. Physiological Role of CFTR as an Apical CI- Channel in Epithelial Tissues CFTR residcs in part at the apical membranes of epithelial cells that line the pancreatic duct (Marino el al., 1991), airways (Puchelle et al., 1992),
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large intestine (Cohn et al., 1992), and reabsorptive sweat duct (Kartner et al., 1992). Most of the epithelial cells that express CFTR engage in regulated fluid and electrolyte secretion (Fig. 6). CFTR C1- channels at the apical surfaces of secretory cells serve as regulatable conduits for CI- efflux from the cell. This efflux is driven by an electrochemical potential gradient that favors passive C1- transport from cell to lumen, which in turn is generated by the operation of two parallel active transport processes at the basolateral membrane (Frizzell and Halm, 1990). Transepithelial CI- secretion electrically obliges Na' secretion via the generation of a lumen-negative transepithelial voltage and osmotically obliges fluid secretion via the generation of a transepithelial osmotic pressure difference. Apical CFTR CI- channels constitute the rate-limiting step in CAMP-stimulated electrolyte secretion by airway and intestinal epithelial cells, as evidenced by the defects in CAMP-dependent secretion exhibited by these cells in cystic fibrosis. CFTR C1- channels also come into play in cholera, another devastating disease of secretory epithelial cells. Cholera toxin promotes massive secretory diarrhea by markedly and chronically stimulating cAMP production in secretory epithelial cells within the colonic crypt (Field et al., 1972). Such elevations in cAMP activate apical CFTR C F channels and thereby fluid
4
4
+
Lumen
lnterstitium
FIG. 6 Model of secretory epithelial cell. CFTR C1- channels reside at the apical plasma membrane in series with three relevant basolateral transport processes: (i) the N a ' / K ' ATPase, (ii) the Na'l2CI-/K- cotransporter, and (iii) K' channels through which K ' that is transported into the cell by the Nat/Ki ATPase and the cotransporter recycles back into the interstitium. Water secretion is shown to be paracellular (i.e., between cells) in this schematic, although fluid secretion probably also occurs across the cells (i.e., via a transcellular pathway).
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secretion within the crypt, as explained previously. On this basis it has been argued that there may exist a heterozygote advantage for subjects that harbor a single defective CF allele (Quinton, 1994; Guggino, 1994). Heterozygotes presumably exhibit a partial loss of CFTR functional activity and, therefore, may be less sensitive to cholera toxin and other factors that promote secretory diarrhea. This notion might help explain the relatively high carrier frequency of mutant alleles among populations whose ancestors experienced cholera epidemics in the Middle Ages. However, apparently conflicting results have been obtained in studies of the effects of cholera toxin on intestinal fluid and electrolyte secretion by transgenic mice that lack one or both CF alleles (Gabriel et al., 1994; Cuthbert et al., 1995). CFTR is also expressed in the human reabsorptive sweat duct in which it facilitates NaCl reabsorption from the primary sweat secretion (Fig. 7). An elevated sweat C1- concentration has been the most reliable clinical indicator of CF since the 1950s (Gibson and Cooke, 1959). Quinton (1083) established that the CF sweat duct exhibits a reduced transepithelial C1
Lumen
ci
1I
ci
N.a+
Na'
+
lnterstitium FIG. 7 Model of reabsorptive sweat duct cell. The sweat duct epithelium has a low water permeability such that NaCl is reabsorbed in excess of water and the final sweat secretion is dilute with respect to plasma. For simplicity the epithelium is depicted as a single cell layer, although the sweat duct consists of two layers that function as a syncytium because the two layers are connected by gap junctions (Jones and Quinton, 1989).
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21 1
conductance that is attributable at least in part to a reduced CI- conductance of the plasma membrane ( Reddy and Quinton, 1989; Ram and Kirk, 1989). The C1- conductance of this tissue serves to shunt the transepithelial voltage that is generated by active Na' reabsorption. A reduced CI-conductance results in the development of a large transepithelial voltage that opposes Na' reabsorption by the CF sweat duct. As a result, the reabsorption of both ions is reduced and the NaCl concentration within the final sweat secretion, which is normally hyposmotic with respect to plasma, is elevated. One of the more interesting unresolved issues regarding the sweat duct is the observation that the CFTR-dependent CI- conductance of this tissue is tonically stimulated, unlike in other CFTR-expressing epithelia. To what extent this difference is attributable to tissue-specific variations in cyclic nucleotide metabolism versus differences in the CFTR molecule itself, such as alternative splicing, remains to be determined. Can a defective apical CI- channel in epithelial tissues explain the pathology in CF? For many but not all of the pathologies in CF there appears to be a relatively straightforward connection to a defect in apical C1conductance. Elevated sweat NaCl levels in CF subjects are easily explained by a reduced CIAconductance in the reabsorptive sweat duct, as noted previously. Pancreatic insufficiency, which associates with the more severe cases of CF, may be attributable to a defective apical CI- channel in pancreatic duct epithelial cells. CFTR C1- channels normally function in parallel with an apical CI-/HC03- cotransporter to secrete HC03- into the pancreatic duct lumen (Gray et al., 1988, 1994; Novak and Greger, 1988). CFTR channels may also participate in HC03- secretion by directly conducting HC03- ions across the apical membranes of pancreatic duct cells, as discussed in Section 11. HC03- secretion serves to neutralize acid emptied into the small intestine from the stomach and to provide an appropriate pH for pancreatic enzymes. A defect in HC03- secretion by the pancreatic duct could contribute to this aspect of the disease. Impaired mucociliary clearance in the Iung may be attributable at least in part to poor hydration of the mucous due to defective fluid secretion by airway epithelial cells. Despite the fact that certain pathologies in CF can be explained on the basis of a defective apical CI- channel in epithelial tissues, other clinical manifestations of this disease are not so easily explained in this way. These include an exaggerated inflammatory response in the CF lung in the apparent absence of infection (Section I), altered sialylation of secreted proteins in the meconium (Duthel and Revol, 1993), decreased ingestion of P. aeruginosa by airway epithelial cells (Pier et al., 1996), and elevated Na' reabsorption by CF airway epithelial cells (Boucher et al., 1986).The apparent lack of connection between these clinical manifestations and a defective apical CI- channel may simply reflect our incomplete appreciation of the physiological role that apical CI- channels play in modulating mucosal
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homeostasis. However, there is accumulating evidence that the function of CFTR extends beyond that of an apical C1- channel in ways that may better explain some of these other clinical abnormalities. In Sections VI and VIII we discuss two other putative functions of CFTR: (i) CFTR as a regulator of other apical ion channels (Section VI), and (ii) CFTR as a regulator of organelle composition, membrane traffic, and the biosynthetic pathway (Section VIII).
V. Regulation of CFTR Function by the Cytoskeleton A. Microtubules The activity of CFTR C1 channels at the surfaces of colonic epithelial cells is dependent to some extent on the microtubule cytoskeleton. Long-term exposure (2.5-3 h) of TX4cells (Fuller et a/., 19Y4) and airway epithelial cells (Schwiebert et al., 1994b) to inhibitors of microtubule polymerization, such as nocodazole and colchicine, moderately reduces CAMP-dependent halide permeability. Microtubule disruption has no effect on Ca2' -activated halide permeability (Fuller et al., 1094); thus, the inhibition appears to be specific for the CFTR-dependent C1 permeability pathway. How such microtubule inhibitors reduce CFTR-dependent halide pcrmeability is currently unclear, although it is tempting to speculate that they disrupt CFTR delivery to the cell surface via the recycling and/or biosynthetic pathways. Microtubule disruption dramatically inhibits trans-Golgi network (TGN)to-apical membrane traffic in epithelial cells (Matter et al., 1090b). Thus, if these inhibitors disrupt CFTR delivery to the cell surface from the TGN and/or recycling endosomes (see Section VII), they would be expected to downregulate the numbers of CFTR C1- channels at the cell surface.
B. Actin The actin cytoskeleton regulates CFTR C1- channel activity in hcterologous expression systems, although the mechanistic basis for this regulation is somewhat controversial. Fischer et al. (1995) have reported that cytochalasin D, a disrupter of microfilaments, rapidly (<2 min) stimulates CFTR C1 currents in transfected mouse fibroblasts without elevating cAMP levels. This stimulatory effect was markedly blunted by inhibiting PKA with the cAMP antagonist RpcAMPS or the inhibitory peptide, PKI. These investigators detected no effects of either cytochalasin D or purified actin on CFTR C1 channel activity in excised membrane patches. Accordingly, they
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conclude that polymerized actin (F-actin) indirectly regulates CFTR C1current activity, possibly by tethering a phosphatase near CFTR C1- channels in intact cells that would compete with PKA for channel phosphorylation/dephosphorylation. In contrast, Prat et al. (1995) have reported that purified actin does directly activate CFTR C1- channels independently of PKA stimulation in membrane patches excised from CFTR-transfected mammary adenocarcinoma cells. This effect of purified actin was blocked both by DNase 1, an inhibitor of actin polymerization, and by filamin, which cross-links actin into long filaments. O n the basis of these observations, Prat el al. (1995) have argued that CFTR C1- channels are directly regulated by short actin filaments, as also reported for epithelial Na' channels by these investigators (Cantiello et al., 1991). The reasons for the different effects of purified actin on CFTR channels in excised membrane patches reported by these two groups is unknown, although it may be related to the different cell types and actin concentrations (0.4 vs. 1.0 rng/ml) that were examined. These investigators do agree that the actin cytoskeleton is an important modulator of CFTR function, at least in the context of a heterologous expression system. Whether or not actin also plays this role in polarized epithelial cells remains to be determined.
VI. CFTR as a Regulator of Other Channels A. Na' Channels CFTR directly or indirectly modulates apical Na+ channels in airway epithelial cells. Boucher and colleagues (1986) reported a decade ago that Na' transport across airway epithelia is also abnormal in CF. In particular, the basal rate of net Nat reabsorption across short-circuited CF respiratory epithelium is elevated several-fold relative to control tissue. Maneuvers that increase intracellular CAMP levels such as isoproterenol treatment further increase the rate of Na+ reabsorption across CF, but not normal, tissue. Nasal epithelia isolated from transgenic mice that are homozygous for a targeted disruption of the murine CFTR gene also display abnormally elevated baseline Nat reabsorption (Grubb et al., 1994). More recent heterologous expression studies indicate that the increased Na' reabsorption exhibited by CF respiratory epithelium is likely due to the loss of a negative modulation of apical Na' channels by CFTR rather than to secondary effects related to disease pathology (Stutts ef aL, 1995). Specifically, the coexpression of CFTR with epithelial Na' channel subunits in MDCK renal epithelial cells or in mouse fibroblasts downregulates CAMP-stimulated Na channel activity. The functional coupling between these two channels +
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is independent of CI- transport through CFTR because it is observed in C1-free solutions. Whether or not this functional interaction between CFTR CIchannels and epithelial Na' channels is attributable to protein-protein interactions or to less direct means (e.g., a soluble intermediate) remains to be determined. The physiological implication of this interaction is that CFTR may control the balance between fluid secretion and fluid absorption both by serving as the rate-limiting step for CAMP-stimulated electrolyte secretion (Fig. 6) and by negatively modulating the rate-limiting step for electrolyte absorption-the apical Nat channel (Fig. 7). The pathological implication of this interaction is that the poor hydration of CF airways may be attributable as much or more to chronically elevated fluid reabsorption that is mediated by hyperactive Nat channels than it is to defective fluid secretion.
6. Outwardly Rectifying CI- Channels CFTR also appears to function as a positive modulator of another type of CI- channel, the outwardly rectifying CI- channel (ORCC). The ORCC is a second CAMP-activated CI- channel that is expressed in many of the same epithelial tissues that express CFTR (Schwiebert et al., 1994a). The ORCC has a distinct biophysical and pharmacological profile; i.e., rectifying current-voltage relationship, halide permeability sequence of I > Br- > CI-, larger single channel conductance, and sensitivity to disulfonic stilbenes (Li et al., 1988; Schoumacher et d., 1987). The ORCC and CFTR clearly are distinct molecules because the ORCC is present in tissues isolated from transgenic mice that lack CFTR protein (Gabriel el al., 1993). The most direct evidence for an interaction between these two CI- channels comes from the work of Jovov et al. (199Sa,b), in which CFTR C1- channels and ORCC were coreconstituted into lipid bilayers. Both channel types could be simultaneously activated in bilayers by the addition of PKA and ATP to the side representing the cytosolic face of each channel. However, ORCC activation by PKA and cytosolic ATP was prevented by immunodepleting CFTR from the starting material, by blocking CFTR C1- channels with a CFTR blocking antibody, or by coreconstituting the ORCC with a CFTR mutant that exhibits negligible CI- channel activity (GSSlD). Activation of the ORCC by PKA could then be restored by the subsequent addition of 0.1 mM ATP to the opposite side of the bilayer either when CFTR was blocked with an antibody or when the ORCC was coreconstituted with CFTR G551D. Importantly, under no conditions could the ORCC be activated by PKA plus ATP added to both sides unless either wild-type CFTR (plus or minus blocking antibody) or CFTR GS51D protein was present in the bilayer. This evidence that the GS51D mutant can support ORCC
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activation by PKA and symmetrical ATP implies that CFTR can modulate ORCC function independently of its C1- channel function. The additional requirement for ATP on the trans side has been interpreted as evidence for a second mode of regulation, namely, channel coupling via ATP transport possibly through CFTR channels. In support of this second mode of regulation, Jovov et al. (1995b) observed that activation of the ORCC by PKA and ATP in the presence of wild-type CFTR could be inhibited by adding hexokinase and glucose to the trans side in order to deplete ATP on this side. As noted in Section 11, the ability of CFTR to conduct ATP is controversial; thus, it remains to be rigorously shown if CFTR could regulate ORCC activity in this manner. An interesting analogy to the coupling between CFTR and ORCC may be the recently described coupling between the sulfonylurea receptor (SUR) and ATP-sensitive K' channels in pancreatic p cells. The SUR is an ABC transporter that appears not to function as an ion channel itself but confers sulphonylurea sensitivity and possibly ATP sensitivity on inwardly rectifying K' channels (Ammala et al., 1996; Inagaki et al., 1995). It will be interesting to determine if the coupling between CFTR and ORCC is mechanistically similar to SUR-K' channel coupling in the p cell.
VII. Itinerary of CFTR Traffic within Epithelial Cells A. CFTR Traffic from the Golgi to the Apical Cell Surface
The route taken by CFTR from the biosynthetic pathway to the apical poles of polarized epithelial cells is currently unknown and could be celltype specific. The delivery of this integral membrane protein from the trunsGolgi network to the apical cell surface could involve at least three different routes: (i) direct transport from the TGN to the apical membrane via Golgi-derived transport vesicles; (ii) initial transport from the TGN to the basolateral membrane followed by transcytosis to the apical membrane, as reported for apically destined proteins in hepatocytes (Bartles et al., 1987) and in some colonic epithelial cell lines (Matter et al., 1990b); and (iii) initial traffic from the TGN to apical recycling endosomes and from there to the apical cell surface. The third possibility (i.e., TGN to endosomes to apical surface) is particularly intriguing given the evidence to be described below that CFTR constitutively recycles between endosomes and the apical surfaces of colonic epithelial cells. To our knowledge there is currently no definitive evidence for the traffic of newly synthesized apical proteins from the Golgi to apical endosomes prior to their delivery to the apical surface. However, two groups have shown that the polymeric immunoglobin recep-
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tor is delivered first to apical endosomes and then to the apical cell surface as it is transcytosed from the basolateral membranes of transfected MDCK renal epithelial cells (Apodaca et al., 1994; Barroso and Sztul, 1994). In addition, the H1 subunit of the asialoglycoprotein receptor is transported from the TGN to endosomes prior to being delivered to the basolateral surfaces of MDCK cells,and newly synthesized transferrin receptors can first appear in endosomes before being delivered to the plasma membrane (Futter et al., 1995). As argued by Apodaca et al. (1994), endosomes may constitute important sorting compartments for routing membrane proteins to their appropriate intracellular domains, as will be discussed in the following section.
6. Dependence of CFTR Targeting on Epithelial Polarization CFTR targeting to the plasma membranes of colonic epithelial cells is dependent on the state of epithelial polarization. Morris et al. (1 992) have shown that undifferentiated HT29 colonic epithelial cells exhibit negligible CAMP-dependent CI- currents, even though they express as much mature CFTR protein as differentiated colonic epithelial cells that do exhibit CFTR CI- currents. The delivery of CFTR to the plasma membrane does not absolutely depend on acquisition of a polarized epithelial phenotype because thc heterologous expression of recombinant CFTR in a wide variety of nonepithelial cells does lead to the appearance of functional ClF channels at the surfaces of these unpolarized cells (Anderson et al., 1991; Dalemans et al., 1991). The retention of mature CFTR within unpolarized HT29 colonic epithelial cells is reminiscent of the retention of apical membrane markers within subapical compartments in dissociated MDCK renal epithelial cells, as originally described by Vega-Salas et al. (1988). It is unknown if the retention of mature CFTR within an intracellular compartment normally occurs during epithelial polarization in viva If so, it will be of interest to define the nature of this putative intracellular staging area for apically destined CFTR. These results also speak to the differences between epithelial cells and nonepithelial cells in terms of the organization and regulation of their membrane traffic pathways. Such differences must be kept in mind as we interpret the results of experiments in which the function of CFTR and its possible role in regulating intracellular membrane traffic are assessed in nonepithelial expression systems (see Section VIII). C. CFTR Recycling between Endosomes and the Apical Surface
CFTR resides in endosomes as well as at the apical membrane on the basis of a variety of morphological, biochemical, and biophysical criteria. Using
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immunoelectron microscopic methods, Webster et al. (1994) have localized CFTR to subapical compartments that are also positive for endosomal markers (i.e., rab4 and transferrin receptor) in submandibular gland duct cells. In addition, Bradbury et al. (1994) have shown that CFTR cofractionates with clathrin-coated vesicles (CCVs) purified from TE4colonic epithelial cells and coimmunoprecipitates with wadaptin, a specific component of the AP-2 complex that mediates coated pit formation at the plasma membrane. CFTR within this CCV preparation appears to be functional, namely, CCVs that are stripped of clathrin and then fused with lipid bilayers exhibit PKA-activated C1- channels with properties identical to those of cell surface CFTR. Functional CFTR also targets to endosomes as well as to the plasma membrane when heterologously expressed in fibroblasts (Lukacs et al., 1992; Biwersi and Verkman, 1994). The presence of CFTR both at the plasma membrane and within endosomes, and the physical association of this protein with wadaptin, imply that CFTR constitutively recycles between intracellular endocytic compartments and the cell surface. Prince et al. (1994) have provided functional evidence for this hypothesis using a two-step biotinylation procedure to monitor the kinetics of CFTR internalization and recycling in Tg4cells. These investigators observed that approximately 50% of the cell surface CFTR that could be derivatized at 4°C was rapidly internalized (<2 min) from the surface upon warming the cells to 37°C. The majority of this internalized pool of derivatized CFTR then reappeared at the cell surface over the course of 10-15 min. Recombinant CFTR protein, when heterologously expressed in pancreatic epithelial cells and COS fibroblasts (Copeland et a[., 1995), is similarly internalized from the cell surface into a recycling compartment, although with somewhat slower kinetics in the nonepithelial COS cells. A possible caveat regarding the interpretation of these results is that the reported experiments were not performed using epithelial cells that were cultured on permeable supports to promote the development of a fully polarized epithelial phenotype. One cannot exclude the possibility that the characteristics and quantitative importance of CFTR recycling are different for polarized epithelial monolayers compared to epithelial cells cultured on impermeable supports such as plastic dishes. Despite this caveat, the existing data support a model in which CFTR constitutively recycles between the cell surface and endosomes in a manner analogous to recycling membrane proteins such as the transferrin receptor (see Fig. 8). What is the functional significance of the recycling of CFTR between the cell surface and endocytic compartments? Three possibilities, which are not mutually exclusive, are worth considering: (i) sorting defective CFTR molecules from the cell surface pool for delivery to degradative compartments (i.e., lysosomes); (ii) routing CFTR C1- channels to intracellular
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plasma membrane
endosome
Lysosome FIG. 8 A schematic diagram of the endocyticlrecycling pathway. See text for discussion of the four steps in this pathway.
