304

CURRENT COMMENT

GROWTH FACTORS, EXTRACELLULAR MATRIX, AND ONCOGENES IN SCLERODERMA STEFFEN GAY, ANDREAS TRABANDT, LARRY W. MORELAND, and RENATE E. GAY Pathologic conditions in which fibrosis and structural tissue alterations occur arise through fibroproliferative responses and/or excessive accumulation of connective tissue matrix (I). Fibrosis in the skin of scleroderma patients is readily recognized by biopsy, and skin thickness has been correlated with the content of the major matrix component, collagen (2). Although the molecular mechanisms responsible for the excessive deposition of collagenous matrix are unclear, it has been demonstrated that the increase in procollagen production by fibroblasts obtained from fibrotic sclerodermatous skin is associated with elevated levels of corresponding procollagen messenger RNA (mRNA), suggesting pretranslational control (3). With regard to the types of collagen genes expressed in sclerodermatous skin, alterations in the expression of several collagen genes, including those encoding collagen types I, 111, and VI, have been reported (4). In vitro studies concerning the regulation of collagen biosynthesis, especially the influence of growth factors on fibroblasts, have indicated that platelet-derived growth factor (PDGF) has a mitogenic effect on cultured skin fibroblasts (3,while transforming growth factor P (TGFP) induces increases in collagen and Presented in part at the 54th Annual Meeting of the American College of Rheumatology, Seattle, WA, October 1990. From the Division of Clinical Immunology and Rheumatology, The University of Alabama at Birmingham. Steffen Gay, MD: Professor of Medicine; Andreas Trabandt, MD: Postdoctoral Fellow; Larry W. Moreland, MD: Assistant Professor of Medicine; Renate E. Gay, MD: Research Associate Professor of Medicine. Address reprint requests to Steffen Gay. MD, The University of Alabama at Birmingham, Division of Clinical Immunology and Rheumatology, UAB S t a t i o f l H T 433, Birmingham, AL 35294. Submitted for publication April 25, 1991; accepted in revised form October 23, 1991.

Arthritis and Rheumatism, Vol. 35, No. 3 (March 1992)

fibronectin mRNA in normal fibroblasts (6). TGFP also increases gl ycosaminoglycan synthesis in scleroderma fibroblasts (7).

Growth factors In order to examine the involvement of growth factors in situ, the appearance and localization of TGFa, TGFP, and PDGF in sclerodermatous skin were determined (8). PDGF was associated predominantly with the endothelial lining of capillaries and some perivascular macrophages in the lower dermis of early lesions. In contrast, in lesions that were not of recent onset, PDGF was detected only in dense interstitial matrix. TGFP appeared rather faintly throughout the fibrotic dermal lesions, while no significant deposition of TGFa was observed in sclerodermatous skin. What does it mean if PDGF is present in the vascular and perivascular extracellular matrix of the lower dermis, and resident tissue fibroblasts become exposed to this factor? Since PDGF up-regulates type V collagen production in fibroblasts (9), these findings may explain the earlier observation that explant cultures from the lower dermis of patients with early systemic sclerosis (SSc) accumulate significantly more type V collagen than fibroblast cultures from the upper dermis of patients, or from the upper or lower dermis of normal skin (10). The significance of early expression of PDGF in sclerodermatous skin is enhanced by the observation that there is also increased expression of the receptors for PDGF type B in the skin of patients with SSc (11). The notion that PDGF may play a major role in the pathogenesis of scleroderma is further supported by the findings that elevated plasma