organelles where they could regulate the compositions and functions of these compartments; and (iii) regulating the numbers of CFTR C1- channels at the apical cell surface. The first possibility would constitute a quality control mechanism by which the integrity of the surface pool of CFTR would be maintained. One can only speculate as to how defective CFTR molecules would be recognized as such within the endocytic pathway, although it is possible that covalent modifications such as ubiquitination could target internalized CFTR molecules for degradation in lysosomes (e.g.,by analogy to STE2p in yeast; Hicke and Riezman, 1996). The second possibility would be analogous to the recycling of membrane proteins such as TGN 38/41 from the plasma membrane to intracellular compartments (in this case, the TGN) where they regulate intracellular membrane traffic and protein targeting (Jones et al., 1993). The emerging notion that CFTR has such an intracellular function will be discussed further in Section VIII. The third plausible function that is worth considering is that the CFTR recycling pathway serves as a control point for regulating the number of CFTR C1- channels at the apical cell surface. CFTR is only one of a number of transport proteins that recycle between intracellular endocytic compartments and the cell surface. Other examples include the insulinresponsive glucose transporter (GLUT4) in adipocytes (Shibata et al., 1995) and vasopressin-responsive water channels in distal renal epithelia (Brown and Sabolic, 1993). For these transport proteins the numbers of transporters in the plasma membrane are acutely upregulated by hormone-induced alterations in the kinetics of internalization and/or externalization. In this regard, it is interesting to note that CAMP regulates not only CFTR C1channel activity but also endocytosis and exocytosis in colonic epithelial cells and airway epithelial cells (see Section VIII for details). This coordinate regulation of endocytosis and exocytosis in CFTR-expressing epithelial
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cells raises the interesting possibility that cAMP modulates CFTR function at two levels: (i) by directly activating CFTR C1- channels via PKA phosphorylation and (ii) by increasing the numbers of CFTR C1- channels at the cell surface. Currently, the quantitative importance of the second mode of regulation is unknown. Indeed, early attempts to estimate the surface pool of CFTR in TS4cells using immunofluorescence methods (Denning et af., 1992a) or surface biotinylation (Prince et al., 1993) failed to detect an appreciable short-term effect of cAMP (<30 min) on the size of this pool. To what extent the PKA pathway participates in controlling the surface expression of CFTR over longer time periods or in combination with other potential modulators of CFTR recycling (e.g., cytokines, growth factors, etc.) remains to be determined. The probability that CFTR recycles between endosomes and the cell surface raises two questions: (i) What is the nature of this pathway? and (ii) What are the molecules that control this process and, ultimately, the number of CFTR molecules at the plasma membrane? Regarding the first question, CFTR recycling probably involves a series of at least four distinct membrane traffic events, by analogy to the transferrin receptor (Ghosh et al., 1994; Fig. 8): (i) the initial formation of CFTR-containing endosomes at the plasma membrane, (ii) the subsequent fusion of these endosomes to form sorting endosomes, (iii) the formation of recycling endosomes by membrane budding, and (iv) the fusion of recycling endosomes with the plasma membrane. It is also possible that some fraction of CFTR recycles through the TGN via a longer-range recycling pathway, as shown for a subset of apical membrane proteins in MDCK cells (Brandli and Simons, 1989). Although currently we know nothing about the molecular details regarding CFTR internalization and recycling, we can make educated guesses regarding the kinds of molecules that control the putative steps in CFTR recycling. The initial formation of CFTR-containing endosomes at the cell surface conceivably involves AP-2 and clathrin [as for the transferrin receptor (Draper et af., 1990; Chin et al., 1989)], given the biochemical evidence that CFTR coprecipitates with a-adaptin and cofractionates with clathrincoated vesicles in TS4cells (Bradbury et al., 1994). It is also reasonable to propose that molecules that control the fission of coated pits from the plasma membrane, such as the dynamin GTPases (Damke et af., 1994), may participate in CFTR internalization. Downstream steps in CFTR endocytosis and recycling presumably involve membrane fusion and vesicle budding reactions that are probably regulated by a host of factors; in particular, members of the ADP ribosylation factor (ARF) family, rab monomeric GTPase family, and the recently described SNAPS and SNARES (SNAP receptors). ARFs have been implicated as regulators of vesicle formation in the Golgi as well as in endosomes, where they probably
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function in concert with phospholipase D to facilitate membrane budding (Moss and Vaughan, 1995; Ktistakis et al., 1995). Zerial and colleagues (Bucci et al., 1992; Stenmark et al., 1994) have established that rab5 is a rate-limiting factor in endocytosis in a variety of cell types in which it serves as a positive regulator of endosome-endosome fusion. Each of these factors conceivably regulates apical CFTR endocytosis. Abundant evidence indicates that the soluble SNAPS and the membrane-bound SNARES (c.g., syntaxins and VAMPS) form molecular complexes that facilitate fusion between donor and acceptor membranes in cells ranging from yeast to mammalian neurons [see Rothman (1994) and Siidhof (1995) for excellent reviews]. Components of these SNAP-SNARE complexes are present in the apical endocytic pathway of colonic epithelial cells (e.g., syntaxins 1 A and 3; Naren et al., 1996) where they conceivably regulate fusion events in the recycling pathway. In this regard, Jo et al. (1995) have reported that a VAMP homolog (i.e., SNAKE) is an abundant protein in distal renal endosomes in which it regulates homotypic endosome fusion in vitro. This protein is positioned to regulate the recycling of vasopressin-sensitive water channels between the apical plasma membrane and endosomes, although direct evidence for this notion is lacking. In summary, we know very little about the molecular basis for CFTR recycling at the apical membrane; however, we expect that there exist many factors, such as ARFs, rabs, and SNARES, that control the various steps in this pathway. All are potential targcts for rcgulation (e.g., by PKA) and their characterization will be important for defining the molecular machinery that controls the numbers of CFTR C1- channels at the apical cell surface.
VIII. Regulation of Membrane Traffic by CFTR? Before the cloning of the CF gene and the identification of CFTR, two observations implied a connection between the regulation of membrane traffic and the function of the CF gene product. First, Sorscher et al. (1988) observed that CAMP regulated outbound membrane traffic as well as C1 permeability in TR4colonic epithelial cells. These investigators proposed that the regulation of chloride conductance in epithelial cells could take place via a CAMP-induced recruitment of C1- channels to the apical plasma membrane. Second, due to the accumulation of mucus in CF airways, an abnormality of mucin secretion by CF airway cells has been proposed (Frates et al., 1983; Quissell et al., 1983; Rudick et al., 1984) as a possible underlying mechanism for the obstructive pulmonary disease that is a hallmark of this disease.
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Initial studies that examined the connection between the regulation of membrane traffic and C1- permeability determined the effects of cAMP on endocytosis (i.e., the mechanism by which C1- channels can be removed from the plasma membrane), exocytosis (i.e, the mechanism by which CIchannels can be inserted into the plasma membrane), and membrane capacitance (i.e., an indicator of membrane surface area that increases as a result of increased insertion of membrane vesicles into the surface membrane or decreased endocytic retrieval of plasma membrane). Following the cloning of CFTR, subsequent studies addressed the traffic of CFTR itself and also the function of CFTR C1- channels in endosomes and in the TGN. A number of both negative and positive findings regarding the functional role of CFTR in intracellular membranes have generated a great deal of controversy. Our discussion of these data will attempt to resolve this controversy by pointing out the differences in model systems and membrane traffic markers that have been used in various studies of regulated membrane trafficking. In general, the majority of studies that have been conducted using epithelial model systems have supported the idea that CFTR regulates endocytosis, membrane recycling, the acidification of the TGN, and the CAMP-dependent regulation of epithelial macromolecule secretion. On the other hand, studies that have addressed the functional role of CFTR in membrane traffic or vesicular acidification in nonepithelial model systems have indicated that CFTR does not influence either vesicular acidification or the regulation of membrane traffic in these systems.
A. Endocytosis The regulation of endocytosis by cAMP in CFTR-expressing cells has been analyzed using both fluid phase and absorptive endocytic markers in combination with enzymatic as well as optical detection methods (Bradbury et al., 1992b). Using either type of endocytic tracer or either detection method, it was observed that treatment of TS4colonic epithelial cells with f o r s k o h (10 p M ) significantly inhibited (by 30-60%) the intracellular accumulation of the endocytic markers. Endocytosis was also inhibited by cpt-CAMP, a cell permeant analog of CAMP, and by vasoactive intestinal peptide, which activates adenylate cyclase by a receptor-dependent mechanism. Dideoxyforskolin, a forskolin analog that exhibits some of the non-CAMPdependent effects of forskolin but has no effect on cellular cAMP production, was without effect on endocytosis. These data confirm that the inhibitory effect of forskolin on endocytosis was a CAMP-specific effect. It should be noted that the available data do not distinguish between effects of cAMP on the initial formation of endocytic vesicles at the plasma membrane versus regulation of downstream steps in the endocytic pathway such as
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endosome-endosome fusion. It is also conceivable that the reduced accumulation of endocytic tracer in the presence of cAMP is due in part to stimulation of a rapid recycling of internalized material back to the cell surface (see below). Nevertheless, by using two independent methods of detection, several laboratories reached the same conclusion that cAMP inhibits the accumulation of endocytic markers in TX4colonic cells (Bradbury et al., 1992a,b; Prince et al., 1994; Santos and Reenstra, 1994). A cystic fibrosis pancreatic adenocarcinoma epithelial cell line (CFPAC1) has been used to specifically assess a possible role for CFTR in the regulation of endocytosis. These cells were generated from a pancreatic ductal carcinoma of a cystic fibrosis patient (Schoumacher et id., 1990) who was homozygous for the most common CF mutation, AFSOX. Consequently, these cells do not produce any detectable CFTR and are widely used for complementation experiments in which heterologously expressed wild-type CFTR complements the loss of function. For studies addressing the role of CFTR in the regulation of membrane traffic, CFPAC-I cells stably transduced with wild-type CFTR (CFPAC-PLJ-CFTR) or with retroviral vector alone (CFPAC-PLJ) were used (Drumm et al., 1990). The production of functional CFTR in the CFPAC-PLJ-CFTR cells was verified by multiple laboratories using various assays of CAMP-activated halide permeability. Such CAMP-stimulated C1- permeability is absent from the mocktransduced CFPAC-PLJ cells. Similar to TX4cells, cAMP inhibited the accumulation of endocytic markers in CFPAC-PLJ-CFTR cells, as determined by both the enzymatic and the microscopic assays of endocytosis (Bradbury et al., 1992b). Cyclic AMP had no effect on endocytosis in CFPAC-PLJ cells that do not produce functional CFTR. A recent study of these cell lines using a different assay of endocytosis failed to demonstrate a correlation between CFTR expression and endocytic membrane traffic (Dunn ef al., 1994). However, these results should be interpreted with caution because transferrin, i.e., a basolateral endocytic marker (Barroso and Sztul, 1994; Fuller and Simons, 1986; Podbliewicz and Mellman, 1990), was used to investigate the regulation of endocytosis by CAMP. Because there is no evidence for the presence of CFTR in the basolateral endocytic pathway, the lack of regulation of transferrin endocytosis in CFTRexpressing cells is perhaps not surprising. 6. Does CFTR Regulate Its Own Traffic? If CFTR enters thc endocytic recycling pathway, as discussed in Section VII, and if CFTR regulates the movement of membranes in this recycling pathway, then it is feasible that CFTR can autoregulate its own trafficking. The fact that CFTR is glycosylated on its fourth predicted extracellular loop
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allows the specific testing of this prediction. Namely, site-specific labeling of CFTR molecules that are on the cell surface by domain-selective biotinylation of these carbohydrate moieties enables the monitoring of CFTR movement between the surface membrane and intracellular vesicles. Such experiments have shown that CFTR is rapidly internalized from the cell surface at 37"C, with 50% of cell surface CFTR internalized within 2 min (see Section VII) (Prince et al., 1994). This rapid internalization of CFTR resembles the internalization rates observed for the LDL receptor and for the transferrin receptor, both of which are internalized in a clathrindependent manner (as also proposed for CFTR; Section VII). Subsequent to its endocytosis, CFTR recycles to the cell surface. Prince et al. (1994) have used this method to show that the elevation of intracellular cAMP levels results in a decrease in the rate of CFTR internalization from the cell surface. This inhibitory effect of cAMP on CFTR internalization was comparable to the degree to which cAMP inhibited the endocytosis of fluid phase markers in the experiments described previously. Furthermore, the inhibition of CFTR internalization by cAMP was dependent on the ability of CFTR to conduct C1-. Specifically, the G551D mutant was internalized at rates comparable to wild-type (WT) CFTR under baseline conditions, but cAMP did not decrease the rate of internalization compared to wild-type CETR. Unlike processing mutants such as AF508 CFTR, G551D CFTR escapes the E R to become fully processed but has a defect in its channel activation due to a mutation of a critical glycine residue in the ATP-binding region of the first NBD. The lack of regulation of G551D CFTR recycling by cAMP is consistent with the notion that the regulation of CFTR trafficking by cAMP is related to its CAMP-regulated C1- channel activity. As noted previously, it is currently unknown if this CAMP-dependent regulation of endocytic membrane traffic and CFTR recycling is due to an inhibition of endosome formation at the plasma membrane, an inhibition of endosome fusion, or a stimulation of recycling from early endosomes to the cell surface. Nevertheless, the preceeding findings that were obtained in several studies using independent assays of apical endocytosis and CFTR traffic point to a role for CFTR in the regulation of a step or steps in the apical endocytic/recycling pathway. C. Exocytosis
In the strictest sense, exocytosis represents the fusion of an exocytic transport vesicle with the plasma membrane and the subsequent dumping of the vesicle contents into the extracellular space. As we discuss the role of CFTR in exocytosis we will use a broader interpretation of this term that
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includes all the steps in outbound membrane traffic from the cell interior to the cell surface. This outbound membrane traffic can be dissected into three main components: (i) transport vesicle formation at the TGN or at sorting/recycling endosomes, (ii) delivery of the transport vesicles to the cell surface via the involvement of cytoskeletal elements, and (iii) bona fide exocytosis, i.e., vesicle fusion with the plasma membrane. Several findings indicate that CFTR probably plays a role in the CAMP-dependent regulation of outbound membrane traffic, but again there is limited information available as to which of the previously mentioned three steps of the exocytic pathway are sites of regulation by cAMP in a CFTR-dependent manner. Bradbury et al. (1992b) used the same heterologous expression system that was used to investigate the role of CFTR in the regulation of endocytosis (i.e., CFPAC-1 cells stably transduced with wild-type CFTR and corresponding mock-transfected controls) to examine the role of CFTR in the recycling of endocytic markers to the cell surface. The lectin wheat germ agglutinin (WGA) was used to label glycoproteins in the recycling pathway. Following the loading of the recycling pathway with WGA-biotin and blocking the biotin remaining on the cell surface with unlabeled avidin, the recycling of WGA-biotin from intracellular compartments was monitored based on the binding of fluorescently labeled streptavidin to the cell surface. It was found that both forskolin and cpt-CAMP stimulated the recycling of biotin-WGA to the cell surface by 2.5- to 3-fold in CFPAC-PLJ-CFTR cells. Neither forskolin nor cpt-CAMP had any effect on WGA-biotin recycling in the mock-transfected CFPAC-PLJ cells that do not express functional CFTR. The regulation of exocytic membrane traffic by CFTR has also been analyzed in human airway epithelial cell lines that were isolated and immortalized from surgical specimens obtained from CF and non-CF individuals (Schwiebert et al., 1994b). The rate of exocytosis was assayed by measuring changes in membrane capacitance with whole cell patch clamp analysis and by the measurement of the recycling of previously endocytosed FITCdextran, i.e., a fluid phase endocytic marker. Membrane capacitance measurements indicated that treatment of airway epithelial cells from non-CF individuals with cpt-CAMP (100 pM) resulted in a significant increase in membrane capacitance, indicative of a net addition of membrane to the plasma membrane. This cAMP stimulation of cell surface area coincided with the stimulation of FITC-dextran recycling from intracellular membranes t o the extracellular medium, which implies that the CAMPdependent increase in whole cell capacitance was due in part to the fusion of exocytic transport vesicles with the surface membrane. Under the same experimental conditions, cAMP had no effect on either whole cell capacitance or dextran exocytosis in airway cells derived from CF individuals.
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Mergey et al. (1 995) have provided similar evidence for a positive regulation of exocytosis by CFTR using a marker of the secretory pathway in immortalized airway epithelial cells derived from CF and non-CF individuals. The secretion of glycoconjugates was monitored in CF and non-CF airway epithelial cells following the labeling of complex carbohydrates with a [ ''C]glucosamine precursor. These investigators observed that stimulation of PKA with isoproterenol (10 p M ) or forskolin (10 p M ) , as well as the stimulation of PKC with PMA, resulted in an enhanced secretion of 14Clabeled glycoconjugates in non-CF cells. Conversely, the stimulation of glycoconjugate secretion by PKA was absent in CF cells, whereas the stimulation via PKC was similar to that in non-CF cells. The defective regulation of glycoconjugate secretion by cAMP in the CF cells was corrected by adenovirus-mediated transfer of WT CFTR to these cells, indicating that the defect in the CAMP-dependent regulation of glycoconjugate secretion in the CF cells was due to the absence of functional CFTR. In summary, all three of these studies found a close correlation between the expression of functional CFTR C1- channels and the regulation of exocytosis in epithelial cells. The specific mechanism(s) by which CFTR might regulate exocytic membrane traffic is unknown, although CFTR C1- channel function in intracellular membranes has been proposed as a potential basis for this regulatory activity, as will be discussed later. It should be noted that contradictory results regarding the role of CFTR in regulating exocytosis have been obtained in studies of CHO fibroblasts transduced with CFTR. Dho et al. (1993) observed no regulation of membrane recycling by cAMP in CHO cells using the previously mentioned assay of WGA-biotin recycling, regardless of the expression of CFTR. These negative data for CHO cells might reflect differences between epithelial cells and unpolarized fibroblasts regarding the nature and regulation of membrane traffic pathways. Indeed, according to our unpublished observations, endocytosis and protein secretion are also not regulated by cAMP in mouse L cells, and this lack of regulation is unaffected by the heterologous expression of WT CFTR. In a later section we discuss the possible reasons why fibroblasts might differ from epithelial cells regarding the participation of CFTR in the regulation of membrane traffic.
D. The TGN and Recycling Endosomes as Possible Sites of CFTR Regulation of Membrane Traffic Direct membrane traffic from intracellular membranes to the cell surfaces of epithelial cells can occur from at least three intracellular membrane domains: (i) the TGN, (ii) secretory granules, and (iii) recycling endosomes. The direct pathway from the TGN to the cell surface is often referred to
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as the constitutive secretory pathway. Secretory granules receive input from the TGN and store the secretory material until the stimulus-induced release of this material to the extracellular space. Recycling endosomes receive input from both the cell surface and the TGN. Although two studies (Kuver et al., 1994; Mergey et al., 1995) have found a close correlation between the expression of WT CFTR and the CAMPdependent secretion of glycoconjugates, the relative involvement of the constitutive secretory pathway (i.e., direct traffic from the TGN to the cell surface) and the regulated secretory pathway (i.e., the acute release of secretory material from secretory granules) was not specifically addressed. In a recent study in our laboratory we investigated such mechanistic details regarding the CAMP-regulated protein secretory pathway in polarized monolayers of colonic epithelial cells (Jilling and Kirk, 1996). We observed that in HT29-CL19A cells, a tissue culture model of the colonic crypt that expresses endogenous CFTR, newly synthesized proteins are secreted predominantly via the constitutive secretory pathway. The rate of apical but not basolaterai constitutive secretion was stimulated two- to fourfold by cAMP in a manner that required the presence of C1- ions, supporting the hypothesis that a CAMP-regulated C1- channel is involved in the regulation. Furthermore, cAMP also stimulated the rate by which one of the secreted proteins, cwl-antitrypsin, was sialylated in the TGN, indicating that the TGN is at least one site of the CAMP-dependent regulation of constitutive protein secretion. Although this study did not specifically investigate the role of CFTR in the regulation of the secretory pathway, the data are consistent with the previously mentioned evidence for a role for CFTR in the regulation of apical exocytic membrane traffic.
E. A Model of the CAMP-and CFTR-Dependent Regulation of Membrane Traffic Endosomes, lysosomes, and the TGN have acidic intraorganellar pH. This acidic interior is required for the functions of enzymes with acidic p H optima in the lysosomes and in the TGN, for the dissociation of ligands from their receptors in recyclinghorting endosomes, and for coat formation on membranes. Intraorganellar acidification in the TGN and in endosomes is generated by the vacuolar H+-translocating ATPase and requires the presence of a C1- conductance in the organellar membrane to shunt the dectrical potential difference that is generated by the movement of positive charges (see Fig. 9). In the absence of a permeable anion or an anion conductance in the organellar membrane, acidification is limited by the generated membrane potential that serves as a thermodynamic barrier to proton transport (Bae and Verkman, 1990; Zeuzem et al., 1992).
BIOGENESIS, TRAFFIC, AND FUNCTION OF CFTR
Mechanism of TGN acidiacation
ADPiP
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\
v.
chCFTR?
TGN acidification promotes:
I
Budding of secretow vesicles
FIG. 9 Putativc mechanism by which CAMPcould regulate both sialylation and vesicle budding at the TGN via a CFTR-dependent mechanism.
Because CFTR is present and functional in endosomes (Lukacs et al., 1992) (see Section VII), it could fulfill this requirement for a C1- conductance pathway for acidification.The results of several studies have indicated a correlation between the expression of WT CFTR and the degree of endosomal acidification in epithelial cells using various measures of endosoma1 acidification (Al-Awqati, 1995; Barasch and Al-Awqati, 1993; Barasch et al., 1991; Dosanjh et al., 1994). Using the accumulation of weak bases in acidifying organelles as a marker of acidification, Barasch et al. (1991) provided evidence that endosomal acidification is defective in the TGN of airway epithelial cells cultured from CF patients when compared to airway epithelial cells cultured from non-CF individuals. The same group has also demonstrated a close correlation between endosomal acidification and CFTR expression in the previously described CFPAC-PLJ 2 CFTR model. Sialylation of glycoproteins, an acidification-dependent function of the TGN, has also been shown to be dependent on WT CFTR expression in airway epithelial cells and in C127 mammary epithelial cells (Dosanjh et ab, 1994; Barasch et al., 1991).
1. Connection between Acidification, Membrane Traffic, and Protein Sialylation? Yilla et al. (1993) observed an intimate relationship between endosomal acidification, the sialylation of secretory proteins in the TGN, and the rate
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of constitutive protein secretion in hepatocytes. Inhibition of the vacuolar H+-ATPase by concanamycin in HepG2 cells resulted in a reduced rate of the sialylation of al-antitrypsin (i.e., one of the secreted proteins) and a reduced rate of constitutive secretion. Our studies of colonic epithelial cells have shown that cAMP stimulated both the rate of constitutive secretion and the rate by which al-antitrypsin was sialylated in the TGN. The regulation of constitutive secretion required the presence of C1-, thus suggesting the involvement of a C1- channel in the regulation. Considering the proposed role of CFTR in TGN acidification, all these data are consistent with the hypothesis that a CAMP- and CFTR-dependent regulation of TGN acidification might underlie the regulation of both constitutive protein secretion and protein sialylation by CAMP.The stimulation of sialylation by cAMP is likely due to enhanced TGN acidification that favors sialyltransferase activity and/or the uptake of sialic acid precursors by the TGN. The mechanistic coupling between TGN acidification and the rate of constitutive secretion is conceivably related to the acidification dependence of coat formation on TGN membranes (Fig. 9). In particular, Zeuzem et al. (1993) demonstrated that the binding of A R F to microsomal membranes was enhanced by vacuolar acidification. ARF binding to microsomal membranes was inhibited by removing ATP or C1-, by a C1- channel blocker, and by an inhibitor of the vacuolar proton ATPase (bafilomycin A,). ARF binding is required for coat formation and, therefore, for secretory vesicle formation at the TGN. The regulation of A R F binding by PKC has been shown to regulate the rate of constitutive secretion by rat basophilic leukemia cells (De-Matteis ef al., 1993). Therefore, if TGN acidification is regulated by cAMP in a CFTR-dependent manner, this could lead to a regulation of coat formation and secretory vesicle budding from the TGN. The budding of recycling endosomes from sorting endosomes probably occurs via a similar coat-mediated mechanism (Matter et al., 1993). Because sorting endosomes are also acidifying organelles, the regulation of membrane recycling by cAMP and the regulation of secretory vesicle generation at the TGN could be mediated by similar mechanisms, namely, a CAMP- and CFTR-dependent regulation of acidification that could modulate coat formation and membrane budding. 2. Polarity of Regulation
Given this model of the regulation of the biosynthetic pathway by CFTR C1- channels, how do we explain the fact that cAMP regulates the rate of apical but not basolateral constitutive protein secretion in polarized colonic epithelial cells? The TGN is the first known polarized compartment of the biosynthetic pathway. This polarity has been demonstrated by both functional and morphological criteria (Simons and Wandinger-Ness, 1990;
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Ladinsky et al., 1994; Mostov and Cardone, 1995). Functionally, it has been shown that transport vesicles deriving from the TGN can target to at least four distinct membrane compartments-the apical and the basolateral plasma membranes, lysosomes, or nascent storage vesicles. High-voltage electron microscopic studies of the TGN of normal rat kidney cells have indicated that there are two morphologically distinct coat structures that can be observed on budding vesicles on the tubules that comprise the TGN (Ladinsky et at., 1994). Each tubule bears only one of the two kinds of coats. The exact relationship between the structural and functional polarity at the TGN is yet to be determined. However, it is interesting to speculate that a single TGN tubule generates only transport vesicles destined for one of the previously mentioned destinations. Thus, apically destined CFTR could target to TGN tubules from which only apical secretory vesicles are generated and could regulate the pH and the rate of budding of this compartment in a CAMP-dependent manner. This speculation would also imply that CAMP-independent Cl- channels would target to the other TGN tubules, from which vesicles destined for the basolateral plasma membrane and lysosomes would form. An alternative mechanism for the polarized regulation of the secretory pathway could involve the recruitment of direction-specific components (Lafont et al., 1994), such as direction-specific motor proteins (i.e., dynein or kinesin), onto apically destined transport vesicles in a pH-dependent manner. Such a mechanism would allow the generation of a greater number of apically destined vesicles from a single, unpolarized compartment upon enhanced acidification of this compartment. Such regulated budding of apical but not basolateral transport vesicles from a single TGN compartment would predict that the stimulation of apical secretion corresponds to an inhibition of basolateral secretion via a competition for substrate. However, during the course of our studies of the regulation of polarized protein secretion in HT29-CL19A cells we failed to observe a reduced basolateral secretion that coincided with the stimulation of apical secretion. Consequently, we believe that this latter mechanism of polarized regulation of the secretory pathway is less likely. 3. Importance of the Proper Model System to Study the Role of CFTR in Regulated Membrane Traffic
The Cl- conductance of an intracellutar organelle plays only a permissive role in acidification; thus, the presence of CFTR may not be limiting for acidification in membranes in which other C1- channels are present. To date, more than a dozen distinct C1- channels have been identified, some of which are expressed in a tissue-specific manner (Valverde et al., 1995). This latter point might be crucial for resolving some of the controversies
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that exist in CF research regarding the various proposed cellular roles of CFTR in general and in endosomal acidification in particular. When CFTR is heterologously expressed in cells that do not normally produce this C1channel, the appearence of a novel CAMP-regulated C1- conductance at the cell surface can be verified by applying a driving force for C1 movement (i.e., an electrical potential difference in patch clamp studies or a concentration gradient in flux assays) and testing for the regulation of Cl- permeability by CAMP. The presence of a CFTR CI- conductance in endosomes of fibroblasts that are heterologously expressing CFTR has been demonstrated by similar principles (Lukacs etal., 1992). In the latter experiments vesicular C1 conductance was made rate limiting by imposing concentration driving forces and by imposing parallel conductances via proton ionophores. Under normal conditions, including those conditions under which membrane traffic assays are typically conducted, the vacuolar Ht-ATPase provides the driving force for acidification and, consequently, for C1 transport across vesicular membranes. In a cell that does not normally produce CFTR but has competent vesicular acidification, such as the majority of nonepithelial cells, a non-CFTR C1- conductance pathway is presumably expressed in endosomal membranes to support acidification. Under these conditions the C 1 conductance ~ may not be rate limiting; thus, the heterologous expression of CFTR in such cells might have no effect on endosomal acidification. On the other hand, in the apical recycling pathway and perhaps in a polarized subcompartment of the TGN of airway and intestinal epithelial cells CFTR might be the major C1- conductance. On this basis the controversy between data supporting the role of CFTR in vesicular acidification in epithelial cells and the negative data obtained in heterologous expression studies in fibroblasts could be resolved. On the same basis, any functional attribute of CFI'R that is presumably downstream of its role in vesicular acidification (i.e., modulating sialylation or membrane vesicle budding) could not be evoked in fibroblasts by the heterologous expression of CFTR. For this reason, CFTR knockout studies in epithelial cells that normally express CFTR, or genetic complementation studies in CF epithelial cells, will be necessary to elucidate the role of CFTR in membrane traffic processes.
4. The Significance of the CAMP- and CWR-Dependent Regulation of Apical Membrane Traffic As discussed in Section VII, many proteins that are expressed o n the surfaces of polarized epithelial cells continously recycle between the plasma membrane and various intracellular membrane domains (e.g., endosomes and the TGN). It is intriguing to speculate that the regulation of apical membrane traffic in a CAMP-and CFTR-dependent manner might influence the traffic and distribution of CFTR as well as of other transporters and/
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or receptors. For example, epithelial sodium channels (ENaCs) have been shown to possess tyrosine-based internalization signals that when mutated alter ENaC expression on the cell surface (Snyder et al., 1995); a result that implies that ENaC may recycle in an apical recycling pathway. The coexpression of CFTR with ENaC in polarized MDCK cells alters the regulation of ENaC by cAMP (Stutts et al., 1995). Conceivably, this regulatory relationship between the two channels could be related in part to the CAMP-dependent regulation of internalization, sorting, or recycling of ENaC. The CAMP- and CFTR-dependent regulation of membrane traffic could also impact on the processes of mucosal immunity and inflammation. Airway and intestinal epithelial cells transcytose polymeric immunoglobulins via a receptor-mediated process (Mostov, 1991). The polymeric immunoglobulin receptor (pIgR) binds IgA and IgM on the basolateral surface, which subsequent to its endocytosis enters the apical recycling pathway from where it is exocytosed at the apical surface (Apodaca el al., 1994). The regulation of the apical recycling pathway by cAMP and/or CFTR could modulate the rate of polymeric immunoglobulin transcytosis. In addition, airway epithelial cells secrete a host of proteins that play crucial roles in mucosal homeostasis. Many of these proteins, such as surfactant protein A, al-antitrypsin, extracellular superoxide dismutase, defensins, and interleukins, act as antibacterial or antiinflammatory mediators. The regulated secretion of these factors could play a role in the protective response of mucosal surfaces to environmental and microbial challenges. A defect in the baseline or regulated secretion of such factors in CF could contribute to the exaggerated inflammatory state of the airway mucosa or the susceptibility of airways to pathogens. In summary, although a role for CFTR in the regulation of epithelial membrane traffic is yet to be rigorously established, several lines of evidence support this hypothesis. This will be an important issue t o resolve, given that such a function of CFTR could profoundly influence the physiology and pathology of epithelial tissues.
IX. Summary and Conclusions The field of CF research has moved at a rapid pace since the identification of the C1- transport defect in the early 1980s and the subsequent cloning of the CF gene in 1989. In our review we attempted to highlight areas of CFTR biogenesis that appear to be most significant for understanding disease pathogenesis. We also explored controversial areas in which consensus building might lead to a better understanding of the molecular and cellular functions of CFTR. Regarding this latter point, it is worth noting
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that the resolution of earlier controversies in the CF field has led to some of the most significant discoveries in this area. For instance, a debate over the C1- channel activity of AF508CFTR that was observed in some, but not all, heterologous expression systems led to the discovery that the processing defect of AF508CFTR is temperature sensitive. In a similar way, future research that addresses the role of CFTR in regulating membrane traffic in polarized epithelial cells versus nonpolarized cells might provide seminal information regarding the mechanisms that underlie the development and regulation of such polarized membrane traffic pathways. In conclusion, we have learned much about the biosynthetic processing of the CFTR and the C1- channel properties of this protein. Ongoing structure-function studies of CFTR are expanding our general understanding of how ion channels can be configured and regulated. Developing efforts to correct the lung abnormalities in CF patients by pharmacological and genetic means are helping advance two additional fields: protein processing within the E R and gene therapy. Other aspects of CFTR biology that are less well understood include (i) the mechanisms and regulation of CFTR traffic beyond the biosynthetic pathway, (ii) the functional interactions between CFTR and other epithelial ion channels, and (iii) the involvement of CFTR C1- channels in regulating organelle composition and intracellular membrane traffic. The latter two issues relate to the emerging notion that CFTR is a multifunctional protein that is capable of regulating epithelial physiology at multiple levels. Elucidating these various functions should improve our understanding of the pathogenesis of cystic fibrosis and help us better design therapies for this devastating disease. Acknowledgments The authors thank Drs. David Bedwell, James Collawn, Eric Sorschcr, and Charles Venglarik for critically reading the manuscript. We also thank Dr. Venglarik for Fig. 2 and Mary Nelle Shelton and Kelli Folgman for their valuable secretarial support. Stably transduced UCPK cells were provided by PR Seng Cheng (Genzyme Corp.)
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Regulation of Phenylpropanoid Metabolism in Relation t o Lignin Biosynthesis in Plants Mark S. Barber* and Heidi J. Mitchell? *School of Biological Sciences, University of Southampton, Southampton SO16 7PX, United Kingdom; and ?Research School of Biological Sciences, Australian National University, Canberra ACT 0200, Australia
Lignin is a group of complex phenolic polymers that provide important strengthening and waterproofing properties to plant cell walls. Considerable progress has been made in the study of the regulation of phenylpropanoidmetabolism in relation to lignification. The prospect of altering plant lignins to improve intrinsic properties, such as biomass digestibility or pathogen resistance, by manipulating phenylpropanoid metabolism represents a rational approach toward plant improvement. The feasibility of artificially manipulating lignin and lignin-dependentprocesses is largely reliant on our understanding of the regulatory mechanisms controlling lignin biosynthesis. This chapter focuses on the enzymic steps of phenylpropanoidmetabolism, namely the general phenylpropanoid and lignin branch pathways, describing attempts to manipulate these steps and alter lignin deposition and lignin-dependentprocesses. The prospect of attaining control over plant lignification via the manipulation of phenylpropanoidmetabolism is addressed, and the potential problems and pitfalls relating to the multifunctionalityof lignin, the complexity of the biosynthetic pathways, and the lack of specificity in the enzymes are examined. The problem of judging the significance of the controls that operate at the level of phenylpropanoidmetabolism with those that operate at other levels, such as monolignol transport and the polymerization steps, is highlighted. KEY WORDS: Plant cell wall, Lignin, Monolignol, Phenylpropanoid.
1. Introduction Lignin is the second most abundant plant polymer after cellulose and forms a major component of the terrestrial biomass (McKie et aZ., 1993). Yet lnlernlrtronal Review of CyroloRy. Vol. I72 0074-7696197 $25.00
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Copyright Q 1997 by Academic Press All rights of reproduction in any form reserved.
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surprisingly there are no definitive criteria established to verify the existence of lignin and there is still some debate regarding the distribution of lignin in the plant kingdom. Although it is generally accepted that terrestrial higher plants contain lignin, the debate as to its existence in aquatic or lower plants is still unresolved (Lewis and Yamamoto, 1990). Lignin is the term given to a group of complex phenolic polymers that provide important strengthening and waterproofing properties to plant cell walls. It is not surprising, therefore, to find lignin playing fundamental roles in mechanical support, solute conductance, and disease resistance in higher plants. The evolution of lignin is believed to be one of the key factors in the appearance and diversification of land plants. Only with lignified cell walls was it possible to build the rigid stems of woody plants and provide efficient waterproofing for water transport (Grisebach, 1981). Lignified cell walls are considered to be very effective at limiting the progress of microorganisms, as demonstrated by the slow biodegradation of highly lignified tissues and the negative correlation between lignin content and digestibility. In addition to this passive role in defense, lignification also underpins many of the inducible plant defense responses raised against microbial pathogens. The prospect of manipulating the quantity and composition of lignin in plants has several attractions, particularly in the modification of the digestibility of plant material and in the development of resistance to microbial pathogens. Lignification is ultimately dependent on phenylpropanoid metabolism for the supply of the basic building blocks of lignin and this dependency has led to the targeting of phenylpropanoid metabolism as a suitable point at which to manipulate lignin biosynthesis. The intent of this chapter is to survey the enzymes of phenylpropanoid metabolism, namely the general phenylpropanoid pathway and the lignin branch pathway, focusing attention on their suitability for manipulation and describing the effects of manipulation on lignin deposition and lignin-dependent processes. Limited coverage will be given to the monolignol transport processes (Grisebach, 1981) and the final polymerization steps (Dean and Eriksson, 1992) that have been reviewed elsewhere. We also draw the reader’s attention to the excellent reviews published in this area (Sederoff ef aL, 1994; Boudet ef nl., 1995; Whetten and Sederoff, 1995; Douglas, 1996; Boudet and GrimaPet tenati, 1996).
A. Structural Features of Lignin Lignin may be viewed as a metabolically inert matrix polymer enclosing celfulosic and other cell wall materials, rendering the cellulose fibers inaccessible to microbial enzymes (Walter, 1992). The polymer is ill-defined at the chemical level, its composition varying between species, and at the
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tissue and cellular level within a single species. Strictly speaking, it is derived principally from three hydroxycinnamyl alcohols: p-coumaryl, coniferyl, and sinapyl alcohols. These hydroxycinnamyl alcohols occur in either the trans (E) or cis ( Z ) configuration. Although it is widely accepted that most lignins are derived exclusively from E monolignols, there is evidence to suggest that in some plants Z monolignols are involved in lignification (Yamamoto et al., 1989). Oxidation of the hydroxycinnamyl alcohols leads to the formation of free radicals that undergo spontaneous polymerization into lignin. However, this is a gross oversimplification of the structure of lignin because other phenolics, usually hydroxycinnamic acids or hydroxycinnamaldehydes, are often incorporated at low levels. Additional complexity arises at the structural level due to the nonenzymic polymerization of the hydroxycinnamyl alcohols, giving rise to an optically inactive polymer containing a network of nonrepetitive units linked by many different types of stable C-C and C-0 bonds. The polymerization of monolignols is no longer thought to be a random process and the composition of the underlying polysaccharide matrix is thought to be a factor influencing the type of lignin deposited. The use of cellulose synthesis inhibitors to disrupt the deposition of cellulose in Zinnia elegans tracheary elements was seen to lead to dispersed lignin deposition in contrast to the normal precise pattern of secondary thickening (Taylor et al., 1992). Furthermore, computational models have suggested that the nonrandom arrangement of lignin bonding is due to the cellulose component of the wall (Houtman and Atalla, 1995). The three hydroxycinnamyl alcohols, p-coumaryl, sinapyl, and coniferyl, that form the basic building blocks of lignin are collectively termed monolignols. Incorporation of these monolignols into lignin gives rise to p hydroxyphenol (H), guaiacyl (G), and syringyl (S) lignin residues, respectively. The relative proportions of the monolignols incorporated into lignin are highly variable, allowing lignins to be classified according to their residue composition. Guaiacyl, or type I lignin, is derived principally from coniferyl alcohol, whereas guaiacyl-syringyl, or type I1 lignin, is derived from coniferyl and sinapyl alcohol units. Generally, gymnosperms produce guaiacyl lignins, whereas angiosperms produce mainly guaiacyl-syringyl lignins. A n exception to this rule is found in grasses that contain lignin derived from all three hydroxycinnamyl alcohols. Although it is possible to generalize about the type of lignin found in an angiosperm or gymnosperm, within an individual plant different types of lignin may be deposited at different developmental stages or in response to environmental stresses, although the significance of this phenomenon is unknown. For instance, the defenserelated lignin of wheat has a high proportion of syringyl residues (Ride, 1Y75), whereas the defense-related lignins of other plants tend to be rich in p-hydroxyphenol residues (Asada and Matsumoto, 1972; Robertsen and Svalheim, 1990; Hammerschmidt, 1984; Hammerschmidt et aL, 1985; Lange
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et al., 1995). It has been suggested that the incorporation of the unmethylated monolignol, p-coumaryl alcohol, into lignin may occur more quickly and be more efficient in terms of energy and resources utilized (HammerSchmidt, 1984). In addition, the lignin may also be more condensed and stronger by virtue of more sites available for cross-linkage. Putting the functional significance issue aside, the mechanism by which plants achieve this variation in lignin composition is likely to be of immense value in the artificial modification of lignin in plants.
6. Overview of Lignin Biosynthesis Lignin biosynthesis involves the coordinated regulation of three major biosynthetic pathways, the shikimate, general phenylpropanoid, and lignin branch pathways (Fig. 1). The shikimate pathway is essentially primary metabolism, providing building blocks for a variety of plant products (Dewick, 1994). Although the production of lignin is ultimately dependent on the shikimate pathway and several shikimate genes are expressed during lignification (Gorlach el al., 1995), it will only be discussed briefly in this review. The reliance of lignification of the shikimate pathway is illustrated by the dramatic reduction in lignin content of potato plants antisensed for the shikimate enzyme 3-deoxy-~-arabino-heptulosonate-7-phosphate synthase (EC 4.1.2.15) (Jones et al., 1995). Similarly, creating a metabolic sink for tryptophan by introducing the gene for tryptophan decarboxylase (EC 4.1.1.28) into potato reduced L-phenylalanine pools and lowered lignin levels (Yao el al,, 1995). The shift from the shikimate pathway to the general phenylpropanoid pathway begins with the deamination of L-phenylalanine to cinnamic acid. This irreversible step effectively represents a switch from primary to secondary metabolism in the plant. Cinnamic acid then undergoes further reactions leading to the formation of various hydroxycinnamic acids and hydroxycinnamoyl-CoA esters, varying in their degree of hydroxylation and O-methylation. The hydroxycinnamoyl-CoA esters are the precursors of numerous phenolic compounds in plants including the monolignols. The shift from the general phenylpropanoid pathway to the lignin branch pathway begins with the reduction of the hydroxycinnamoyl-CoA esters to their corresponding hydroxycinnamaldehydes. These are further reduced to generate hydroxycinnamyl alcohols, the immediate precursors of lignin. The hydroxycinnamyl alcohols may become glycosylated for transport or storage purposes subsequent to their deglycosylation and polymerization. The final polymerization of the hydroxycinnamyl alcohols into lignin, although dependent on cell wall oxidases to generate free radicals, is essentially a spontaneous nonenzymic reaction. The enzymes responsible for the major catalytic reactions of the general phenylpropanoid and lignin-
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specific pathways will be considered in more detail in the following sections. Limited coverage will be given to the storage and transport of monolignols and their polymerization into lignin.
II. General Phenylpropanoid Pathway The general phenylpropanoid pathway links the shikimate pathway to the lignin branch pathway. The pathway leads to the formation of a series of hydroxycinnamic acids and hydroxycinnamoyl-CoA esters varying in their degrees of hydroxylation and methylation (Fig. 1). This is not a linear pathway, but rather better considered as a biochemical grid, and there is much debate as t o the level at which the hydroxylation and O-methylation reactions occur. Research in this area is complicated by extensive enzyme polymorphism, incomplete analysis of substrate specificity, and inappropriate enzyme nomenclecture. In the following sections the characteristics of the enzymes responsible for the major catalytic steps of the general phenylpropanoid pathway are described.
A. Deamination Reactions The general phenylpropanoid pathway begins with the deamination of Lphenylalanine to cinnamic acid catalyzed by phenylalanine ammonia lyase (PAL; EC 4.3.1.5). In grasses, the deamination of tyrosine may occur directly to p-coumaric acid, catalyzed by tyrosine ammonia lyase (TAL; EC 4.3.1S ) .
1. Phenylalanine Ammonia Lyase Due to the position of PAL at the entry point of phenylpropanoid metabolism it has the potential to play a regulatory role in lignin production. Its importance is illustrated by its high degree of regulation during development and in response to environmental stimuli. PAL catalyzes the first step in the phenylpropanoid pathway, the elimination of NH3 from Lphenylalanine to give cinnamic acid. This step is important because it represents a shift from primary to secondary metabolism and is the first committed step into phenylpropanoid metabolism. The two most commonly employed methods for assaying PAL rely on either the spectrophotometric detection of cinnamic acid (Havir and Hanson, 1968) or the radiometric detection of cinnamic acid from labeled L-phenylalanine (Amrhein et al., 1976). Although PAL is largely absent from prokaryotes and has not been
248
MARK S. BARBER AND HEIDI J. MITCHELL l.-phenylalminc
tyrosiiic
TAL caffcic acid
HO
4CI.