GROWTH FACTORS, MATRIX, AND ONCOGENES IN SSc levels of PDGF activity are observed in patients with SSc (12) and are associated with increased visceral organ involvement (1 3). Another growth factor implicated in the pathogenesis of SSc is TGFP (14). It is increasingly clear that TGFp interacts closely with other growth factors, including PDGF, and plays a major role during inflammatory and repair processes, as discussed earlier (14). Indeed, it not only stimulates the production of collagens and fibronectin as well as their integrin receptors, but also inhibits the action of proteolytic enzymes that degrade newly formed tissue matrix (15). However, despite interest in the role of TGFP, the results of in situ hybridization studies of skin biopsy material from patients with SSc have been inconsistent. One study revealed TGFPl and TGFP;! mRNA in dermal and subcutaneous infiltrating cells in patients with both acute and chronic SSc, but also in patients with other inflammatory skin disorders (16). Nevertheless, in the vicinity of infiltrates, TGFPlpositive fibroblasts could be found only in patients with acute SSc. Conversely, a comparative evaluation of TGFp and procollagen type I gene expression in situ demonstrated that, although expression of the proa,(I) collagen gene was detected, TGFPl mRNA was not expressed at a detectable level in affected skin from patients with SSc (17). Another study, however, found colocalization of type I collagen and TGFP;! gene expression around blood vessels during inflammatory stages of SSc, but not in the inflammatory infiltrates of systemic lupus erythematosus or dermatomyositis (18). Therefore, the cellular origin of the TGFP found to be associated with the extracellular matrix in scleroderma skin (8) requires further exploration. A recent study conducted to examine further the involvement of growth factors in SSc revealed that in addition to the deposition of PDGF, basic fibroblast growth factor (bFGF) could be detected in endothelial cells and some mononuclear cells present in perivascular infiltrates (19). It is well established that bFGF is synthesized by endothelial cells and binds to heparan sulfate proteoglycans (20). The binding of bFGF to heparan sulfate proteoglycan may thereby limit its distribution and increase its concentration in the subendothelial microenvironment (2 1). However, some skin cells, such as keratinocytes and fibroblasts, also express bFGF mRNA in normal skin (22). It is of interest that PDGF increases the rate of bFGF gene transcription in normal fibroblasts (23) and that bFGF appears to be responsible for the growth-promoting

305

activity released by wounded endothelial cells (24). Based on these findings, it is suggested that the deposition of bFGF and PDGF observed in scleroderma (8,18) is related to vascular injury, e.g., endothelial cell damage.

Extracellular matrix It is now well established that the local regulatory signals that modulate bFGF action and control cell growth and differentiation are influenced by the extracellular matrix (25). With regard to the composition of the extracellular matrix in normal skin, it is of interest that the majority of the interstitial collagen fibers throughout the dermis consist of fibrillar types I and 111 collagen and that type V collagen appears predominantly within the vascular matrix (26). In view of the significant increase in pericellular type V collagen associated with fibroblasts derived from the lower part of sclerodermatous skin (lo), it is of interest that of collagen types I-VI, laminin, and fibronectin, only collagen type V binds specifically to heparan sulfate (27). This finding suggests that cell-associated heparan sulfate, previously shown to participate in cell adhesion, is likely an important receptor in the adhesion of cells to type V collagen. Until recently, the extracellular matrix was thought to serve mainly as a relatively inert scaffolding that stabilized the physical structure of tissues. However, it is now well recognized that the intricate network of macromolecules constituting the extracelMar matrix plays an active and complex role in regulating the behavior (i.e. , migration, proliferation, and metabolic function) of cells that come into contact with it (28,29). As illustrated in Figure 1, the extracellular matrix is thought to exert its influence on gene expression via transmembrane receptors, by changing the association of the cytoskeleton with the mRNA and by altering the interaction of the nuclear matrix with chromatin (30). In this regard, it has been shown that interstitial collagens may influence the cytoskeleton by binding to integrins or cell surface receptors, such as heparan sulfate proteoglycans (31). Taken together, these data indicate that the extracellular matrix may not only provide a dynamic milieu which directly influences cellular behavior, but may also serve as a reservoir of growth factors involved in regulation of proliferation and matrix biosynthesis and degradation. Although it is obvious that the pathways involved in signaling, resulting eventually in the development of skin lesions in SSc, are very

GAY ET AL

306

complex, it is possible to schematically depict a potential pivotal interaction of growth factors (PDGF and bFGF) with the extracellular matrix components type V collagen and heparan sulfate proteoglycan, leading to an excessive deposition of extracellular matrix (Figure 2).