1-
4CI.
$.
p-counl aroyl-CoA
CCR
I
cullcoyl-CoA
COSCoA ____3
HO
CCoAOMT
OH CCR
rcH: J.
HO
OMT 01-1
C*"
\
1
p-coiunaraldehyde
*
H O U
OH
CAI)
inonolignoi glycosides
UI'G
GLS
*
FIG.1 An overview of lignin biosynthesis. The enzymes are phenylalanine ammonia lyase (PAL), tyrosine ammonia lyase (TAL), cinnamate 4-hydroxylase (C4H), coumarate 3-hydroxylase (C3H), ferulate 5-hydroxylase (FSH), coumaroyl-CoA 3-hydroxylase (CCoA3H), 0methyltransferase (OMT), caffeoyl-CoA 3-0-methyltransferase (CCoAOMT), 4-coumarateCoA ligase (4CL), cinnamoyl-CoA reductase (CCR), cinnamyl alcohol dchydrogenase (CAD), I JDP-glucose: cinnamyl alcohol glucosyltransferase (UTG). cinnamyl alcohol 0-glucosidasc (GLS), peroxidase (PO), and laccase (LAC).
REGULATION OF PHENYLPROPANOID METABOLISM ferulic acid
HO
F5 1-1
OM
HO
+
4CL
\
~
c
sinapoylCoA
Me
.+ COSCoA ?
HO
OMe
rqL
-OMe ..
sinapaldehyde
5-Iiydroxyconile1aldehyd~
coniferaldehyde
HO -OMe .
rco OMe
femloyl-CoA
\
Meo HO
OMe
OMe 4CL
249 sinapic acid
5-hydroxyfemlicacid
rz Me
1-10 \OMe
?
polymerization PO or LAC
NO
\OMe
* LIGNIN
FIG. 1 (continued)
detected in animals, its distribution in both plants and fungi has been widely reported. In plants, the enzyme is located in both the cytoplasm and cell organelles including plastids, microbodies, mitochondria, glyoxysomes, and peroxisomes (Hanson and Havir, 1981). Characterization of PAL from a number of plants has revealed consistencies in the enzyme structure. The enzyme exists as a high-molecular-weight tetramer composed of identical or very similar subunits. In wheat the tetramer is made up of pairs of two different subunits (Nari et al., 1972), although later work indicates that the tetramer units may actually be identical and the smaller units are generated by proteolytic modification (Zimmermann and Hahlbrock, 1975). Polymor-
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MARK S. BARBER AND HEIDI J. MITCHELL
phism in PAL appears to be a fairly widespread phenomenon in plants (Bolwell et al., 1985; Liang et al., 1989). Multiple forms of PAL have been identified and in some cases these have been found in different locations within the cell. Analysis of DNA and genomic clones points to the existence of three PAL genes in bean (Cramer et al., 1989) and up to 11 isoforms of bean PAL (Bolwell et at., 1985; Liang et at., 1989). Direct measurement of PAL during development has demonstrated a strong correlation of activity with areas of active lignification in various plants including buckwheat (Yoshida and Shimokoriiyama, 1965), bamboo (Higuchi, 1966), asparagus (Goldstein et al., 1971; Hennion et al., 1992), and maize (Morrison and Buxton, 1993). Expression of PAL has been examined using molecular techniques and has revealed a complex pattern of tissue-specific developmental expression in tobacco (Bevan et al., 1989; Liang et nl., 1989), potato (Bevan et al., 1989) and Arabidopsis thaliana (Oh1 et al., 1990). In bean, three PAL genes have been characterized, PALl, PAL2, and PAL3 (Cramer et al., 1989), that show differential expression during development and in response to environmental stimuli (Liang et al., 1989). This differential expression is possibly attributable to differences in the 3' and 5' flanking regions of the PAL2 and PAL3 genes. The expression of PAL2 has been investigated in both potato and tobacco using a bean PAL2 promotor-GUS fusion (Bevan et al., 1989). The GUS activity was associated with various tissues and organs in which phenylpropanoids accumulate such as flowers, petioles, and the epidermis. Interestingly, GUS activity was also observed in tissues such as xylem that actively undergo lignification. Although GUS activity was absent from the mature xylem, it was detected in both developing xylem and in the xylem parenchyma cells adjacent to mature vessels. The presence of PAL in xylem parenchyma cells has subsequently been confirmed by histochemical staining techniques (Smith et al., 1994) and is thought to provide precursors to enable continued lignification of nonliving mature vessels (Bevan et al., 1989). In a comparative study of bean PAL2 and PAL3 promotor-GUS fusion in A. thaliana, potato, and tobacco, the pattern of GUS expression differed between the two gene promotors (Shufflebottom et al., 1993). In some tissues both genes were expressed, whereas in differentiating xylem and vascular tissues only the PAL:! promotor was active, suggesting that the PAL3 gene is not involved in developmental lignification. Direct measurement of PAL activity has clearly demonstrated a strong correlation with areas of active lignification in response to environmental stimuli in plants. Although the responsiveness of PAL to several environmental stimuli, including wounding (Tanaka and Uritani, 1977; Shields et al., 1982) and light (Hadwinger, 1972; Zimmerman and Hahlbrock, 1975; Ragg et al., 1981; Wilkinson and Butt, 7992), have been reported, most work in this area has concentrated on the effect of microbial challenge.
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The activity of PAL has been seen to change following fungal infection or treatment with fungal elicitors in bean (Hadwinger, 1972; Bolwell et af., 1985), wheat (Green et al., 1975), soybean (Ebel et af., 1976), reed canary grass (Vance and Sherwood, 1976), parsley (Ragg et al., 1981; Lois et uf., 1989), alfalfa (Dalkin et ul., 1990), vanilla (Funk and Brodelius, 1990), and pearl millet (Nagarathna et af., 1993). Studies in wheat have shown that PAL activity increases in response to infection with Puccinia graminis fsp. fritici (Moerschbacher et af., 1988). In resistant plants an early increase in PAL activity was seen corresponding to the hypersensitive lignification response. In susceptible plants PAL induction was greater, although the response was considerably delayed. In interactions between Erysiphe graminis f.sp. tritici race 21 and wheat isolines carrying genes for resistance, PAL induction occurred in all inoculated isolines, whereas lignification was only detectable in those genotypes possessing the Pm2 gene. In the wound margin response of wheat PAL induction is transient following fungal challenge or elicitor treatment (Maule and Ride, 1976; Mitchell et al., 1994; Thorpe and Hall, 1984). An early study showed PAL induction to be localized to sites of lignin accumulation (Maule and Ride, 1976), although later work indicated that PAL induction also occurred in the nonlignifying surrounding tissues (Thorpe and Hall, 1984;Mitchell et af.,1994).Molecular studies have also been used to investigate PAL. In potato infected with Phytophthora infestans, PAL mRNAs were seen to accumulate in cells surrounding sites of fungal penetration (Cuypers et af., 1988). In parsley, four PAL genes have been identified that show different responses to ultraviolet (UV) light, fungal elicitor, and wounding (Logemann et al., 199%). Three of the parsley PAL genes are expressed following exposure to any of the stimuli, but PAL4 mRNAs only accumulate in roots following wounding or treatment with a fungal elicitor. Analysis of transcriptional activation of PAL genes after environmental and pathogen stress showed that de novo mRNA synthesis is the major regulatory mechanism in both cases (Cramer et af., 1985a,b;Edwards et af.,1985; Lawton and Lamb, 1987; Lois et af., 1989). Three commonly utilized chemical inhibitors, L-a-aminooxy-P-phenylpropionic acid (AOPP), a-aminooxy acetic acid (AOA), and (l-amino-2phenylethy1)phosphonic acid, have been used in studies to alter the activity of PAL in pfanfa. Although these compounds have been used to inhibit lignification, they also affect other processes reliant on general phenylpropanoid intermediates such as flavonoid and simple phenolic production (Amrhein, 1981; Massala et al., 1980). Additionally, these compounds suffer from a lack of specificity toward PAL and also inhibit pyridoxal phosphate enzymes involved in ethylene and amino acid synthesis such as 1aminocyclopropane-1-carboxylatesynthase (De Laat and Vanloon, 1981) and aminotransferases (Amrheim et al., 1976; John et af., 1980). Despite
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these shortcomings the compounds have been used to reduce lignin in several plants. In tobacco, inhibition of PAL activity by AOPP had dramatic consequences on the formation of lignin causing severe impairment of the vascular system and increased susceptibility to fungal infection (Amrhein et al., 1983; Smart and Amrhein, 1985). AOPP was also found to be a potent inhibitor of the accumulation of insoluble lignin-like polymers in tobacco cells reacting hypersensitively to viral infection (Massala et nl., 1987). Both AOPP and AOA were found to reduce lignin deposition when added to cucumber hypocotyls treated with an oligogalacturonide elicitor (Robertsen and Svalheim, 1990) or to reed canary grass during fungal penetration (Sherwood and Vance, 1982). The use of PAL inhibitors to examine resistance in cereals has also resulted in plants with altered resistance (Moerschbacher et al., 1990; Carver and Zeyen, 1993; Carver et al., 1994a,b; Zeyen et al., 1995). However, the results are difficult to interpret due to the multifunctionality of PAL, lack of specificity in the PAL inhibitors, and the complexity of plant resistance. Recently, studies have used genetic manipulation to understand the role of PAL in lignification. Introduction of a PAL gene from bean into tobacco (Elkind ef al., 1990) led to a number of morphological changes including altered flower pigmentation and, paradoxically, a decrease in PAL and a decrease in phenylpropanoid products. This decrease in PAL activity and concomitant reduction in the accumulation of phenylpropanoids led the authors to conclude that PAL is a key regulatory step in this pathway. However, despite a large amount of data on the number and structure of PAL genes in different systems, it has not been possible to associate one specific PAL gene with lignification (Cramer et al., 1989, Lois et al., 1989). It therefore seems unlikely that PAL alone could exert effective control over lignification.
2. Tyrosine Ammonia Lyase TAL activity, whereby L-tyrosinc is deaminated to p-coumaric acid, has been demonstrated mainly in grasses (Neish, 1961). This creates a potential second entry point into the general phenylpropanoid pathway, effectively bypassing the 4-hydroxylation of cinnamic acid to p-coumaric acid. The assay methods for PAL may be adapted for TAL, but care must be taken, particularly for the spectrophotometric assay, to avoid artifactual data (Hanson and Havir, 1981). Although PAL and TAL are clearly two measurable enzyme activities, the general consensus i s that both reactions are catalyzed by a single enzyme. The flux of material via the TAL route is thought to be secondary and most material enters the general phenylpropanoid pathway via PAL. Not surprisingly, there is very little data relating to this novel entry point into the general phenylpropanoid pathway.
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6. Hydroxylation Reactions
Hydroxylation of cinnamic acid at the 4-carbon to form p-coumaric acid is catalyzed by cinnamate 4-hydroxylase (C4H; E C 1.14.13.11), sometimes referred to as cinnamate 4-monooxygenase. Further hydroxylation at the 3- and 5-carbons is catalyzed by coumarate 3-hydroxylase (C3H) and ferulate 5-hydroxylase (F5H; no E C numbers assigned), respectively. Potentially, these activities can operate at two levels, hydroxylating both the hydroxycinnamic acids and the hydroxycinnamoyl-CoA esters. The enzyme catalyzing the hydroxylation of hydroxycinnarnoyl-CoA esters is sometimes specifically referred to as coumaroyl-CoA 3-hydroxylase (CCoA3H; E C 2.1.1.104).
1. Cinnamate 4-Hydroxylase C4H catalyzes the second step in the general phenylpropanoid pathway. This step may be bypassed in grasses if tyrosine is converted directly to pcoumaric acid by TAL. Some evidence suggests that C4H may play a central role in the regulation of the phenylpropanoid pathway because its substrate, cinnamic acid, may modulate PAL activty (Bolwell et al., 1988, Mavandad et al., 1990). The reaction catalyzed by C4H occurs via the addition of a hydroxyl group at the 4-carbon of cinnamic acid to form p-coumaric acid. C4H is a cytochrome P450-dependent mixed function oxygenase that requires NADPH to cleave molecular oxygen, adding one oxygen atom to the aromatic ring and reducing the other t o water. C4H occurs coupled with NADPH-cytochrome P450 reductase, its electron transfer partner. The assay for C4H activity may employ an artificial substrate such as tetrahydrofolic acid, although it is more commonly detected radiochemically using labeled cinnamic acid ( M a d e and Ride, 1983). Suprisingly, C4H has been purified from relatively few plants and the extent of C4H polymorphism in unknown. In contrast, isolation of cDNA clones has been reported from a number of plants including Jerusalem artichoke (Teutsch et al., 1993), mung bean (Mizutani et al., 1993), alfalfa (Fahrendorf and Dixon, 1993), and Catharanthus roseus (Hotze et ul., 1995). In pea C4H is coded by a single gene (Frank et al., 1996), whereas in alfalfa two genes are present (Dixon et al., 1995). In developing bean plants C4H has been localized, using immunogold labeling, to the prexylem and phloem tissues (Smith et al., 1994). Later in development the label was seen to be localized in the cells adjacent to the metaxylem. Within the cell, the enzyme is found as an integral membrane protein on the endoplasmic reticulum (Steffens et al., 1989). There is some evidence that C4H is associated with PAL and that metabolic channeling occurs between the two enzymes (Steffens et al., 1989). Experiments have
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shown that exogenously supplied cinnamic acid is used less readily by C4H than that generated by PAL in sifu, suggesting that some transfer mechanism exists to channel the cinnamic acid to the C4H (Czichi and Kindl, 1977; Hrazdina and Wagner, 1985). Structurally, C4H exists as a highly glycosylated monomer of molecular weight 58 kDa (Gabriac et al., 1991), although glycosylation was not required for enzyme activity (Teutsch et ul., 1993). An investigation into the substrate specificity of C4H showed a very high affinity for cinnamic acid and an inability to catalyze any of the other hydroxylation reactions in the general phenylpropanoid pathway. In addition, C4H was able to detoxify a number of xenobiotic compounds including the herbicide diclofop, suggesting it may have multiple functions in plants (Pierrel et al., 1994). The activity of C4H is reported to be induced following wounding in peas (Stewart and Schuler, 1989), dark treatment of mung bean (Mizutani et al., 1993) and elicitor treatment in peanut (Steffens et ul., 1989) and old man cactus (Liu et al., 1995). Interestingly, in elicitor-treated bean cell suspension cultures C4H activity increases concomitantly with PAL (Bolwell et al., 1985). The two enzymes were also seen to be localized to the wound sites, especially concentrated in the epidermal cells (Smith et al., 1994). In wheat leaves, C4H is reported to increase in activity following fungal infection (Maule and Ride, 1983). This increase was localized to the area surrounding the infection site, corresponding to the area actively undergoing lignification. Analysis of C4H mRNA levels has indicated that the changes in activity observed in peas and Jerusalem artichoke following wounding, and in alfalfa following elicitor treatment, are due to increases in transcription (Fahrendorf and Dixon, 1993; Frank et al., 1996; Teutsch et al., 1993). To date there are no reports of the effects of C4H inhibition on lignification in plantu using either inhibitors or molecular techniques. A useful compound for the inhibition of C4H is tetracylis, which reportedly prevents elicitor-induced increases in the phytoalexin medicarpin (Orr et al., 1993). Two other compounds, l-aminobenzotriazole and 3-(2,4-dichlorophenoxy)1-propyne, have been identified as mechanism-based suicide inhibitors of C4H (Pierre1 et al., 1994) and thus would be useful for examination of the role of C4H in lignification. 2. Coumarate 3-Hydroxylase
C3H catalyzes the hydroxylation of p-coumaric acid at the 3-carbon to form caffeic acid. Very little information exists about this step to the extent that C3H has yet to be isolated. In spinach, phenolases with a broad specificity range arc suggested to be responsible for the hydroxylation (Vaughan and Butt, 1969; Bolwell and Butt, 1983). However, the phenolase inhib-
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itor tentoxin had no effect on caffeic acid production in mung bean (Duke and Vaughn, 1982). A phenolase from mung bean with a high specificity for p-coumaric acid has been characterized (Kojima and Takeuchi, 1989). A currently unresolved question relates to the level at which the 3hydroxylation step occurs. In addition to the hydroxylation of p-coumaric acid it is also possible that C3H could hydroxylate p-coumaroyl-CoA. A separate enzyme, termed CCoA3H, may be responsible for this step. This hypothesis is supported by a mutation in Silene dioica that prevents the conversion of p-coumaroyl-CoA to caffeoyl-CoA, blocking anthocyanin pigment production (Kamsteeg et al., 1981), and by the ability of microsomal fractions to hydroxylate hydroxycinnamoyl shikimate esters in parsley (Heller and Kuhnl, 1985) and hydroxycinnamoyl quinate esters in carrot (Kuhnl et al., 1987). More directly, enzymes have been reported to be capable of hydroxylating both p-coumaric acid and p-coumaroyl-CoA in potato (Boniwell and Butt, 1986) and parsley (Kneusel et al., 1989). Collectively, these observations suggest that hydroxylation may occur at the level of both hydroxycinnamic acids and hydroxycinnamoyl-CoA esters, although the relative importance of each is unknown. 3. Ferulate 5-Hydroxylase
F5H catalyzes the hydroxylation of ferulic acid at the 5-carbon to form 5hydroxyferulic acid. To date, F5H activity has only been reported in poplar microsomal fractions, in which a cytochrome P450-dependent mixed function oxygenase requiring oxygen and NADPH was identified (Grand, 1984). Comparison of F5H and C4H in poplar suggested that the activities were dependent on two distinct cytochrome P450 systems. The A. thaliana sin1 mutant, later renamedfahl, in which F5H is dysfunctional produces a lignin deficient in syringyl residues (Chapple et al., 1992). In fact the lignin of this angiosperm mutant more closely resembles the guaiacyl lignins of gymnosperms. This emphasizes the potential importance of F5H in the production of sinapyl alcohol and the typical guaiacyl-syringyl lignins of angiosperms. Indeed, the anomalous formation of guaiacyl lignin in the angiosperm Erythrina cristu galli was attributed to low levels of F5H (Kutsuki and Higuchi, 1978). C. Methylation Reactions
Methylation reactions of the general phenylpropanoid pathway are catalyzed by S-adenosyl-L-methionine-dependentdiphenol-0-methyltransferases (OMT; EC 2.1.1.6). The widespread use of caffeic acid as the substrate to detect OMT activity has led to many OMTs being specifically
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named as caffeate 3-O-methyltransferase (C-OMT; E C 2.1.1.68). This is somewhat unfortunate because several of these C-OMTs also O-methylate 5-hydroxyferulic acid to sinapic acid, and in some cases S-hydroxyferulic acid is the preferred substrate (Kuroda et al., 1981). Unlike the hydroxylases that appear to operate at the level of both hydroxycinnamic acids and hydroxycinnamoyl-CoA esters, a distinct form of OMT, termed caffeoylCoA 3-O-methyltransferase (CCoAOMT; E C 2.1.1.104), seems to be responsible for the O-methylation at the level of the hydroxycinnamoylCoA esters.
1. O-Methyltransferase OMT catalyzcs the O-methylation of caffeic acid and S-hydroxyferulic acid using S-adenosyl-L-methionine as the methyl donor and may be assayed either spectrophotometrically (Shimada et al., 1970) or radiochemically (Shimada et nf., 1972). Several OMTs have been purified from plants including spinach (Poulton and Butt, 1975), soybean (Poulton et ul., 1976), Thuja orientalis (Kutsuki et af., 1981), tobacco (Hermann et al., 1987), and alfalfa (Edwards and Dixon, 1991; Gowri et af., 1991). Distribution studies in T. orienralis using crude tissue extracts showed that the OMT was predominantly located in the xylem adjacent to the cambium, with no activity in thc phloem (Kutsuki el al., 1981). A similar distribution was found in aspen using both tissue printing with an antibody to the OMT and Northern blot analysis (Bugos et af., 1991). Slightly different results were obtained in differentating Z . efegans tracheary elements using tissue printing (Ye and Varner, 1995). In the young internodes of the stem the OMT mRNA was present in the developing phloem but not in the xylem, whereas in older tissues the mRNA could be detected in both the xylem and phloem fibers. Polymorphism in OMT appears to be a fairly widespread phenomenon. In aspen two forms have been detected (Kuroda et al., 1981), whereas alfalfa and tobacco have three forms (Gowri et al., 1991; Hermann et nl., 1987). The substrate specificity of the OMT has been seen to vary depending on the origin of the plant material, and three major types have been identified (Kuroda et a1.,1981). The gymnosperm-type OMT catalyzcs only the O-methylation of caffeic acid, the angiosperm-type OMT preferentially O-methylates 5-hydroxyferulic acid over caffeic acid, and the bambootype OMT O-methylates both substrates with equal efficiency. The substrate specificities of the three OMT types generally correlates with the degree of O-methylation of the lignin produced by these different plant groups. There are some notable exceptions such as in Thuja spp., which produce typical gymnosperm guaiacyl-type lignins but have OMTs capable of O-methylating both caffeic acid and 5-hydroxyferulic acid (Kutsuki et
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al., 1981). Not suprisingly, OMTs have been postulated to explain the differences in lignin composition (Gross, 1985; Higuchi, 1985). Developmental changes in OMT have been reported in aspen, in which analysis of OMT activity and mRNA levels revealed two peaks of activity in the growing season-one in early June and the second in late July (Bugos et al., 1991). This is thought to correspond to the change in the type of xylem produced from early to late wood. In bamboo, the syringyl content of the lignin increases with plant age, but the activity of the OMT toward 5-hydroxyferulic acid remains constant (Shimada et al., 1973). In the early stages of seedling development in wheat, prior to lignin deposition, there is a transient increase in caffeic acid OMT activity, whereas later in development, following the onset of lignification, 5-hydroxyferulic acid OMT activity predominates (Tuyet et al., 1996). The early burst of caffeic acid OMT activity may be associated with the production of ferulic acid destined for cell wall esterification, whereas the later 5-hydroxyferulic acid OMT activity may be more closely associated with lignification. Following fungal challenge in wheat leaves OMT activity increased to the same extent using both caffeic acid and 5-hydroxyferulic acid as substrates (Maule and Ride, 1976). Similarly, in alfalfa the OMT was responsive to elicitor treatment (Gowri et al., 1991). In tobacco undergoing the hypersensitive response, following infection with tobacco mosaic virus, OMT levels were also elevated (Pellegrini et al., 1993). The expression of the constitutive OMT gene was not affected by the tobacco mosaic virus infection, but two new forms of OMT appeared that were the product of one induced gene. To date there are no inhibitors available that are specific enough for OMT to warrant their use inplanta and thus alteration of OMT has focused on molecular techniques. Investigations into a brown rib mutant (bm3) of maize revealed a decreased lignin content, increased digestibility, and lignins containing more p-hydroxyphenol residues and less guaiacyl residues than the wild type (Grand et al., 1985a). These changes were attributed to a reduction in the level of the OMT and recent work has identified the mutation as occurring in the gene encoding the OMT (Vignols et al., 1995). Following the work on the maize mutant, OMT has been manipulated in tobacco by introducing antisense constructs from lucerne (Ni et al., 1994), aspen (Dwivedi et al., 1994), and tobacco (Atanassova etal., 1995). Introduction of an lucerne OMT gene in the antisense orientation led to a reduction in OMT enzyme levels, a reduction in total lignin content, and no change in the composition of lignin (Ni et al., 1994). The aspen antisense gene similarly led to a decrease in total lignin but also changed the composition of the lignin, reducing the syringyl content (Dwivedi et al., 1994). Introducing an antisense tobacco gene led to no change in the quantity of lignin, but again altered the composition of lignin. The syringyl content was reduced and incorporation of a new monomer derived from 5-
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hydroxyferuloyl-CoA was observed (Atanassova et al., 1995). Similar results were also found in poplar plants expressing a poplar OMT antisense gene (Van Doorsselaere et ul., 1995). The reduction in syringyl units is desirable for the papermaking industry because the decreased O-methylation of lignin reduces the production of polluting mercaptans during processing (Atanassova et al., 1995). The reduction in syringyl units is also thought to be a way of improving forage crop digestibility (Jung, 1989). However, the differing conclusions of the three studies, possibly due to the heterologous nature of the gencs used, make it difficult to evaluate the role of the OMT as a target for manipulation (Halpin et al., 1994). Further studies on the biochemistry and substrate specificity of the OMTs would help to resolve the differential effects obtained by antisensing OMT.
2. Caffeoyl-CoA 3-O-Methyltransferase CCoAOMT is a distinct form of OMT that specifically utilizes hydroxycinnamyl-CoA esters and has been reported in carrot (Kuhnl et a/., 1989), parsley (Pakusch et al., 1989; Schmitt et al., 1991), and 2. elegans (Ye e f al., 1994). The parsley CCoAOMT is induced following elicitor treatment (Pakusch et al., 1991; Schmitt et al., 1991), and 2. elegans CCoAOMT expression has been shown to correlate both spatially and temporally with the appearance of lignin (Ye et al., 1994). In light of these observations an alternative pathway for methylation has been suggested (Ye et al., 1994). This involves the O-methylation of the hydroxycinnamoyl-CoA esters instead of the hydroxycinnamic acids as had previously been assumed. The alternative methylation pathway is probably best considered as a rerouting of the general phenylpropanoid pathway rather than a separate pathway in itself and had been proposed as the most probable route in an earlier study in wheat (Ride, 1983). The existence of CCoAOMT most likely accounts for the lack of lignin reduction observed in some OMT antisense plants (Atanassova et al., 1995), although similar molecular techniques have yet to be applied to CCoAOMT.
D. CoA-Esterification Reactions The formation of the CoA esters of hydroxcinnamic acids is catalyzed by hydroxycinnamate-CoA ligases, often referred to as 4-coumarate-CoA ligase (4CL; EC 6.2.1.12). The hydroxycinnamoyl-CoA esters formed by 4CL are key intermediates in phenylpropanoid metabolism from which numerous plant products, including lignin, are ultimately derived.
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1. 4-Coumarate-CoA Ligase 4CL catalyzes the formation of activated CoA thioesters of hydroxycinnamic acids. This involves the formation of an intermediate acyl adenylate that reacts with CoA to form a thioester and requires adenosine triphosphate, magnesium, and a thiol source for activity (Maule and Ride, 1983). 4CL is commonly measured spectrophotometrically using p-coumaric acid as the substrate (Gross and Zenk, 1974). A number of 4CLs have been purified from plants including forsythia (Gross and Zenk, 1974), spruce (Luderitz et al., 1982), poplar (Grand et al., 1983), loblolly pine (Voo et al., 199.5), and maize (Hipskind et al., 1993). Characterization of 4CL identified the enzyme as a monomer with a molecular weight of between 4.5 and 60 kDa. Work on potato has indicated that expression of the 4CL gene occurs in both lignified tissues, including stems and tubers, and young developing tissues such as roots and young leaves (Becker-Andre and Hahlbrock, 1989).This work has been confirmed and extended by a study in A. thaliana using the parsley 4CL promotor fused to a GUS reporter gene (Lee et al., 199.5). From this study GUS activity was detected in the developing vascular system, but no data were available on the cellular localization of 4CL. Multiple forms of 4CL have been identified in several plants including soybean (Knobloch and Hahlbrock, 197.5), pea (Wallis and Rhodes, 1977; Wilkinson and Butt, 1992), oat (Knogge et al., 1981), poplar (Grand et al., 1983),parsley (Lozoya et al., 1988), and potato (Becker-Andre et al., 1991). In some cases the 4CL forms exhibited different substrate specificities, leading to the suggestion that their expression pattern could influence the type of lignin produced in a given tissue (Knobloch and Hahlbrock, 197.5; Kutsuki et al., 1982a; Grand et al., 1983). The substrate specificity of 4CL has been found to differ between angiosperms and gymnosperms. Circumstantial evidence was obtained from feeding experiments that indicated that only angiosperms were capable of reducing sinapic acid to sinapaldehyde (Nakamura et al., 1974). Unfortunately, because these experiments did not directly measure 4CL activity, it was not possible to determine whether 4CL or cinnamoyl-CoA reductase (CCR), a later enzyme in the ligninspecific pathway, was responsible for the observed results. Direct evidence from the measurement of 4CL in several gymnosperms revealed a general inability to utilize sinapic acid (Higuchi, 1981; Luderitz et al., 1982), consistent with the lack of syringyl units present in gymnosperm lignin. However, there are a number of instances in which the substrate specificity of the 4CL does not appear to correspond with the composition of the lignin produced. Although angiosperms tend to produce a typical guaiacylsyringyl lignin, their 4CLs are often unable t o utilize sinapic acid (Knogge et al., 1981; Kutsuki et al., 1982a). In some angiosperms, such as parsley
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(Lozoya et ul., 1988) and potato (Becker-Andre et al., 1991), the various isoforms have almost identical substrate specificities. In cultured soybean cells, which produce a typical angiosperm guaiacyl-syringyl lignin, two 4CL forms are present that differ in their abilities to utilize hydroxycinnamic acids. Form 4CL-2 poorly utilizes methylated hydroxycinnamic acids and has been linked to flavonoid biosynthesis. In contrast, form 4CL-1 utilizes 0-methylated hydroxycinnamic acids and has been linked to lignification (Knobloch and Hahlbrock, 1975). A similar situation has been found in pea seedlings, in which only one of the 4CLs was reported to utilize sinapic acid. In pea (Wallis and Rhodes, 1977) and Petunia (Ranjeva et ul., 1976) 4CL forms have been loosely associated with various processes based on their substrate specificities. Although it is tempting to ascribe functions to various 4CL forms based on substrate specificity, the validity of this type of interpretation is questionable. This arises from the potential of O-methylation reactions to occur at the level of the hydroxycinnamoyl-CoA esters, after 4CL has acted. Thus, it is entirely possible that a 4CL with a preference for unmethylated hydroxycinnamic acids may be involved in the production of highly methylated lignins. In tobacco, a study using parsley 4CL promotor fragments fused to the GUS reporter gene showed detectable GUS activity in flowers, roots, and vascular tissue (Hauffe et al., 1991). This work was expanded by producing a transgenic tobacco plant containing a full copy of the parsley 4CL gene (Reinold et al., 1993). Gene expression showed a complex pattern of developmental regulation in all tissues, for example, in anthers the timing of mRNA was seen to correspond with secondary wall development. In situ hybridization and gene-specific probes were used to show that GUS expression using the 4CL promotor generally matched 4CL mRNA accumulation during development, thus indicating that the gene regulation was promotor driven. However, in petal development this correlation was not absolute, suggesting that downstream sequences must also play a role in 4CL regulation. There are many more studies of 4CL concerning its response to environmental factors, most of which concentrate on the effects of microbial attack. In wheat, innoculation of resistant and susceptible varieties with P. graminis gave a different pattern of 4CL activity. Both isolines showed an initial increase in the activity of 4CL following germination of fungal spores. The resistant isoline then showed a second increase corresponding to the induction of the hypersensitive resistance response, whereas the 4CL activity in the susceptible isoline declines to basal levels (Moerschbacher et al., 1988). A further study in wheat following innoculation of wounds with nonpathogenic fungal spores showed that the increase in 4CL activity was localized to the lignifying tissues (Maule and Ride, 1983). The substrate specificity of the 4CL was determined for both healthy leaves and those responding t o a nonpathogenic fungi. Under both conditions, p-coumaric
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acid was the preferred substrate and the enzyme showed low affinity for sinapic acid, despite the high syringyl content of the defensive lignin (Maule and Ride, 1983). Following elicitor treatment in parsley the activity of 4CL increases concommitently with PAL activity (Kombrink and Hahlbrock, 1986) and lignification (Farmer, 1985). This is due to changes in transcription rates that increase dramatically after elicitor or UV light treatment (Chappell and Halhbrock, 1984; Douglas et al., 1987; Lois et al., 1989). The 4CL mRNA is detected first in localized regions of leaf cells surrounding sites of hypersensitive cell death. Wounding of the tissues induces 4CL mRNA accumulation that is induced sequentially from the site of damage out into the leaf tissue (Schmelzer et al., 1989; Dangl, 1992). In A. thaliana, a GUS reporter gene fused to a parsley 4CL promotor has been used to study the effects of wounding and innoculation with a virulent and a nonvirulent bacteria on 4CL induction (Lee et al., 1995). Both the 4CL transcripts and GUS activity were induced following wounding and innoculation with the nonvirulent strain of the pathogen. Conversely, innoculation with the pathogenic bacterial strain revealed no changes in 4CL. Different forms of the 4CL enzyme have been reported to be affected differently following environmental changes. In pea seedlings two forms of 4CL have been detected with different substrate specificities (Wallis and Rhodes, 1977). Both forms are reported to increase during lignogenesis, although to different extents (Wilkinson and Butt, 1992). Several 4CL cDNA clones have been isolated from soybean that are thought to code for at least three 4CL forms. Examination of elicitor-treated tissues showed that only one 4CL form was induced and the increased activity was due to mRNA synthesis (Uhlmann and Ebel, 1993). In poplar cell cultures, elicitor treatment causes an increase in both 4CL enzyme activity and mRNA accumulation (De Sa et al., 1992). The induced 4CL had a different substrate specificity to that found in the untreated cells preferentially utilizing ferulic acid and p-coumaric acid. The authors concluded from this that activation of a subset of 4CL forms that prefentially use these substrates may occur following elicitor treatment. The induced 4CL forms have a similar substrate specificity as the 4CL-2 from poplar (Grand et al., 1983), which was found in the lignifying xylem tissue. Differential activation of 4CL forms with distinct catalytic properties also occurs during fungal infection of reed canary grass (Vance and Sherwood, 1976) and maize (Vincent and Nicholson, 1987). Differences in the substrate specificity have been observed between infected resistant and susceptible varieties, which suggests different roles for products of phenylpropanoid metabolism in each case (Vincent and Nicholson, 1987). It has been proposed that the existence of 4CL forms with different substrate specificities, which show differential regulation of expression during defense responses, supports the involvement of 4CL in metabolic chan-
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neling into different phenylpropanoid branch pathways (Uhlmann and Ebel, 1993). Considering the interest 4CL has attracted it seems a little surprising that there is little or no work on the chemical or genetic manipulation of 4CL, despite the availability of cDNA clones from various plants.
111. Lignin Branch Pathway The flux of material passing through the general pathway phenylpropanoid seems likely to be one of the controlling factors of plant lignification. However, despite extensive study it has not been possible to exclusively link any of the general phenylpropanoid pathway enzymes to lignin formation. In the search for suitable target sites within phenylpropanoid metabolism attention has turned to the lignin branch pathway, which is considered to be highly specific for lignification and channels general phenylpropanoid intermediates toward monolignol production.
A. Reductive Reactions The lignin branch pathway consists of two enzymes, cinnamoyl-CoA reductase (CCR; EC 1.2.1.44) and cinnamyl alcohol dehydrogenase (CAD; E C 1.1.1.195), that sequentially reduce the hydroxycinnamoyl-CoA esters via their hydroxycinnamaldehydes to their corresponding hydroxycinnamyl alcohols.
1. Cinnamoyl-CoA Reductase CCR is the first enzyme in the lignin branch pathway and is able to divert general phenylpropanoid intermediates toward the accumulation of monolignols. As an entry point enzyme in the lignin branch pathway it has been hypothesized to play a key regulatory role in lignin biosynthesis (Goffner et af., 1994). CCR reduces the hydroxycinnamoyl-CoA esters to their corresponding hydroxycinnamaldehydes with the concomitant oxidation of NADPH and is commonly measured spectrophotometrically (Luderitz and Grisebach, 1981). Mainly as a consequence of technical problems, such as nonavailability of the natural substrates, low enzyme activity, and poor enzyme stability, studies of CCR have been infrequent compared with other enzymes of phenylpropanoid metabolism. CCR has been purified from soybean (Wegenmayer et al., 1976; Luderitz and Grisebach, 1981), spruce (Luderitz and Grisebach, 1981), poplar (Sarni et al., 1984), and eucalyptus (Goffner et al., 1994). Due to the lack of detailed characterization, it is
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difficult to generalize about the structural features of CCR, although it would seem to be a monomeric protein with a molecular weight in the range of 36-40 kDa. It is possible to obtain a partial correlation between the type of lignin, guaiacyl or guaiacyl-syringyl, commonly deposited by plants and the substrate specificity of CCR. Sinapoyl-CoA is utilized by the CCR of most angiosperms, including soybean (Luderitz and Grisebach, 1981), poplar (Sarni et al., 1984), and eucalyptus (Goffner et al., 1994), although it is not usually the favored hydroxycinnamoyl-CoA ester. Similarly, extracts of isolated xylem tissue from the angiosperms cherry and poplar were both reported to reduce ferulic and sinapic acids to the corresponding hydroxycinnamaldehydes and hydroxycinnamyl alcohols (Nakamura et al., 1974). Thus, the ability of these plants to utilize sinapoyl-CoA or reduce sinapic acid is in accordance with the guaiacyl-syringyl lignin typically associated with angiosperms. In contrast, the CCR of spruce, a gymnosperm that produces a guaiacyl lignin, does not utilize sinapoyl-CoA but favors feruloyl-CoA (Luderitz and Grisebach, 1981). Similarly, extracts of isolated xylem tissue from the gymnosperms Japanese red pine and ginkgo could only reduce ferulic acid to coniferaldehyde and coniferyl alcohol (Nakamura el al., 1974). The nonavailability of the natural substrates has severely limited studies on CCR, such that there is little information regarding its induction during development or in response to environmental stimuli. In spruce hypocotyls, CCR activity peaks early in the development of seedlings and subsequently declines (Luderitz and Grisebach, 1981), and in asparagus the levels of CCR rapidly declined during the first few hours of postharvest storage (Hennion et al., 1992). Manipulation of the levels of phytohormones was seen to stimulate CCR activity and other lignin biosynthetic enzymes in bean (Grima-Pettenati er al., 1989) and parsley (Hose1 et al., 1982) cell cultures. A temporal increase in CCR following elicitor treatment preceded the formation of lignin-like material in spruce suspension cultures (Messner and Boll, 1993). In order to obtain more information regarding the mechanism of CCR action its activity has been inhibited in vitro using a number of compounds (Goffner et al., 1994). In addition to inhibition by CoA and NADP', the most effective compounds were protein-modifying agents specific for lysine and cysteine residues. The low specificity of these inhibitors effectively rules out their use in planfa, although they may prove to be of value in the structural analysis of CCR. The only other inhibitor shown to possess activity against CCR is the CAD inhibitor N-( O-aminopheny1)sulfinamoyltertiobutyl acetate (NH2PAS) (Grand et al., 1985b). In vitro NHzPAS inhibits both CCR and CAD, and in planta NH2PAS treatment leads to a dramatic decrease in the incorporation of label into lignin from applied [14C]cinnamicacid. Whether this effect is due to the inhibition of CCR or
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CAD or a combination of both activities is unknown. Manipulation at the molecular level has yet to be reported in full, although the isolation of fulllength eucalyptus cDNA and genomic clones encoding the CCR has been cited (Boudet etal., 1995;Boudet and Grima-Pettenati, 1996). Very preliminary results suggest that significant phenotypic changes occur following downregulation of CCR in tobacco. An orange-brown coloration was associated with the xylem and the effects on the content and composition of lignin are currently in progress (Boudet and Grima-Pettenati, 1996).