Oncogenes

HSPa

PDGF has been shown to regulate expression of genes that are important in the mitogenic response (32). Specifically, these genes code for transcription factors, such as c-fos, c-jun, c-myc, and c-myh, which have been implicated in the regulation of DNA synthesis and other events linked to cell division (32). The c-myc gene is one of a set of genes that respond rapidly to growth stimulation. In studying the rapid induction of the c-myc gene by PDGF in BALB/c-3T3 mouse fibroblasts, it was shown that an 81-basepair sequence is the growth factor-responsive element within c-myc (33). To probe the basis of proliferation in scleroderma fibroblasts, Trojanowska et al have investigated c-myc gene expression and found that under low serum conditions (1% serum), scleroderma fibroblasts ex-

Microtubules Actin

0-

Actinin

Intermediate Filaments

Vinculin Talin lntegrins

Cell membrane Extracellular Matrix

Figure 1. Influence of the extracellular matrix on gene expression via transmembrane junctions between matrix and nucleus. This refined model for the ultrastructural interaction of cells with the extracellular matrix is based on a model of “dynamic reciprocity,” where the extracellular matrix is postulated to exert an influence on gene expression via transmembrane proteins and cytoskeletal components, proposed originally by Bissell and Carcellos-Hoff (30). (Reproduced, with permission, from Cecil Textbook of Medicine [ I ] . )

Figure 2. Schematic representation of a possible pivotal mechanism of connective tissue fibrosis in the pathogenesis of scleroderma. The hypothesis is based on the fact that vascular-derived platelet-derived growth factor (PDGF) both up-regulates collagen biosynthesis, in particular the synthesis of type V collagen (9), and induces expression of basic fibroblast growth factor (bFGF) in fibroblasts (23). This model is further based on the notion that heparan sulfate proteoglycan (HSPG)plays a crucial role as a bFGF modulator (281, by binding to bFGF (20) and specifically to type V collagen (27). The net result of these interactions between growth factors and the pericellular extracellular matrix is the formation of new extracellular matrix in the perivascular compartment of the lower dermis (10).

press a 2.5-3 times higher level of c-myc message than do normal fibroblasts (34). Based on the evidence that certain cellular proto-oncogenes play key roles in the pathways controlling cell proliferation and oncogenes encoding nuclear proteins (e.g., c-myc) and have the ability to cooperate with an activated rus gene in the transformation of cells ( 3 3 , we investigated for the expression of rus in scleroderma skin. Figure 3 illustrates that rus-encoded oncoproteins can be found associated with mononuclear cell infiltrates in early SSc skin lesions. The product of the rus proto-oncogene is a guanine nucleotide-binding protein that associates with the inner surface of the plasma membrane and appears to relay intracellular signals via the rus guanosine triphosphatase activator protein, GAP (36). In cells expressing PDGF receptors, it was shown that binding of PDGF causes approximately one-tenth of the total GAP molecules to complex with the receptor, suggesting that regulation of GAP function may participate in mediating the biologic functions of PDGF (37). Binding of GAP to the PDGF receptor may thereby decrease the ability of GAP to promote hydrolysis of guanosine triphosphate (GTP) bound to rus product and consequently increase the effective concentration of the activated form of GAP that relays the intracellular signal to proliferate (37). The early expression of PDGF in scleroderma skin (8). later to

GROWTH FACTORS, MATRIX, AND ONCOGENES IN SSc

A

307

B

Figure 3. lmmunohistologic appearance of skin biopsy sections from patients with scleroderma. Sections were stained with a monoclonal antibody raised against the p21 protein encoded by the v-H-ras oncogene (A) and a polyclonal antibody against cystatin C (B),and visualized utilizing the peroxidase ABC technique and a hematoxylin counterstain. A, Black immunoreaction products of ras protein are localized in the endothelial lining of vessel walls and perivascular mononuclear cell infiltrates (arrows). B, Cystatin C is found in certain cells of mononuclear cell infiltrates (arrows), similar to the localization of ras protein in these lesions. (Magnification x 50.)

be associated with increased expression of PDGF receptors (1 1) and rus proto-oncogene products, may epitomize certain molecular interactions in the pathogenesis of scleroderma. The role of the rus oncogene was further elucidated by the finding that fibroblast cell lines transformed by the rus oncogene express increased levels both of the commonly found 18-kd form of bFGF, and, in even greater amounts, of a 22-kd cell-associated form of bFGF (38). Thereby, bFGF may play an autocnne role contributing to uninterrupted growth stimulation. Unexpectedly, rus oncogene products may play a key role in the inhibition of proteinases, especially connective tissuedegrading enzymes. In this regard, recent data indicate that the activity of cathepsin L is inhibited by c-Ha-rus gene products (39). Since the amino acid sequence (between positions 24 and 59) of c-Ha-rus is highly homologous to a strongly conserved domain of the cystatins, which are cysteine proteinase inhibitors, it has been hypothesized that the thiol proteinase inhibitor activity of rus gene products might be common among the rus gene family and play an important role in its cellular functions. Interestingly,

c-Ha-rus gene products inhibit cathepsin L-induced degradation of epidermal growth factor receptors, raising the possibility that rus proteins can suppress the degradation of growth-related structures and thereby modulate cell growth and transformation (40). Alternatively, rus proteins may induce cell transformation by inhibiting cysteine proteinases synergistically with GTPase-activating protein, which has a similar amino acid sequence as the cysteine proteinase inhibitors human stefin A, human stefin B, and human cystatin C (41). With regard to the presence of rus oncogene products in patients with scleroderma, it is of interest that immunohistologic analysis of skin specimens by applying an antibody against cystatin C also reveals reactivity with certain cells of mononuclear cell infiltrates present in the lesion (Figure 3). Since cystatin C is a major cysteine proteinase inhibitor (42) and since thiol inhibitors may also inhibit the cysteine proteinases involved in converting procollagenase to its active form (43), we are further exploring the relevance of these observations. It will be interesting to learn whether this potential pathway of collagenase-activation inhibition contributes to an accumulation of collagenous matrix by inhibiting the physiologic

GAY ET AL

308

degradation of newly synthesized matrix, thus playing a role in the pathogenesis of tissue lesions in scleroderma, since we know that scleroderma affects more women than men and that the expression of proteinase inhibitors is regulated by estrogen (44).

Conclusion LeRoy’s original finding that dermal fibroblasts

obtained from sclerodermatous skin maintain a higher level of matrix production in culture for several passages than do normal skin fibroblasts (45) has been confirmed repeatedly (46). Related studies have indicated, however, that a subpopulation of fibroblasts may contribute disproportionately to the accumulation of extracellular matrix in scleroderma skin (47). Indeed, subpopulations of fibroblasts responsible for increased collagen production in scleroderma have been identified by cloning (48) and flow cytometry (49). It should be stressed, however, that fibroblasts cultured from normal human dermis are heterogeneous with respect to morphologic features, growth kinetics, matrix biosynthesis, and hormone responses (50). Therefore, a selective expansion of clonal fibroblast populations with unique patterns of matrix biosynthesis and surface receptors could provide the basis for a pathologic accumulation of matrix. On the basis of the available data, we hypothesize that only the resident fibroblasts associated with the perivascular environment become exposed to vascular-derived growth factors, e.g., PDGF and bFGF, and respond with both increased synthesis and decreased degradation of collagen, resulting in excessive deposition of extracellular matrix. The latter may then serve as a reservoir for these factors and in turn provide the extracellular basis of an autocrine and/or paracrine loop, which perpetuates the disease long after the initiating vascular insult has occurred.