2. Cinnamyl Alcohol Dehydrogenase CAD is the last enzyme in the lignin branch pathway and is directly responsible for the formation of the monolignols. A large body of work on CAD has accumulated in the literature due to its positioning in the lignin branch pathway and the widespread availability of several of its substrates. The reaction catalyzed by CAD reduces the hydroxycinnamaldehydes to their corresponding hydroxycinnamyl alcohols with the concomitant oxidation of NADPH. CAD has a specific requirement for zinc, both as part of the enzyme structure and as a specific cofactor (Mackay et al., 1995). The reaction catalyzed by CAD is reversible and may be measured spectrophotometrically in the forward and reverse directions (Wyrambik and Grisebach, 1975,1979).An early survey of CAD distribution in the plant kingdom revealed the widespread occurrence of this enzyme (Mansell et al., 1976). CAD has been purified from several plant species including soybean (Wyrambik and Grisebach, 1975), wheat (Pillonel et al., 1992), eucalyptus (Goffner et af., 1992), tobacco (Knight et al., 1992; Halpin et al., 1992), and bean (Grima-Pettenati et al., 1994). The cellular localization of CAD has been examined in several plant species using a coupled electron transfer dye technique (Baudracco et al., 1993). In addition to xylem elements and phloem fibers, CAD was localized to the epidermal and subepidermal layers of roots and shoots. Unfortunately, the study was not able to determine the exact subcellular site of CAD activity, although a cytoplasmic location was suggested. The CAD enzyme is part of a large family of aliphatic and aromatic alcohol dehydrogenases. Sequence analysis of CAD has classified it as a long-chain zinc-containing alcohol dehydrogenase. This group, which is found in both prokaryotic and eukaryotic organisms, is identified by 22 highly conserved amino acid residues ( Jornvall et al., 1987). These residues include the structural zinc binding site and the cofactor binding sites for NADP+(H) and zinc. The sequences are generally most conserved in regions that are important for physiological function. Four amino acid residues that might account for CAD enzyme substrate specificity have been identified (McKie el af., 1993). Three of the residues are conserved in all known CAD sequences; however, the fourth amino acid was found to differ
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between the angiosperm species and two gymnosperms, pine and spruce. This led Mackay et al. (1995) to postulate that this residue could be important in determining the CAD enzyme specificity. It has been suggested that CAD might be associated in a multienzyme complex (Walter, 1992). However, it is now generally accepted that CAD exists as a dimeric protein of molecular weight within 63-84 kDa with subunit weights ranging from 38 to 45 kDa. An extensive survey of the plant kingdom, employing lowresolution starch gel electrophoresis, concluded that CAD existed as a single isoform in the majority of plants examined (Mansell et al., 1976). Failure to identify more instances of polymorphism was most probably due to the analysis of crude extracts using a low-resolution separative technique. Work has established that, although the CADs of gymnosperms are generally single isoforms, their angiosperm equivalents exhibit considerable polymorphism. The functional significance of CAD polymorphism in relation to their substrate specificity and the type of lignins deposited in plants has only recently been addressed. In the three gymnosperm species studied, loblolly pine (O’Malley et al., 3 992), Norway spruce (Galliano et al., 1993b), and Japanese black pine (Kutsuki et al., 1982b), only one form of CAD, consisting of two identical subunits, has been identified. The cDNA from both pine and spruce has been isolated and used to probe genomic libraries (Galliano etal., 1993a; Mackay et al., 1995). In both species this has indicated the presence of a single gene encoding CAD. Examination of the substrate specificity of gymnosperm CAD reveals a high specificity for coniferyl alcohol and coniferaldehyde in comparison to sinapyl alcohol and sinapaldehyde (O’Malley et al., 1992; Galliano et al., 1993b; Kutsuki et al., 1982b). Thus, there is good correlation between the substrate specificity of these CADs and the guaiacyl-type lignin found in these gymnosperms. Despite the fairly small number of plant species studied, CAD polymorphism in angiosperms is already very complex (Hawkins et al., 1994). With the exceptions of poplar (Sarni et al., 1984) and forsythia (Mansell et al., 1974), angiosperm CAD is polymorphic and has been reported in soybean (Wyrambik and Grisebach, 1975), wheat (Pillonel et al., 1992), eucalyptus (Goffner et al., 1992; Hawkins and Boudet, 1994), and bean (GrimaPettenati et al., 1994). Attempts to correlate CAD substrate specificity to lignin heterogeneity in angiosperms are compounded by the complexity of CAD polymorphism. In Zelkora serrata, the substrate specificity of the CAD corresponds with the monomer composition of the lignin present (Kutsuki et al., 1982b). However, in poplar, in which sclerenchyma tissue contains a more syringyl-rich lignin in comparison to the lignin of xylem tissue, the CAD substrate specificity of both cell types was identical (Sarni ef al., 1984). A number of authors have suggested that the occurrence of multiple CAD isoforms may arise from allelic variants (Halpin et al., 1992; Pillonel et nl., 1992). This would not appear to be the case in eucalyptus,
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in which a detailed investigation of polymorphism combined with an appropriate kinetic characterization of the substrate specificity of the CAD forms has been performed (Hawkins and Boudet, 1994). In eucalyptus periderm tissue, one of two major CAD forms, CAD2P, can be resolved into three closely related isoforms. The three isoforms of CAD2P arise from different combinations of two nonidentical subunits giving two homodimers and one heterodimer. A kinetic study of the high-molecular-weight homodimer and the heterodimer, using all six potential substrates, revealed significant differences in their utilization of hydroxycinnamaldehydes and hydroxycinnamyl alcohols. The high-molecular-weight homodimer C A D isoform worked most efficiently with sinapaldehyde and relatively poorly with other substrates, including coniferaldehyde. In contrast, the heterodimer CAD isoform utilized coniferaldehyde and sinapaldehyde with roughly equal efficiency (Hawkins and Boudet, 1994). A similar phenomenon has been observed with the alcohol dehydrogenases of maize (Freeling, 1974), rice (Xie and Wu, 1989), and soybean (Newman and Van Toai, 1991). These alcohol dehydrogenases are the products of two separate genes, which combine to give two homodimers and one heterodimer. The three distinct alcohol dehydrogenase isoforms appear to have different physiological roles, exhibited by their different tissue specificity and patterns of induction. Whether similar mechanisms operate at this level of C A D polymorphism to regulate lignin heterogeneity is unknown, although the differential substrate specificity exhibited by some CAD isoforms suggests that this is possible. A number of investigations have shown a high degree of correlation between CAD activity and areas of active lignification. For no apparent reason developmental changes in CAD have generally been investigated using molecular techniques, whereas CAD induction to environmental stimuli has relied more heavily on biochemical analysis. Changes in the levels of CAD activity during developmental processes have been reported in developing maize stalks in which the activity correlates spatially with lignin deposition (Morrison and Buxton, 1993). During development in maize, CAD activity and lignin deposition move in a wave-like fashion up the stalk (Morrison et al., 1994). In gymnosperm compression wood, which has a higher than normal lignin content, C A D activity is seen to be increased (Kutsuki and Higuchi, 1981). A number of plant C A D genes have been identified including tobacco (Knight et al., 1992), eucalyptus (GrimaPettenati et al., 1993), loblolly pine (MacKay et al., 1995), and A. thaliana (Baucher et al., 1995; Somers et ul., 1995). Northern blot analysis in Aralia cordata using a full-length CAD cDNA clone from the same plant showed that the CAD mRNA was detected primarily in the lower stem tissues where the most lignification was occurring (Hibino et al., 1993). In eucalyptus plants CAD2 mRNA was detected in the developing xylem but also in young stems and leaves (Grima-Pettenati et al., 1993). A similar pattern
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was seen in loblolly pine, with the CAD mRNA also detected in the nonlignifying gametophyte tissue (Mackay et al., 1995). Reporter genes have also been used to assess the spatial and developmental expression of the CAD gene. The eucalyptus CAD promotor region was fused to the GUS reporter gene and stably introduced into poplar. GUS activity was prefentially expressed in the vascular tissues, specifically associated with those tissues undergoing active lignification. During the early stages of xylem differentiation GUS activity was detected in parenchyma cells adjacent to xylem vessel, whereas later, during secondary thickening, GUS activity was expressed in the radial files of xylem parenchyma cells (Feuillet el al., 1995). Changes in the levels of CAD activity following exposure to environmental factors, particularly microbial challenge, are common and often precede lignification.CAD activity is seen to increase in response to ozone treatment in spruce (Galliano et al., 1993b) and following microbial challenge or elicitor treatment in wheat (Moerschbacher et al., 1988), spruce (Messner and Boll, 1993), bean (Grand et al., 1987; Walter et al., 1988), and pine (Campbell and Ellis, 1992). However, in a number of cell suspension cultures, such as pine (Campbell and Ellis, 1992) and poplar (De Sa et al., 1992) treated with fungal elicitor or pea exposed to UV light (Wilkinson and Butt, 1992), CAD induction does not precede lignification. Although there are many possible explanations to account for the lack of CAD induction in these systems, it is clear that the use of CAD as a reliable marker for lignification is questionable. In wheat, CAD activity increases in response to challenge by the nonpathogen Botrytis cinerea and treatment with fungal elicitors (Mitchell et al., 1994). It was observed that the greatest induction of CAD occured when sinapyl alcohol was used as a substrate. Further work demonstrated that CAD exists as three major forms in wheat (CAD-A, CAD-B, and CAD-C) that vary in their substrate specificity and their responsiveness to elicitors. Comparison of untreated and elicitortreated leaves revealed that although all three CAD forms were present in healthy tissues only CAD-C was responsive to elicitors. The preferred substrate for CAD-C was sinapyl alcohol, which is consistent with the increased syringyl content of defensive lignin in wheat (Ride, 1975). Relatively few molecular studies have been used to investigate CAD induction in plants. One study performed in bean identified the CAD4 cDNA clone, which was transcribed following elicitor treatment (Walter et al., 1988). Unfortunately, this clone was subsequently identified as a malic enzyme (Walter et al., 1990). Since then, several authentic CAD clones have been produced and in spruce CAD mRNAs have been shown to accumulate following elicitor treatment or exposure to ozone (Galliano et al., 1993a). Two compounds have commonly been used to inhibit CAD, N - ( 0 hydroxypheny1)sulfinamoyltertiobutyl acetate (OHPAS) and NH2PAS. Both compounds have a loose structural resemblance to the natural mono-
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lignols and were designed to chelate zinc, which is required by C A D both as a part of the enzyme structure and as a specific cofactor. These inhibitors appear to be relatively specific for CAD, not affecting other zinc metalloenzymes, although the CCR of the lignin branch pathway was inhibited by NH2PAS (Grand et al., 198%). The relatively high degree of specificity exhibited by these compounds has led to their use in planta to investigate various lignin-dependent processes. In poplar stems application of OHPAS and NH2PAS was shown to dramatically reduce the incorporation of label from ['4C]cinnamic acid into lignin (Grand et al., 198%). In both barley (Carver et af.,1994a) and oat (Carver et al., 1994b) OHPAS treatment has been shown an increase susceptibility to powdery mildew infection. This correlates with a reduction in hypersensitive cell death and a lack of cell wall autofluorescence, possibly lignification, at penetration sites. Similar work in wheat showed that treatment with either OHPAS or NH2PASled to increased susceptibility to stem rust infection and a reduced hypersensitive response (Moerschbacher et al., 1990). Manipulation of CAD at the molecular level has already been achieved through the use of brown midrib mutants and genetically engineered antisense CAD mutants. Naturally occurring bm mutants were identified in corn (Jorgenson, 1931) and four recessive genes were identified that would produce the bm phenotype (Kuc et al., 1968). These mutants, which are characterized by a reddish pigmentation in the midrib, exhibit lower lignin levels and show differences in lignin composition (Kuc and Nelson, 1964; Barriere and Argillier, 1993). In terms of their digestibility and feed value, the bm mutants are superior to the normal genotypes (Gallais et al., 1980; Barriere and Argillier, 1993), although this gain is offset by their reduced forage yield (Weller el al., 1985). The brown midrib mutants of sorghum (bmr), which vary in their lignin contents and color, were generated by chemical mutagenesis (Porter et al., 1978). The bmrl2 and bmrl8 mutants produce lignins with few syringyl residues, whereas the bmr6 mutant has a similar syringyl content to the normal genotype (Chabbert et al., 1993). The bmr6 mutant, which has lower CAD activity, has a reduced lignin content and incorporates higher levels of hydroxycinnamaldehydes into its lignin (Bucholtz et al., 1980). Recent work revealed the mutation to result from a single locus genetic lesion affecting both CAD and OMT enzyme activities (Pillonel et al., 1991). Introduction of a CAD antisense gene into tobacco gave rise to mutants with dramatically reduced CAD activities and had little effect on the quantitative levels of lignin deposition, but did alter the composition, structure, and extractability of the lignin (Halpin etal., 1994). The antisense plants incorporate more hydroxycinnamaldehydes into their lignin and show a red-brown coloration in their xylem, similar to the brown midrib mutants of corn and sorghum. Similarly, two reviews have cited reports of poplar CAD antisense mutants with reddish coloration of the wood, strongly suggesting increased
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incorporation of hydroxycinnamaldehydes into lignin (Boudet et al., 1995; Boudet and Grima-Pettenati, 1996). It would appear, therefore, that attempts to manipulate CAD at the molecular level generally result in compensation in terms of incorporation of hydroxycinnamaldehydes into lignin. Commercially, the red-brown phenotype is seen as a way of producing prestained timber (Higuchi et al., 1994) and the associated increased extractability of the lignin is a desirable property in terms of papermaking (Halpin et a!., 1994).
IV. Storage and Transport of Monolignols Our understanding of the transport processes associated with movement of monolignols to sites of lignification or storage is rudimentary. There are two possible fates for the monolignols and monolignol glycosides generated in the cytoplasm. They may either cross the plasma membrane and diffuse through the cell wall to the sites of lignification or cross the tonoplast into the vacuole for storage. The nature of all membrane carriers is unknown and their is no consensus as to whether the free monolignols or their glycosides are transported. Monolignol glycosides can accumulate to relatively high levels in some plant tissues, but their distribution appears to be restricted to gymnosperms and less advanced angiosperms (Terazawa et al., 1984). In gymnosperms there is a good correlation between the tissue distribution of monolignol glucosides and sites of active lignification such as developing xylem (Terazawa and Miyake, 1984; Savidge, 1989; Leinhos and Savidge, 1993). Interestingly, in spruce only a small proportion of the coniferyl alcohol destined for lignin biosynthesis enters the monolignol glycoside pool (Marcinowski and Grisebach, 1978). This suggest that glycosylation of monolignols may not be essential for intercellular transport but could still be required for intracellular transport and storage. Where monolignol glycosides occur in angiosperms they have been associated with the developing xylem and with the nonlignifying phloem (Terazawa and Miyake, 1984), suggesting alternative physiological roles for monolignol glycosides other than lignin precursors. Regulatory controls have been suggested to operate at the level of monolignol and monolignol glycoside transport, but their mechanisms are not known (Terashima and Fukushima, 1989; Terashima, 1990). There is some tentative experimental evidence to suggest that transport across membranes occurs via a vesicle fusion mechanism. This is based on experiments in wheat using labeled cinnamic acid in which the label was found in vesicles derived from the golgi and endoplasmic reticulum and at the sites of active lignification (Pickett-Heaps, 1968). The potential involvement of monolignol glycosides in lignification
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has focused attention on the enzymes responsible for the glycosylation and deglycosylation reactions.
A. Glycosylation and Deglycosylation Reactions The glycosylation of monolignols to form the monolignol glycosides is catalyzed by UDP-glucose: cinnamyl alcohol glucosyltransferase (UTG; EC 2.4.1.11l ) , sometimes referred to as coniferyl alcohol glucosyltransferase. Deglycosylation of the monolignol glycosides is catalyzed by cinnamyl alcohol P-glucosidase (GLS; EC 3.2.1.21). 1. Cinnamyl Alcohol Glucosyltransferase
UTG catalyzes the formation of the P-D-glucosides at the phenolic hydroxyl of hydroxycinnamyl alcohols. The monolignol glycoside derived from p-coumaryl alcohol is usually referred to as y-hydroxycinnamyl alcohol glycoside, whereas the glycosides of coniferyl and sinapyl alcohol are given the trivial names coniferin and syringin. Plant UTGs have been reported in rose (Ibrahim and Grisebach, 1976), spruce (Schmid et al., 1982), and Forsythia orata (Ibrahim, 1977). With regard to the substrate specificity the UTG isolated from lignifying F. oruta stems had a strong affinity for both coniferyl and sinapyl alcohols (Ibrahim, 1977), whereas the spruce UTC had a marked preference for coniferyl alcohol (Schmid and Grisebach, 1982). The spruce UTG was cytoplasmic and localized to the vascular bundles and epidermal and subepidermal layers (Schmid et ul., 1982). The use of inhibitors or molecular techniques to manipulate UTG activity in planta has yet to be reported.
2. Cinnamyl Alcohol P-Glucosidase GLS hydrolyzes monolignol glycosides, releasing free monolignols for polymerization. Although P-glucosidases are widespread in plants, specific GLS activities have been reported in relatively few species (Hose1 et al., 1978, 1982). The problem of identifying specific GLSs associated with lignification is complicated by the presence of nonspecific P-glucosidases. Polymorphism in GLS has been found in Pinus banksiana (Leinhos et al., 1994) and lodgepole pine (Dharmawardhana et al., 1995). In lodgepole pine there is one major GLS isoform, but other P-glucosidases with minor GLS activity are also present. The major GLS activity in pine was located in the differentiating xylem and utilized both syringin and coniferin, suggesting it was unlikely to direct the monolignol composition of lignin. The induction of GLS has been shown to correlate with the induction of other enzymes
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involved in phenylpropanoid metabolism and with sites of active lignification. In parsley GLS increased concomitantly with enzymes such as PAL, C4L, CCR, and CAD and preceded lignification (Hose1 et al., 1982). In elicitor-treated P. banksiana GLS induction correlated with a burst of peroxidase (PO) activity that could be used to polymerize the liberated monolignols (Campbell and Ellis, 1992). The potential importance of the deglycosylation step is illustrated by the effects of the origin of monolignols on the nature of the dehydrogenation polymers produced in vitro. When monolignol glycosides and GLS are used to generate monolignols the dehydrogenation polymer produced more closely resembles natural lignin compared to when free monolignols are provided (Terashima et al., 1993). Enzyme inhibitors and molecular techniques have yet to be applied to manipulate GLS activity in ylanta.
V. Polymerization Process There are major gaps in our knowledge of the mechanisms and enzymology of lignin polymerization. The formation of lignin from monolignols is an oxidative process involving the enzymic generation of free radical intermediates that undergo spontaneous polymerization. In addition, a nonenzymic polymerization reaction, known as chain propagation, has also been suggested (Boudet et al., 1995). A. Oxidative Reactions
A major unresolved question relates to the nature of the oxidative enzymes involved in the generation of these free radical intermediates or phenoxyradicals. Two cell wall phenol oxidases, peroxidases (PO; EC 1.11.1.7) and laccase (LAC; EC 1.10.3.2), have been extensively studied, but their exact role in the enzymology of lignin polymerization remains unclear (O’Malley et al., 1993; Dean and Eriksson, 1994).
1. Peroxidase
POs are a large group of hydrogen peroxide-dependent phenol oxidases that have multiple functions in plants (Van Huystee, 1987). In addition to lignification, POs are also implicated in other cell wall-associated processes including suberin and hydroxyproline-rich glycoprotein deposition (Fry, 1982). The activity of PO in lignin formation is rarely measured following the formation of dehydrogenation polymers from monolignols, but it is
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most commonly assayed by the use of artificial substrates that give rise to colored products. PO requires a hydrogen peroxide cosubstrate for activity and this is unlikely to be generated in the cytoplasm due to its toxicity and rapid degradation. The hydrogen peroxide required by the PO has been detected in plant tissues (Sagisaska, 1976) and systems for the generation of cell wall hydrogen peroxide involving PO have been described (Gross et al., 1977). Interestingly, the activity of the PO involved in the generation of hydrogen peroxide is stimulated by monolignols, suggesting that the hydroxycinnamyl alcohols themselves may be involved in regulating the supply of hydrogen peroxide required for lignin polymerization. POs are widely distributed within the plant kingdom, with numerous forms occurring within a single species. The major fraction of PO is predominantly associated with the cell wall and within this compartment POs are often further subdivided in terms of their form of association with the cell wall. From a survey of PO forms from different tree species (Stich and Ebermann, 1988a,b), it was concluded that no one form was responsible for polymerizing monolignols because most forms detected had activity against a wide variety of substrates. Examination of the substrate specificity of PO toward the various monolignols revealed p-coumaryl alcohol to be dehydrogenated slowly, possibly explaining the low levels of p-hydroxphenyl residues associated with many lignins (Stich and Ebermann, 1988a,b). Several studies have demonstrated a correlation between PO activity and lignification in tissues, including tobacco (Lagrimini et al., 1987), castor bean (Bruce and West, 1989), barley (Kerby and Sommerville, 1989, 1992), and wheat (Flott et al., 1989; Schweizer et al., 1989; Rebmann et al., 1991). There is also evidence suggesting that some PO isozymes, differing in their specificity for monolignols, may influence the composition of lignin (Masuda et nl., 1983; Imberty et al., 1985;Church and Galston, 1988;Stich and Ebermann, 1988a;Tsutsumi and Sakai, 1994). Despite extensive correlative evidence linking PO induction to lignification, the direct assignment of one or more POs to lignification has yet to be shown. Assignment of PO forms to lignification will, in addition to temporal and spatial correlation with lignification, require indisputable subcellular localization and comparative analysis of monolignol specificity. It is hoped that molecular techniques will allow the unambiguous identification of specific POs associated with lignification, although early results suggest a more complex relationship between PO and lignin. Sense and antisense constructs of an anionic PO, reportedly involved in lignification, have been generated in tobacco (Lagrimini, 1987). In the sense constructs, overexpression of PO led to a wilting phenotype and a higher lignin content (Lagrimini, 1991). The antisense constructs had reduced stem strength and were epinastic in severely affected plants (Rothstein et al., 1990). Surprisingly, the tobacco plants with a reduced PO activity did not have a signifi-
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cantly lower lignin content than their wild-type counterparts (Chabbert et al., 1992).
2. Laccases Until recently, POs were considered to be the most likely candidates in the generation of phenoxyradicals, and the involvement of L A C in lignification was generally discounted due to its reported inability to form synthetic lignins (Nakamura, 1967). There is now a renewed interest in LAC and two recent reviews on LAC and lignification have been published (O’Malley et at., 1993; Dean and Eriksson, 1994). LAC catalyzes the oxidation of phenolic substrates using molecular oxygen as the electron acceptor. Typically, LACs are highly glycosylated, monomeric metalloproteins containing four copper ions and of variable molecular weight within the range of 52-1 10 kDa. Several groups have provided evidence for the involvement of laccases in lignification. Indirect evidence has arisen from the reduced lignin content of copper-deficient plants (Downes et al., 1991). More directly, and contrary to earlier reports (Nakamura, 1967), laccases have been shown to oxidize monolignols and generate dehydrogenativepolymers (Sterjiades et al., 1992). Correlative evidence has also been obtained that tightly links LAC activity with areas of active lignification in gymnosperms (Savidge and Udagama-Randeniya, 1992; Bao et al., 1993), sycamore (Driouich ef al., 1992), and Z . elegans (Liu et al., 1994). Whether the LAC specificity for monolignols influences the composition of lignin is unclear, although it appears that LACs utilize p-coumaryl alcohol poorly in comparison to coniferyl and sinapyl alcohols (Sterjiades et al., 1992, 1993; Bao et al., 1993). It has been suggested that LACs could operate in the initial steps of lignification to polymerize coniferyl and sinapyl alcohols in the absence of toxic hydrogen peroxide and that POs might act later to polymerize the cross-linking of the monolignols and oligolignols (Sterjiades et al., 1993). If true, this would suggest that for lignins with high levels of p-hydroxyphenol residues, such as those commonly deposited in response to stress, PO may be the key cell wall oxidase. As with PO, and for similar reasons, it has not yet proved possible to assign a role to a specific LAC in lignification. A cDNA clone encoding the acidic LAC from sycamore maple has been obtained (Lafayette et al., 1995) and an attempt to reduce lignin levels by applying antisense technology in yellow poplar has been cited by Boudet and Grima-Pettenati (1986).
VI. Concluding Remarks Considerable progress has been made in recent years in the study of the regulation of phenylpropanoid metabolism in relation to lignin deposition
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in plants. The prospect of manipulating lignification in plants to improve intrinsic properties, such as biomass digestibility or pathogen resistance, by manipulation of the key enzymes of phenylpropanoid metabolism represents a rational approach toward plant improvement. Developmental lignification and the lignins deposited in response to environmental factors appear to be tightly regulated in terms of their composition and their spatial and temporal distribution. However, the nature of the cues that induce lignification and determine its composition are poorly understood. Clearly, some elicitors modulate lignin deposition and lignification also appears to be under the influence of several plant growth regulators (Miller et al., 198.5; Diaz et al., 1988; Aloni et al., 1990). The mechanisms by which these substances interact with phenylpropanoid metabolism to regulate lignification are slowly being unraveled. The identification of potential protein phosphorylation sites in PAL suggests that some form of posttranslational regulation may operate at the level of the general phenylpropanoid pathway (Bolwell, 1992). However, the key regulatory events appear to involve transcriptional activation of genes encoding phenylpropanoid metabolism. Although the transcription factors controlling the temporal and spatial expression of genes have not yet been identified, there is good evidence to suggest they exist. A number of cis-acting elements with plant Myb protein binding sites have been found in PAL (Bevan et al., 1989; Logemann et aL, 199Sa), 4CL (Hauffe et al., 1991), and CAD (Feuillet et af., 1995) genes. In addition, ectopic expression of a M y b gene in tobacco led to the expression of the PAL promoter (Sablowski et al., 199.5).
A. Problems and Pitfalls of Lignin Manipulation At first sight this task might seem a relatively simple problem of applying biotechnological methods to the regulation of phenylpropanoid metabolism. The appropriate biochemical pathways have been elucidated and, with a few notable exceptions, sequence information for the enzymes catalyzing the major steps is generally available. This is, of course, a gross oversimplification of the task, and although plants with altered lignins have been produced, the prospect of manipulating lignification in plants with any degree of fine control is beset with potential problems and pitfalls.
1. Multifundionality of Lignin Potential problems arise from the multiple functions of lignin in various plant processes that may be spatially and temporally separated. This is illustrated in wheat, in which, in addition to the developmental lignification associated with the xylem, stomata1 guard cells, and surface hairs, lignifica-
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tion also underpins three separate defense responses, papilla and halo formation, the hypersensitive cell death response, and the wound margin response (Ride et al., 1989). The degree to which these defense responses and developmental lignification processes share the common biosynthetic machinery is unknown. The involvement of lignin in key plant processes, such as water transport, cell wall strengthening, and defense, dictates that a blanket approach to its manipulation is likely to have detrimental effects on the plant. A possible scenario would be the inadvertent lowering of resistance in reduced lignin plants manipulated for the purpose of increased biomass digestibility. This has been reported in alfalfa; two lines were selected with high and low lignin content. After two field seasons only 34% of the low-lignin plants had survived compared to 64% of the high-lignin plants as reported in Buxton and Casler (1993).
2. The Nature of Phenylpropanoid Metabolism Several potential problems arise from the nature of phenylpropanoid metabolism itself. Both the general phenylpropanoid pathway and the lignin branch pathways are not simple linear routes, but are better considered as enzyme nets. Thus, the likelihood of compensation following inhibition or downregulation of individual components of the enzyme net is high. Although compensation effects tend to alleviate attempts to quantitatively reduce lignin levels, the consequence of compensation on the composition of lignin can be dramatic. This is illustrated in tobacco, in which downregulation of CAD by antisense techniques leads to the accumulation of lignins with abnormally high incorporation of hydroxycinnamaldehydes (Halpin et al., 1994; Hibino et al., 1995). The lack of specificity for lignification in the enzymes of phenylpropanoid metabolism also gives rise to further problems, particularly pleiotropic effects. This is especially true for the general phenylpropanoid pathway enzymes but also applies to a lesser extent to the enzymes of the lignin branch pathway. In the case of the general phenylpropanoid pathway the problem stems from the multiplicity of plant products derived from its intermediates. Although lignification is ultimately dependent on the general phenylpropanoid pathway, genetic manipulation at this level is highly likely to have pleiotropic effects. This is illustrated by the F5H-deficient sin1 or fuhl mutation of A. thulium, often described as lignin specific, which has an impact on both lignin composition and the accumulation of soluble sinapic acid esters (Chapple, 1992). Similarly, downregulation of PAL by antisense leads to a number of morphological changes including alteration in flower pigmentation (Elkind et al., 1990). Awareness of this problem has focused attention on the enzymes of the lignin-specific pathway as better target sites for the manipulation of lignin in plants. The two steps of the
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lignin-specific pathway, CCR and CAD, are considered t o be highly specific for lignin and are regarded as reliable markers for lignification in plant tissues. Although the number of products other than lignin derived from the lignin-specific pathway intermediates is greatly reduced in comparison to intermediates of the general phenylpropanoid pathway, the fate of monolignols cannot be totally ascribe to lignification. In addition to lignin, monolignols are also required for the formation of “lignin-like” material in suberized tissues (Borg-Olivier and Monties, 1993) and in the formation of simple dilignols, collectively termed lignans. Thus, although the risk of pleiotropic effects may be greatly reduced by manipulation of the lignin branch pathway, they cannot be totally ruled out. Another aspect to be considered is the functional significance of the various molecular forms of the pathway enzymes. Polymorphism has been reported for most of the major enzymes of the general phenylpropanoid and lignin-specific pathways, yet the functional significance of this phenomenon is poorly understood. There is also the possibility that some of the reactions thought to be catalyzed by specific pathway enzymes may also be performed by nonspecific enzymes with broad range substrate specificities. The involvement of these activities in lignification and their potential to compensatc following attempts to manipulate lignification are unknown.
B. Chemical Manipulation of Phenylpropanoid Metabolism Chemical inhibitors of the general phenylpropanoid pathway and ligninspecific pathway enzymes have proved to be excellent tools for investigating lignin-dependent processes in plants, particularly those related to defense. The use of chemical inhibitors has been largely superseded by the emergence of molecular techniques. In addition to the problems and pitfalls outlined in the previous section, chemical inhibitors have their own set of unique problems associated with their use. Inhibitors may be excellent at reducing activity in vitro, but their effectiveness in pfuntu is often lessened due to permeability problems. Other problems include phytotoxicity, metabolism by the plant, and the transitory nature of their effccts. However, the most important deficiency shown by the currently used inhibitors relates to their specificity of inhibition. This is illustrated by one of the most commonly used CAD inhibitors, NH2PAS. This inhibitor was designed to chelate zinc ions from CAD and has a very loose structural resemblance to the monolignols. Although NH2PAS is clearly a potent inhibitor of CAD irz vitro, there are serious questions relating to the specificity of its inhibition. Potentially, any zinc-requiring enzymes are subject to inhibition by NH2PAS.Thus, in addition to CAD, the zinc-requiring C3H of the general phenylpropanoid pathway is potentially
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susceptible to inhibition (Kneusel et al., 1989). Furthermore, the CCR of the lignin-specificpathway, although not zinc requiring, has also been shown to be affected by NH2PAS (Grand et al., 1985b). The next generation of inhibitors is far less likely to suffer from the problems of specificity, due to their closer structural analogy to the natural monolignols in respect to the substitution patterns of their phenolic rings. The latest CAD inhibitors are mechanism-based suicide inhibitors that, in addition to utilizing the binding specificity of their target enzyme, specifically utilize their catalytic apparatus for activation with the result that a normally innocuous reversible inhibitor is converted into a powerful irreversible inhibitor. The two inhibitors illustrated (Fig. 2) have close structural homology to sinapyl alcohol and have been designed specifically to inhibit the elicitor-induced form of CAD in wheat. The acetylenic alcohol (Fig. 2a) and the cyclopropane analog (Fig. 2b) have the 4-hydroxy and 3,5-O-methoxy pattern preferred by the inducible form of CAD. In both cases, when oxidized to the aldehyde by CAD, reactive electrophilic species are generated with the potential to attack active site nucleophiles and irreversibly bind and inhibit CAD.
C. Molecular Manipulation of Phenylpropanoid Metabolism
The potential of mutants-naturally occurring, induced by mutagenesis, or genetically engineered-for applied aspects of plant improvement cannot be overstated. In addition to their applied value, they also provide insights into the fundamental regulatory mechanisms controlling lignification in plants. In comparison to wild-type plants, major phenotypic differences in growth and morphology have not been encountered in mutants of phenylpropanoid metabolism. Considering the multifunctionality of lignin in plants, the lack
a
CH,OH
Me0
Me0
HO
b
\
OMe
HO
\
CH,OH
OMe
FIG. 2 Mechanism-based suicide inhibitors of cinnamyl alcohol dehydrogenase (CAD), with close structural homology to the monolignol sinapyl alcohol. The acetylenic alcohol (a) and the cyclopropane analog (b), when oxidized to the aldehyde by CAD, generate reactive electrophilic species with the potential to attack active site nucleophiles and irreversibly bind and inhibit CAD.
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of pleiotropic effects is surprising until the degree of compensation exhibited by these mutants is revealed. Manipulation of phenylpropanoid metabolism enzymes has already resulted in plants with reduced lignin and increased incorporation of nonmonolignol components into the polymer. This is illustrated by the sorghum brown midrib mutant bmr6 with depressed CAD (Bucholtz, 1980) and in genetically transformed tobacco with downregulated CAD (Halpin, 1994). The sorghum bmr6 mutant has less lignin compared to the wild-type, whereas the CAD antisense tobacco mutant, with only 10% of normal CAD activity, has normal lignin levels. In both mutations analysis of the lignins revealed substantial compensation by the plants in terms of incorporation of hydroxycinnamaldehydes into the polymer. Where antisense techniques are used to downregulate enzymes, such as CAD in tobacco (Halpin, 1994), the nature of the mutation is obvious. This is not the case for mutations arising naturally or from mutagenic treatments. For instance, the sorghum brown midrib mutant, although depressed in CAD, also has lower OMT activity. The mutation is reported to result from a single locus genetic lesion, suggesting that the mutation could correspond to a regulatory gene (Pillonel, 1991). As a way to manipulate the quantity and quality of plant lignins and as tools to investigate fundamental questions relating to lignin biosynthesis, mutants have tremendous potential. The ability of plants to compensate for manipulations suggests that alteration of lignin composition may be a more easily attainable goal that the quantitative control of lignin levels. Quantitative regulation is far more likely to require manipulation at more than one point of phenylpropanoid metabolism and may be better approached by manipulation of regulatory genes. The requirement for better understanding of the regulatory processes at both the molecular and biochemical levels is of paramount important if progress is to be achieved.
D. Future Directions How realistic is the prospect of attaining control over plant lignification via the manipulation phenylpropanoid metabolism? In addition to the major potential pitfalls described earlier, such as the multifunctionality of lignin, the complexity of the pathway, and the lack of specificity in the enzymes, the underlying approach itself may be limited. Manipulation of the general phenylpropanoid pathway and the lignin branch pathway enzymes is unlikely to allow the precise control of lignification in plants. If these pathways are merely considered as providing the building blocks for lignification, then manipulation of the flux of material passing through them might modulate the amount and type of building block available but have little control over the temporal and spatial polymerization of lignin. This fine
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control is far more likely to be exerted by regulatory genes acting on these common pathways and probably involves differential induction of specific molecular forms of key biosynthetic enzymes. In addition to the controls operating on the biosynthetic pathways leading to monolignol accumulation, regulatory mechanisms could also be applied at other leveis of the lignification process. Potentially, controls may operate at the level of monolignol transport and at the polymerization stage. Unfortunately, our knowledge of the cell biology of lignification is very poor, to the extent that the mechanisms of monolignol transport have barely been addressed. The polymerization process is better understood, and the emerging importance of the polysaccharide template over the control of polymerization is becoming apparent. The major focus of attention in the future will inevitable have to address the question of judging the significance of these controls in relation to the regulation of lignification in plants.
Acknowledgments We thank Dr. M. J. Ord (School of Biological Sciences, University of Southampton) for her encouragement during the preparation of the manuscript and Professor J. L. Hall (School of Biological Sciences, University of Southampton) and Dr. J. P. Ride (School of Biological Sciences, University of Birmingham) for their comments on the text.
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Wilkinson, E. M., and Butt, V. S . (1992). Enzyme changes during lignogenesis in pea shoots induced by illumination. J. Exp. Bot. 43, 1259-1265. Wyrambik, D., and Grisebach, H. (1975). Purification and properties of isoenzymes of cinnamyl-alcohol dehydrogenase from soybean-cell-suspension cultures. Eur. J . Biochem. 59, 9-15. Wyrambik. D., and Grisebach, H. (1979). Enzymic synthesis of lignin precursors. Further studies on cinnamyl-alcohol dehydrogenase from soybean-cell-suspension cultures. EUY.J. Biochem. 97,503-509. Xie, Y., and Wu, R. (1989). Rice alcohol dehydrogenase genes-Anaerobic induction, organ specific expression and characterization of cDNA clones. Plant Mol. Bid. 13, 53-68. Yamamoto, E., Bokelman, G. H., and Lewis, N. G. (1989). Phenylpropanoid metabolism in cell walls. In “Plant Cell Wall Polymers Biogenesis and Biodegradation” (N. C. Lewis and M. G. Paice, Eds.), pp. 68-88. American Chemical Society, Washington, DC. Yao, K., De Luca, V., and Brisson, N. (1995). Creation of a metabolic sink for tryptophan alters the phenylpropanoid pathway and susceptibility of potato to Phytophthora infestuns. Plant Cell 7, 1187-1799. Ye, Z. H., and Varner, J. E. (1995). Differential expression of 2 0-methyltransferases in lignin biosynthesis in Zinnia elegans. Plant Physiol. 108, 459-467. Ye, Z. H., Kneusel, R. E., Matern, U., and Varner, J. E. (1994). An alternative methylation pathway in lignin biosynthesis in Zinnia. Plant Cell 6, 1427-1439. Yoshida, S . , and Shimokoriyama, M. (1965). Studies on phenylalanine deaminase in buckwheat plant. Bot. hlag. (Tokyo) 78, 14-19. Zeyen, R. J., Bushnell, W. R., Carver, T. L. W., Robbins, M. P., Clark, T. A., Boyles, D. A,, and Vance, C. P. (1995). Inhibiting phenylalanine ammonia-lyase and cinnamyl alcohol dehydrogenase suppresses Mlal (HR) but not mo15 (non-HR) barley powdery mildew resistances. Physiol. Mol. Plant Pathol. 47, 119-140. Zimmermann, A,, and Hahlbrock, K. (1975). Light-induced changes of enzyme activities in parsley cell suspension cultures. Purification and some properties of phenylalanine ammonialyase (EC.4.3.1.5). Arch. Biochem. Biophys. 166, 54-62.
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INDEX
A Accessibility induction by suppressors, 62-65 in plant-parasite interactions, 57-59 Actin, in regulation of CFTR, 212-213 ADP ribosylalion factor in CFTR recycling, 219 in secretory vesicle formation, 228 Agressin, see Suppressors Alpha-adaptin protein, association with CFTR, 217 Alzheimer’s disease, GLUTl exprcssion in, 36 Amelogenesis imperfecta, crystal structure in, 152 Angiosperms cinnamoyl-CoA reductase substrate specificity in, 263 cinnamy! alcohol dehydrogenase polymorphism in, 265 4-coumarate-CoA ligase activity in, 259-260 lignin types in, 245 Apatite, see also Calcified tissue; Calcium phosphates; Carbonate apatite in biomaterials from bovine bone, 162-164 coralline, 161 medical history and uses of, 159-161 in bone crystal structure, 143-145, 166 dissolution, 167 molecular substitutions in, 165-166 organic matrix and, 167 during osteoporosis, 152, 154
295
in bone graft interface, 176-177 biodissolution and, 167, 170-171 bone ingrowth and, 173-174 carbonate apatite deposition and, 171-173 dislocations in, 170-171 in calcified tissue, 130, 175-176 carbonate incorporation, 154-155 chloride incorporation, 159 in dentin, 143 in enamel crystal structure, 139-142 dissolution, 148-149 interactions with protein, 142 physical characteristics, 131 in fish enameloids, 145 fluoride incorporation, 155- 157 magnesium incorporation, 157-158, 165-166 in pathological calcifications, 176 phosphate incorporation, 158 pyrophosphate incorporation, 159 stroritium incorporation, 159 Aqueous humor blood barrier and glucose transport, 19-22 structure of, 17-19 glucose level in, 17 ARF, see A D P ribosylation factor Articular calcification, crystal structure in, 147 Astrocytes GLUTl expression in, 11 in regulation of GLUTl expression, 34 ATP conductance by CFTR, 199,215 hydrolysis by CFTR, 195, 197, 200
296
INDEX
ATPase cell wall-bound effect of suppressors on, 77-80 in fungal signal transduction cascade model, 82-84 in peroxide generation, 81 membrane-bound cell wall regulation of, 79-80 effect of suppressors on, 67-70 functional association with polyphosoinositide metabolism, 76 Autocrine signaling, platelet-derived growth factors and, 104, 105 in blastorneres. 112
B Bacteria, in suppression of plant defenses, 61 Bicarbonate, conductance by CFTR, 199 in pancreatic duct cells, 211 Blastomeres, platelet-derived growth factor activity in, 112 Blood-tissue barriers, see also specific organs and tissues endothelium and epithelium types, 6-8 glucose transporters in, 8 Blood vessels, development, platelet-derived growth factors in, 116-117 Bone crystal structure and composition, 143-145, 165-167 effects of sintering on, 162-164 interactions with bone graft materials, 134 osteoporosis in, 152, 154 Bone grafts calcium phosphate materials in, 133, 159-161 from bovine bone, 162-164 coralline, 161 implant coatings and, 165 interactions with bone, 134 synthetic, 164 ceramic material interface bioactive processes and, 174-175 bone ingrowth and, 173-174 carbonate apatite deposition in, 167, 171-173, 175, 176-117 cell colonization of, 168-170, 174-175
crystal biodissolution in, 167, 170-171 crystal deposition in, 171-173 Botrytis, accessbility induction and, 62, 64-65 Bovine bone, as bone graft material, 162-164 Brain, blood harrier in glucose transport, 10-13 structure, 9
C Caffeoyl-CoA 3-O-methyltransferase, in phenylpropanoid pathway, 258 Calcifications, see Calcified tissue Calcified tissue, see also Apatite analytical techniques in crystal composition, 138-139 crystal localization, 135 crystal morphology, 137-138 crystal phase identification, 135-137 organic phase removal, 135 bone, crystal structure and composition, 143-145, 165-167 crystal composition, 129-130 invertebrates. 130 pathological, 130 dentin, crystal structure and composition, 142-143, 165-166 diseased conditions amelogenesis imperfecta, 152 dentin caries, 149-151 enamel caries, 148-149 fluorosis, 151-152 osteoporosis, 152, 154 enamel, crystal structure and composition, 139-142, 165-166 fish enameloids, 145 pathological dental calculus, 147-148 mineral phases, 145, 147 soft tissue calcifications, 147 urinary calculus, 148 whitlockites in, 157-158 Calcium in fungal signal transduction cascade model, 83 in plant defense transmembrane signaling, 70-71
INDEX
Calcium carbonate activity in bone grafts, 174 in calcified tissue of invertebrates, 730 Calcium hydroxyapatite. see Apatite Calcium phosphates, see also Apatite in amelogenesis imperfecta, 1.52 in biomaterials, 176-177 bone grafts and, 133 from bovine bonc, 162-164 coralline, 161 dental and medical applications, 159- 161 implant coatings, 165 synthetic, 164 in bone graft interface bioactive processes and, 174-175 biodissolution of, 170-171 carbonate apatite formation and, 167, 171-173, 175, 176-177 cell colonization of, 168-170, 174-175 in dental calculus, 147-148 in dentin caries, 140, 151 in fluorotic enamel, 151-152 minor elements and, 154-159 in pathological calcifications, 130, 145, 147 in soft tissue calcifications, 147 in urinary calculus, 148 Calnexin, in folding of CFTR protein, 205-206, 207 Carbonate, incorporation in apatite, 154-155 Carbonate apatite in bone graft interface, 167, 171-173, 175, 176-177 in soft tissue calcifications, 147 Caries in dentin, 149-151 in enamel, crystal dissolution and, 148-149 Cell capacitance, effects of cyclic AMP on, 224 Cell surface area, effects of cyclic AMP on, 224 Cell wall effects of suppressors on ATPase and, 77-80 peroxide generation and, 80-81 in host-parasite specificity, 77-80, 84 peroxidase activity and, 272