ACKNOWLEDGMENTS We are indebted to Drs. Louis W. Heck and William J. Koopman for their helpful criticisms and comments in the preparation of the manuscript.

REFERENCES 1. Gay S, Gay RE: Connective tissue structure and function, Cecil Textbook of Medicine. Nineteenth edition. Edited by JB Wyngaarden, LH Smith Jr, JC Bennett. Philadelphia, WB Saunders, 1991, 1491-14%

2. Rodnan GP, Lipinski E, Luksick J: Skin thickness and collagen content in progressive systemic sclerosis and localized scleroderma. Arthritis Rheum 22: 130-140, 1979 3. Abergel RP, Chu M-L, Bauer EA, Uitto J: Regulation of collagen gene expression in cutaneous diseases with dermal fibrosis: evidence for pretranslational control. J Invest Dermatol 88:727-731, 1987 4. Peltonen J, Kahiiri L, Uitto J, Jimenez SA: Increased expression of type VI collagen genes in systemic sclerosis. Arthritis Rheum 33:1829-1835, 1990 5. LeRoy EC, Mercurio S, Sherer GK: Replication and phenotypic expression of control and scleroderma human fibroblasts: responses to growth factors. Roc Natl Acad Sci USA 79:128&1290, 1982 6. Varga J, Rosenbloom J, Jimenez SA: Transforming growth factor p (TGFp) causes a persistent increase in steady-state amounts of type I and type I11 collagen and fibronectin mRNAs in normal human dermal fibroblasts. Biochem J 247597404, 1987 7. Falanga V, Tiegs SL, Alstadt SP, Roberts AB, Sporn MB: Transforming growth factor-beta: selective increase in glycosaminoglycan synthesis by cultures of fibroblasts from patients with progressive systemic sclerosis. J Invest Dermatol 89:lOO-104, 1987 8. Gay S, Jones RE Jr, Huang G-Q, Gay RE: Immunohistologic demonstration of platelet-derived growth factor (PDGF) and sis-oncogene expression in scleroderma. J Invest Dermatol92:301-303, 1989 9. Narayanan AS, Page RC: Biosynthesis and regulation of type V collagen in diploid human fibroblasts. J Biol Chem 258:1169411699, 1983 10. Gay RE, Buckingham RB, Prince RK, Gay S, Rodnan GP, Miller EJ: Collagen types synthesized in dermal fibroblast cultures from patients with early progressive systemic sclerosis. Arthritis Rheum 23: 190-196, 1980 11. Klareskog L, Gustafsson R, Scheynius A, Hallgren R: Increased expression of platelet-derived growth factor type B receptors in the skin of patients with systemic sclerosis. Arthritis Rheum 33:1534-1541, 1990 12. Pandolfi A, Florito M, Altomare G, Pigatto P, Donati MB, Poggi A: Increased plasma levels of plateletderived growth factor activity in patients with progressive systemic sclerosis. Proc SOCExp Biol Med 91 :1 4 , 1989 13. Feniss JA, Falanga V, Leitzel KE, Lipton J, Bryce W, Tribby I, Bartholomew MJ, Lipton A: Elevated plasma platelet-derived growth factor B chain levels in scleroderma patients (abstract). Arthritis Rheum 33 (suppl 9):S64, 1990 14. LeRoy EC, Smith EA, Kahaleh MB, Trojanowska M, Silver RM: A strategy for determining the pathogenesis of systemic sclerosis: Is transforming growth factor p the answer? Arthritis Rheum 32:817-825, 1989 15. Sporn MB, Roberts AB: Transforming growth factor p:

GROWTH FACTORS, MATRIX, AND ONCOGENES IN SSc

multiple actions and potential clinical applications. JAMA 262:938-941, 1989 16. Gruschwitz M, Miiller PU, Sepp N, Hofer E , Fontana A, Wick G: Transcription and expression of transforming growth factor type beta in the skin of progressive systemic sclerosis: a mediator of fibrosis? J Invest Dermatol94: 197-203, 1990 17. Peltonen J, Kahari L, Jaakkola S, Kahari V-M, Varga J, Uitto J, Jimenez SA: Evaluation of transforming growth factor p and type I procollagen gene expression in fibrotic skin diseases by in situ hybridization. J Invest Dermatol 94:365-371, 1990 18. Kulozik M, Hogg A, Lankat-Buttgereit B, Krieg T: Co-localization of transforming growth factor p2 with al(1) procollagen mRNA in tissue sections of patients with systemic sclerosis. J Clin Invest 86:917-922, 1990 19. Moreland LW, Huang G-Q, Gay RE, Jones RE Jr, Blackburn WD Jr, Alarc6n GS, Gay S: Immunohistologic demonstration of platelet-derived growth factor and fibroblast growth factor in scleroderma skin (abstract). Arthritis Rheum 33 (suppl9):S36, 1990 20. Bashkin P, Doctrow S, Klagsbrun M, Svahn CM, Folkman J, Vlodavsky I: Basic fibroblast growth factor binds to subendothelial extracellular matrix and is released by heparitinase and heparin-like molecules. Biochemistry 28~1737-1743, 1989 21. Rogelj S, Klagsbrun M, Altzmon R, Kurokawa M, Haimovitz A, Fuks Z, Vlodavsky I: Basic fibroblast growth factor is an extracellular matrix component required for supporting the proliferation of vascular endothelial cells and differentiation of PC 12 cells. J Cell Biol 109:823-831, 1989 22 Scott G , Stoler M, Sarkar S, Halaban R: Localization of basic fibroblast growth factor in RNA in melanocytic lesions by in situ hybridization. J Invest Dermatol 96: I

...n

an,..

3163LL. 1YY1

23. Goldsmith WT, Gammon RB, Carver R Jr: Transcriptional regulation of the basic fibroblast growth factor gene by other growth factors (abstract). Clin Res 38: 455A, 1990 24. McNeil PL, Muthukrishman L, Warder E, D’Amore PA: Growth factors are released by mechanically wounded endothelial cells. J Cell Biol 109:811-822, 1989 25. Ingber DE, Folkman J: Mechanochemical switching between growth and differentiation during fibroblast growth factor-stimulated angiogenesis in vitro: role of extracellular matrix. J Cell Biol 109:317-330, 1989 26. Gay S , Martinez-Hernandez A, Rhodes RK, Miller EJ: The collagenous exocytoskeleton of smooth muscle cells. Coll Relat Res I :377-384, 1981 27. LeBaron RG, Hook A, Esko JD, Gay S, Hook M: Binding of heparan sulfate to type V collagen: a mechanism of cell-substrate adhesion. J Biol Chem 264:795& 7956, 1989