297 in plant defenses, 76-77 in regulation of membrane-bound ATPase, 79-80 Central nervous system, platelet-derived growth factors in activity, 113-114 distribution, 108, 100 Cerebrospinal fluid, blood barrier and glucose transport, 13-14 structure, 13 CFTR, see Cystic fibrosis transmembrane conductance regulator Chloride conductance in cystic fibrosis, 21 1 incorporation in apatite, 159 Chloride channels, see also Cystic fibrosis transmembrane conductance regulator in cystic fibrosis, 195-196 in endosome acidification, 230 Cholera, chloride channel activity and, 209-210 Choroid plexus glucose transport in, 13-14 GLUT1 expression in, 11-12 Ciliary body epithelium barrier properties, 18- 19 gap junctions in, 21-22 GLUT1 in, 19-21 structure, 17-18 Cinnamate 4-hydroxylase activity in response to stress, 254 localization, 253, 254 in phenylpropanoid pathway, 253-254 Cinnamoyl-CoA reductase effects of inhibitors, 263-264 expression, 263 in lignin branch pathway, 262 substrate specificity, 263 Cinnamyl alcohol dehydrogenase expression antisense gene, 268-269 correlation with lignification, 266-267 developmental, 266 pathogen stress, 267 inhibitors, 267-268, 276-277 in lignin branch pathway, 264 localization, 264 mutant forms, 268 polymorphism, 265-266 structure, 264-265
298 Cinnamyl alcohol P-glucosidase, in deglycosylation of monolignol glycosides, 270-271 Cinnamyl alcohol glucosyltransferase, in glycosylalion of monolignols, 270 Clathrin-coated vesicles, CFTR recycling and. 217 CoA-esterification, in phcnylpropanoid pathway, 258 Coniferyl alcnhol, see Monolignols Connexins, ill glucose transfer blood-tissue harriers, 8 ciliary body epithelium, 21-22 rat placenta, 31 Constitutivc secretory pathway polarized regulation in, 228-229 trans golgi network in, 226 Coral, in bone grafts, 161, 174 4-Coumarate-CoA ligase exprcssion developmental, 260 pathogen stress, 260-261 wound stress, 261 isoform substrate specificity, 259-260, 26 1-262 localization, 259, 260 in phenylpropanoid pathway, 259, 261-262 Coumarate 3-hydroxylase, in phenylpropanoid pathway, 254-255 p-Coumaryl alcohol, see Monolignols Crystals, biological, see Calcified tissue Cyclic AMP effects on whole cell capacitance, 224 elevated levels in cholera, 209-210 modulation of membrane budding, 228 possible regulation of truns golgi network polarity, 229 protein sialylation and. 228 regulation of CFTR, 218-219 regulation of constitutive secretory pathway, 226 regulation of endocytosis, 221-222 rcgulation of exocytosis, 224 regulation of glycoconjugate secretion, 225 truns golgi network acidification and, 228 Cyclic AMP protein kinase, phosphnrylation of CFTR, 195, J 97, 199
INDEX Cyclic nucleotides, i n plant defense transmembrane signaling, 71 Cystic fibrosis, see also Cystic fibrosis transmembrane conductance regulator anti-inflammatory drugs and, 194-195 clinical manifestations, 194 defective apical chloride conductance and, 211 compound hcterozygotes in, 203 defective chloridc conductance in, 395-196 elevated sweat chloride levels in, 210-21 I genetic basis, 193-195 mucin secretion in, 220-221 mutations of CFTR and, 200-203 respiratory inllammation, CFTR regulatory activity and, 231 sodium chloride reabsorption in, 196 Cystic fibrosis transmembrane conductance regulator, see rrlso Cystic fibrosis activity when heterologously produced. 230 ATP conductance and, 199, 215 ATP hydrolysis and, 195, 197, 200 bicarbonate conductance and, 199 chloride channel activity biophysical properties of, I97 in cholera, 209-210 in cystic fibrosis, 195-196 in epithelial cells, 208-209 functional redundancy in, 200 in human reabsorptive sweat duct, 210-211 ion selectivity and, 199 regulation of, 197-199 cyclic AMP protein kinase phosphorylation and, 195, 197, 200 cytoplasmic nucleotide binding domain, 195, 197-200 cytoplasmic regulatory domain, 195, 197, 199 effect of glycerol on, 208 endoplasmic reticulum and model of protein kinetics within, 206-207 polyubiquitination, 204-205 protein folding in, 205-206 localization in epithelial cells, 208-209, 216-217 modulation of membrane budding, 228
INDEX
299
modulation of outwardly rectifying chloride channels, 214-215 modulation of sodium channels, 213-214 mucin secretion and, 220-221 mutations activity of DeltaF.508, 201-202. 204-208 activity of D836X, 200 chloride channel activity of, 206 classes of, 200-203 second site mutations, 203 tcmperatul-e-sensitive, 208 in organelle acidification, 227 protein sialylation and, 228 recycling autoregulation, 222-223 clathrin-coated vesicles and, 217 cyclic AMP regulation of, 218-219, 223 endosomes and, 216-217 functional significance, 217-219 molecular control of, 219-220 regulation of. 197-200 actin and, 212-213 cyclic AMP and, 218-219 microtubules and, 212 in regulation of endocytosis, 222 in regulation of exocytosis, 223-225 in regulation of membrane traffic, 220-221 in regulation of other transporters and receptors, 230-23 1 trans golgi network acidification and, 228 transport apical endosomes and, 215-216 effect of epithelial polarization on, 216 trans golgi network and, 215 Cytotrophoblasts in human placenta as blood barrier, 26 GLUT1 in, 27 in rat placenta, as blood barrier, 29
D Degl ycosylation, of monolignol glycosides, 270-271 Dental calculus, mineral phases in, 147-148 Dental implants, calcium phosphate coatings and, 16.5
Dentin caries in, 149, 151 crystal structure and composition, 142-143, 165-166 topical fluoride treatment, 157 Diabetes, GLUTl expression in, 35 Dislocations. in apatite crystals, 170-171 DNA, synthesis, platelet-derived growth factors in, 95-96, 115-116
E Electron microscopy, in analysis of calcifications, 137-138 Electron spectroscopy, in analysis of calcifications, 139 Elicitors in accessibility induction, 58 activation of plant defense genes, 70 effects on cell wall-bound ATPase, 78 effects on peroxide generation, 80-81 effects on polyphosphoinositide metabolism, 71-73 interactions with suppressors, 65-67 in Mycosphaerella, 62-63 in phytoalexin theory, 57 pisatin accumulation and, 65-66 stimulation of membrane-bound protein phosphorylation, 74-75 in suppression of plant defenses, 84 Embryogenesis, platelet-derived growth factors in activity, 110- 117 distribution, 304-110 Enamel amelogenesis imperfecta in, 152 caries in, 148-149 crystal/protein interactions in, 142 crystal structure and composition, 139-142, 165-166 fluorosis in, 151-152 topical fluoride treatment, 157 Enarneloids. fish, crystal structure, 14.5 Endocytosis CFTR regulation of, 222 cyclic AMP regulation of, 221-222 Endolymph, blood barrier and, glucose transport, 23-24 Endoneurial blood vessesls, GLUTl in, 25
300
INDEX
Endoplasmic reticulum, CFTR and model of protein kinetics within, 206-207 protein folding in, 205-206 protein processing and degradation in, 204-208 rctcntion of mutant protein in, 201-202 Etidosomes acidification, 226-227 non-CFTR dependent chloride conductance in, 230 CFTR recycling and, 215-220 Endotheliuin, in blood-tissue barriers, 6-8 Epithelial cells, CFTR and chloride channel activity, 208-209 localization in, 208-209, 216-217 Epithelial sodium channels, see atso Sodium channels possible CFTR regulation of, 231 Epithelium, in blood-tissue barriers, 6-8 Exocytosis, regulation of CFTR and, 223-225 cyclic AMP and, 224
F Fatty acids in fungal signal transduction cascade model. 82-84 in plant defense transmembrane signaling, 74 Ferulate 5-hydroxylase, in phenylpropanoid pathway, 255 Fetal membranes, human, GLUTl in, 27 Fish enameloids, crystal structure, 145 Fluoride, incorporation in apatite, 155-1.57 Fluoroapatitc, in fish enameloids, 145 Fluorosis, dental enamel and, 151-152 Fructose transporters, see GLUTS
G Gap junctions, in glucose transfer blood-tissue barriers, 8 ciliary body epithelium, 21-22 rat placenta, 31 Gastrulation, platelet-derived growth factors in activity, 111-1 12 distribution, 104
Giant cells, in bone graft interface, 168 Gliogenesis, platelet-derived growth factors in, t 14 Glucose dietary absorption. 5-6 fetalhaternal concentrations, 28-29 level in aqueous humor, 17 regulation of GLUTl expression, 35 Glucose transport(ers) in blood-tissue barriers aqueous humor, IY-22 'brain, 10-13 cerebrospinal fluid, 13-14 human placenta, 26-29 inner ear, 23-24 intestines, 5-6 iris, 22-23 peripheral nerves, 24-25 rat placenta, 29-31 retina, 15-17 testis, 31-32 thymus, 32-33 facilitated-diffusion family, srr cilro GLUTI-5 forms and distribution, 2-5 regulation developmental, 33-34 effects of degenerative disease on, 36 effects of ischemia and hypoxia on, 35-36 effects of tumors on, 36-37 by glucose, 35 in hypoglycorrhachia, 37 induced expression in blood vessels, 34 sodium-dependent family, see also SGLTl in blood-brain barrier, 13 forms and activity, 5 GLUTl in astrocytes, 11 in blood-brain barrier, 10-12 in choroid plexus, 11-12, 13-14 in ciliary body epithelium, 19-21 in endonuerial blood vessels, 25 in human placenta, 27-28 in inner ear, 23-24 in iris, 22-23 in perineurium, 24-25 in rat placenta, 29-31 regulation developmental. 33-34
301
INDEX
effects of degenerative disease on, 36 effects of ischemia and hypoxia on, 35-36 effects of tumors on, 36-37 by glucose. 35 in hypoglycorrhachia, 37 induction in blood vessels, 34 in retinal blood vessels, 1.5 in retinal pigment epithelium, 15-16 in testis, 32 GLUT2 in dietary absorption of glucose, 6 in retina, 16--17 GLUT3 in blood-brain barrier, 12 in human placenta, 27-28 in neurons, 12 in rat placenta, 29, 31 regulation developmental. 33 effects of ischemia on, 35 effects of lumors on, 36-37 in retina, 17 GLUT4 in human placenta, 27-28 in insulin-stimulated glucose transport, 4 GLUTS, in blood-brain barrier, 12 Glycoconjugates, secretion of, cyclic AMP and CFTR regulation, 225 Glycosylation in CFTR mutants, 201 of monolignols, 270 of platelet-derived growth factors, 97 Golgi, see tram Golgi network GTP-binding protein, in plant defense transmembrane signaling, 73-74 Gymnosperms cinnamoyl-CoA reductase substrate specificity in, 263 cinnamyl alcohol dehydrogenase in, 265 4-coumarate-CoA ligase activity in, 259-260 lignin types in, 245 monolignol gylcoside accumulation in, 269
H Halides, conductance by CFTR, 199 Heart, platelet-derived growth factors in activity, 114-115 distribution, 109, 110
Host-parasite specificity determination of, 63, 64-65 suppressors and, 84-85 plant resistance in, 55-56 role of cell wall in, 77-80 Host-specific toxins, in plant-parasite interactions, 61 hsp70 protein, in folding of CFTR protein, 205, 207 Huntington’s disease, glucose transporter expression in, 36 Hydroxycinnamyl alcohols, see Monolignols Hypoglycorrhachia. GLUTl expression in, 37 Hypoxia, effects on GLUTl expression, 35-36
1 Ibuprofen, effects in cystic fibrosis, 194-1 95 Impedin, see Suppressors Inducers, see Elicitors Infrared absorption, in analysis of calcifications, 136- 137 Inner ear, blood barriers in, glucose transport, 23-24 Inositol 1,4,5-triphosphate, in plant defense transmembrane signaling, 71-73 Insulin-stimulated glucose transport, GLUT4 and, 4 Intestines, glucose absorption in, 5-6 Iridial epithelium, GLUTl in, 22-23 Iridial stroma, GLUTl in, 22-23 Iris, glucose transport in, 22-23 Ischemia, effects on glucose transporter expression, 35
K Kidney, platelet-derived growth factors in, distribution, 110
1 Laccases, in lignin polymerization, 273 Lignin biosynthesis, see also Phenylpropanoid metabolism caffeoyl-CoA 3-0-methyltransferase in, 258
302 Lignin (confinued) cinnamate-4-hydroxylase in, 253-254 cinnamoyl-CoA reductase in, 262-264 cinnamyl alcohol dehydrogenase in, 264-269 4-coumarate-CoA liguse in, 259-262 coumarate 3-hydroxylase in, 254-255 ferulatc 5-hydroxylase in, 255 general phenylpropanoid pathway and, 246 lignin branch pathway and, 246-247 O-methyltransferase in, 256-258 phenylalanine ammonia lyase in, 247-252 shikimate pathway and, 246 tyrosine ammonia lyase in, 252 composition, 245 distribution in plants, 245 inhibitors, effects in p h t a , 251-252 manipulation future directions. 278-279 problems in, 274-276 use of mutants in, 277-278 polymerization laccases and, 273 peroxidase and, 271-273 phenoxyradicals and. 271. 273 residues, 245 rolcs in plants, 244, 274-275 Lignin branch pathway. see also Phcnylpropanoid metabolism in lignin biosynthesis, 246 cinnamoyl-CoA reductase, 262-264 cinnamyl alcohol dehydrogenase, 264-269 Limb bud, platelet-derived growth factor distribution in, 10X-11)s) Lung, platelet-derived growth factors in activity, 115-116 distribution, 109 Lysosomes, acidification of, 226
Macrophages, in colonization of bone graft interface, 174- 175 Magnesium. incorporation in apatite, 157-158, 165-166 0-methyltransferase expression antisense gene, 257-258
INDEX developmental, 257 pathogen strcss, 257 forms, 258 localization, 256 in phenylpropanoid pathway, 255-256 substrate specificity types, 256 Microtubules, in rcgulation of CFTR, 212 Migrating cells, platelet-derived growth factors and, 112 Mitogenesis, platelet-derived growth factors and, 95, 96, 116 Monolignol glycosides accumulation in plant tissues, 269 deglycosylation, 270-271 transport, 269 Mono1ign o1s glycosylation, 270 lignin residues and, 245 non-lignin fates, 276 polymerization into lignin, 271-273 stimulation of peroxidase and, 272 transport, 269 Mouse, platelet-derived growth factors and in embryogensis, 117-1 18 in patch mutation, 110-111 Mucin, secretion in cystic fibrosis, 220-221 Mucin-type suppressors, interactions with ATPase, 69-70 Miiller cells, GLUT2 expression in, 16-17 Murine development, see Mouse Mycosphaerella accessbility induction and, 62-65 activity of elicitors and suppressors in, 65-67 effects on host cell wall-bound ATPase, 77-80 effects on host membrane-bound protein phosphorylation, 75 effocts on host peroxide generation, 80-81 suppressor production, 59-61 suppressor signal transduction cascade model, 82-84
N Neural crest, platelet-derived growth factor distribution in, 105 Neurogenesis, platelet-derived growth factors in, 114
303
INDEX
Nuclear magnetic resonance, in analysis of calcifications. 137
Oligodendrocytes, precursors, plateletderived growth factors in, 113 Optic nerve, platelet-derived growth factor activity in, 113 ORCC, see Outwardly rectifying chloride channels Orthopedic implants, calcium phosphate coatings and, 165 Orthovanadate induction of phytoalexins by, 68-69 inhibition of ATPase activity, 67-69 inhibition of peroxide generation, 81 Osteoclasts, in bone graft interface, 168 Osteogenesis, in bone graft interface, 168 Osteoporosis, crystal characteristics in, 152, 154 Outwardly rectifying chloride channels, CFTR modulation of, 214-215
P Pancreatic insufficiency, in cystic fibrosis, 211 Paracine signaling, in platelet-derived growth factors, 104, 105 PDGF, see Platelet-derived growth factors Periarticular calcification, crystal structure in, 147 Perilymph, blood barrier and, glucose transport. 23-24 Perineurium GLUT1 in, 24-25 as permeability layer, 24 Peripheral nerves, blood barrier in glucose transport, 24-25 structure, 24 Peroxidase, cell wall bound effects of suppressors on, 80-81 in lignin polymerization, 271-273 Peroxide, generation cell wall-bound ATPase and, 81 effects of suppressors on, 80-X1 in fungal signal transduction cascade model, 82-84
Phenol oxidases, in lignin polymerization, 27 1-273 Phenoxyradicals, in lignin polymerization, 271, 273 Phenylalanine ammonia lyase expression correlation with lignification. 250-251 environmental stress, 250 pathogen stress, 250-251 genes, 250 inhibitors, effects in planfa, 251-252 localization, 249 in phenylpropanoid pathway, 247 polymorphism, 249-250 structure, 249 Phenylpropanoid metabolism, see also Lignin branch pathway; Phenylpropanoid pathway manipulation chemical inhibitors and, 276-277 compensation effects, 275 enzyme specificity and, 275-276 future directions, 278-279 mutations and, 277-278 Phenylpropanoid pathway, see also Phenylpropanoid metabolism in lignin biosynthesis, 246 caffeoyl-CoA 3-0-methyltransferase, 258 cinnamate 4-hydroxylase, 253-254 cinnamyl alcohol dehydrogenase, 264-269 CoA-esterification, 258 4-coumarate-CoA ligase, 259-262 coumarate 3-hydroxylase, 254-255 ferulate 5-hydroxylase, 255 0-methyltransferase, 255-258 phenylalanine ammonia lyase, 247-252 tyrosine ammonia lyase, 252 regulation of, 274 Phosphate, incorporation in apatite, 158 Phosphatidylinositol bisphosphate, in plant defense transmembrane signaling, 71, 73 Phospholipase, in plant defense transmembrane signaling, 71, 73. 74 Phytoalexins effects of suppressors on, 67 induction by orthovanadate, 68-69 Phytoalexin theory accessibility induction in, 58
INDEX Phytoalexin theory (continued) elicitors in, 57 in plant-parasite interactions, 56-57 Phytopathogens elicitors in, 57, 58-59 host plant accessibility and, 58-59 qualities, 81-82 suppressor production, 59-61 supprcssors in spore germination fluids, 62-65 Phvi~p~~hf)r~i activity of glucan suppressors in, 65 suppressor production, 59-60 PI metabolism, see Polyphosphoinositide mcta bolisrn Pisatin effects of elicitors and suppressors on, 65-66 effects ol rnucin-type suppressors on, 69-70 Placenta human, blood barrier in bidirectional glucose transport, 29 glucosc transport, 26-29 structure, 25-26 rat, blood barrier in gap junctions, 31 glucosc transport, 29-31 structure, 29 Plant defenses, w e trlso Plant-parasitc interactions accessibility and, 58-59 in host-parasite interactions, 55-56 nuclear protein phosphorylation and, 75-76 phytoalexin theory and, 56-57 role of cell wall in, 76-77 transmembrane signaling and, 70-76 Plant-parasite interactions, see also Elicitors; Suppressors accessibility in, 57-59 bactcrial, 61 phytoalexin theory in, 56-57 plant resistance in, 55-56 specificity in, 63, 64-65, 84-85 suppressors in, 59-61 Platelet-derived growth factors activity in embryogenesis angiogensis, 116-117 central nervous system, 113-1 14 early stagcs, 111-113
heart, 114-115 lung, 115-116 patch mouse mutation and, 110-111 targeted gene disruption and, 117-118 autocrine signaling in, 104, 105 cellular activities and, 95-96, 102-103 distribution in embryogenesis central nervous system, 108 early developmental stages, 104-105 heart, 109 kidney, 110 limb, 108-109 lung, 109 neural crest, 10s sornites, 105, 108 dominant negative mutants in, 103 intracellular signaling molecules and, 100- I03 ligands dimerization. 96-97 extraccllular localization, 98 genetic conservation, 97 isoforms, 96-97 post-transcriptional modification, 97-98 splice variants, 98 mitogenic activity of, 95, 96 paracine signaling in. 104, 105 receptors chimeric constructs, 102-103 chromosomal organization, 99- LOO dimeric forms, 100, 101 SH2 protein binding and, 100 tyrosine kinase activity or. 99 Polyphosphoinositidc metabolism functional association with membranebound ATPasc, 76 in fungal signal transduction cascade model, 82-84 in plant defense transmembrane signaling, 71-73 Proteasomes, in degradation of immature CFTR protein, 204-205 Protein, interactions with apatite in enamel, 142 Protein phosphorylation in activation of plant defense genes, 75-76 in plant dcfense lransmeinbrane signaling, 74-76 Pseiidr~mrmrsuerrigitwsu, in cystic fibrosis, 194
INDEX
305
Pyrophosphate. incorporation in apatite, 159
R Raman spectroscopy, in analysis of calcifications, 137 Respiratory inflammation, in cystic fibrosis, 21 1 Retina. blood barrier in glucose transport. 15-17 structure, 14415 Retinal pigment epithelium in glucose transport, 15-16 as permeability barrier, 15
S Scanning electron microscopy, in analysis of calcifications, 137-138 Secretory vesicles, A D P ribosylation factor in formation of, 228 Sertoli cells, GLUTl in, 32 SGLTI, in dietary absorption of glucose, 5-6 Shikimate pathway, in lignin biosynthesis, 246 SH2 proteins, in platelet-derived growth factor signal mediation, 100 Sialylation CFTR and, 227 cyclic AMP regulation of, 228 frans golgi network and, 227-228 Sinapyl alcohol. see Monolignols Sintcring of bone, 162-164 of synthetic bone graft materials, 164 SNAP, in CFTR recycling, 219-220 SNARE, in CFTR recycling, 21 9-220 Sodium channels, see also Epithelial sodium channels modulation by CFTR. 213-214 Sodium chloride, reabsorption in cystic fibrosis, 196, 210-211 Somites, platelet-derived growth factor distribution in, 105, 108 Spectrometry, in analysis of calcifications. 139
Spectroscopy, in analysis of calcifications, 137, 139 Spore germination tluids, suppressors in, 62-65 Stop mutations, in CFTR, 200-201 Strontium. incorporation in apatite, 159 Suppressors accessiblity-inducing activities, 62-65 chemical-nature of, 61 effects on ATPase cell wall-bound, 77-80 membrane-bound, 67-70 effects on GTP-binding protein, 73-74 effects on peroxide generation, 80-81 effects on phytoalexins, 67 effects on plant defense transmembrane signaling, 70-76 in host-parasite specificity, 63, 64-65, 84-85 interactions with elicitors, 65-67 mucin-type, 69-70 in Mycosphaerella, 62-63 origin of term, 58-59 phytopathogen production of, 59-61 pisatin accumulation and, 65-66 possible modes of action, 84 signal transduction cascade model, 82-84 Susceptibility, in plant-parasite interactions, .see Accessibility Sweat duct, reabsorptive, CFTR function in, 196, 210-211 Syncytiotrophoblasts in human placenta as blood barrier, 26 GLUTl in, 27 in rat placenta as blood barrier, 29 GLUTl in, 30,31
T Testis, blood barrier in glucose transport, 31-32 structure, 31-32 Thymus, blood barrier in glucose transport, 32-33 structure, 32-33 Transcytosis, of polymeric immunoglobulins. 23 1
INDEX [runs Golgi network
acidification of, 226, 228 in membrane trafficking, 225-226 polarity of, 228-229 sialylation of glycoproteins and, 227-228 transport of CFTR and, 215 tubule coat structures, 229 Transmission electron microscopy, in analysis of calcifications, 138 Transport proteins, recycling of, 218 Tumors, eliects on glucose transporter expression, 36-37 Tyrosine, in platelet-derived growth factor receptor binding, 100, 101-102 Tyrosine ammonia lyase, in phenylpropanoid pathway, 252 Tyrosine kinase, platelet-derived growth factors and, 99
U Ubiquitination, of CFTR protein, 204-205 Urinary calculus, mineral phases in, 148
w Whitlockites, in pathological calcifications, 157-158
X X-ray diffraction in analysis of calcifications, 135-136 of enamel apatites, 131 X-ray microradiography, in analysis of calcifications. 135