309

28. Ruoslahti E , Yamaguchi Y: Proteoglycans as modulators of growth factor activities. Cell 64:867-869, 1991 29. Bernfield M: Extracellular matrix. Curr Opinion Cell Biol 1:953-1019, 1989 30. Bissell MJ, Carcellos-Hoff MH: The influences of extracellular matrix on gene expression: is structure message? J Cell Sci Suppl 8:327-343, 1987 31. Rapraeger AC, Jalkanen M, Bernfield MR: Cell surface proteoglycan associates with the cytoskeleton at the basolateral cell surface of mouse mammary epithelial cells. J Cell Biol 103:2683-2696, 1986 32. Heldin C-H, Westermark B: Platelet-derived growth factor: mechanism of action and possible in vivo function. Cell Regul 135-566, 1990 33. Sacca R, Cochran BH: Identification of a PDGFresponsive element in the murine c-myc gene. Oncogene 5~1499-1505, 1990 34. Trojanowska M, Wu L, LeRoy EC: Elevated expression of c-myc proto-oncogene in scleroderma fibroblasts. Oncogene 3:477-481, 1988 35. Birrer MJ, Segal S, de Greve JS, Kaye F, Sausville EA, Minna JD: L-myc cooperates with ras to transform primary rat embryo fibroblasts. Mol Cell Biol 8:26682678, 1988 36. Santos E, Nebreda AR: Structural and functional properties of ras proteins. FASEB J 3:2151-2163, 1989 37. Kazlauskas A, Ellis C, Pawson T, Cooper JA: Binding of GAP to activated PDGF receptors. Science 247:15781581, 1990 38. Iberg N, Rogelj S, Fanning P, Klagsbrun M: Purification of 18- and 22-kDaforms of basic fibroblast growth factor from rat cells transformed by the ras oncogene. J Biol Chem 264:19951-19955, 1989 39. Hiwasa T, Yokoyama S, Ha J-M, Noguchi S, Saiyama S: c-Ha-ras gene products are potent inhibitors of cathepsins B and L. FEBS Lett 21 I :23-26, 1988 40. Hiwasa T, Sakiyama S, Yokoyama S, Ha J-M, Fujita J, Noguchi S, Bando Y, Kominami E, Katunuma N: Inhibition of cathepsin L-induced degradation of epidermal growth factor receptors by c-Ha-ras gene products. Biochem Biophys Res Commun 151:78-85, 1988 41. Hiwasa T, Sawada T , Sakiyama S, Kominami E, Katunuma N: Cysteine proteinase-inhibitory activity of ras gene product is not affected by mutations at GTPaseactivating protein-binding sites. Biol Chem Hoppe Seyler 370:1215-1220, 1989 42. Tavera C, Guillemot J-C, Capdevielle J, Ferrara P, Leung-Tack J, CollC A: A rapid two-step purification of rat cystatin C, a major inhibitor of cysteine proteinases. Prep Biochern 19:279-291, 1989 43. Unemori EN, Werb Z: Collagenase expression and endogenous activation in rabbit synovial fibroblasts stimulated by the calcium ionophore. J Biol Chem 263: 16252-16259, 1988 44. Kolar Z, Jarvinen M, Negrini R: Demonstration of

310

proteinase inhibitors “cystatin” A, B and C in breast cancer and in cell lines MGF-7 and ZR-75-1. Neoplasma 36~185-189, 1989 45. LeRoy EC: Connective tissue synthesis by scleroderma skin fibroblasts in cell culture. J Exp Med 135:13511362, 1972 46. Vuorio T, KahihPi V-M, Black C, Vuorio E: Expression of osteonectin, decorin, and transforming growth factor-pl genes in fibroblasts cultured from patients with systemic sclerosis and morphea. J Rheumatol 18:247251. 1991 47. Botstein GR, Sherer GK, LeRoy EC: Fibroblast selection in scleroderma: an alternative model of fibrosis. Arthritis Rheum 25:189-195, 1982

GAY ET AL

48. Whiteside TL, Ferrarini M, Hebda P, Buckingham RB: Heterogeneous synthetic phenotype of cloned scleroderma fibroblasts may be due to aberrant regulation in the synthesis of connective tissues. Arthritis Rheum 3 1: 1221-1229, 1988 49. Needleman BW, Ordonez JV, Taramelli D, Alms W, Gayer K, Choi J: In vitro identification of a subpopulation of fibroblasts that produces high levels of collagen in scleroderma patients. Arthritis Rheum 33:842-852, 1990 50. Goldring SR, Stephenson ML, Downie E, Krane SM, Korn JH: Heterogenecity in hormone responses and patterns of collagen synthesis in cloned dermal fibroblasts. J Clin Invest 85:798-803, 1990