JOURNAL OF CELLULAR PHYSIOLOGY 145:181-186 (1990)

Mitogenic Effect of Transforming Growth Factor pl on Human Fibroblasts Involves the Induction of Platelet-Derived Growth Factor a Receptors OSAMU ISHIKAWA, E. CARWILE LEROY, AND MARIA TROJANOWSKA* Division of Rheumatology and Immunology, Department of Medicine, Medical University of South Carolina, Charleston, South Carolina 29425 Platelet-derived growth factor (PDGF) and transforming growth factor P (TGF-P), potent modulators of mesenchymal cell growth and differentiation, are often colocalizable in vivo. Previous in vitro studies in fibroblastic cell lines have shown variable, even antagonistic effects of TGF-P on the mitogenic action of PDGF. This study demonstrates that in diploid human dermal fibroblasts, TGF-PI i s weakly mitogenic in the absence of serum or purified growth factors, and that TGF-P, potentiates DNA synthesis in PDGF-stimulated fibroblasts with delayed kinetics when compared to stimulation with PDGF alone. TGF-P1 enhances mitogenic potency of all three PDGF isoforms and increases receptor binding of both 1251 PDGF-AA and 1251 PDGF-BB, consistent with the increased expression of the a type PDGF receptor. The induction of PDGF a receptor subunits by TGF-P may play a role in enhancing the proliferative potential of human fibroblasts in certain physiologic and pathologic conditions.

Platelet-derived growth factor (PDGF) and transforming growth factor P (TGF-P)are major components of platelet a granules which are released during the platelet release reaction. These factors are also secreted locally by activated monocytes, endothelial cells, smooth muscle cells, and by various transformed and neoplastic cells in vivo and in vitro (Ross et al., 1986). The precise receptor-ligand interactions which regulate these immunehnflammatory and neoplastic microenvironments remain largely unknown. The PDGF molecule (=30 kDa) consists of two peptide chains (denoted A and B) assembled as either heterodimers or homodimers [AB, AA, BBI (Johnsson et al., 1982; Betsholtz et al., 1986). Human platelet PDGF consists of PDGF-AB (70%), PDGF-BB, and small amounts of PDGF-AA (Hammacher et al., 1988). Two PDGF receptor subunits (a and P) have been identified and the corresponding genes cloned (Escobedo et al., 1988; Seifert et al., 1989; Claesson-Welsh et al., 1988; Matsui et al., 1989). A recently proposed model postulates that these receptor subunits either exist separately or form rapidly reversible complexes in the absence of PDGF but associate into stable dimers when PDGF is present. Depending upon the isoform of exogenous PDGF present, aa,ap, or pp receptor dimers are formed. PDGF-BB can bind to all three receptor subunit combinations, PDGF-AB binds either to aa and af3 receptors, and PDGF-AA binds only to a01receptors. In non-transformed human fibroblasts there is a 20fold excess of p over a subunits, resulting in sparse aa receptors consistent with low constitutive mitogenic effects of the PDGF-AA homodimer in these cells (Seifert et al., 1989). 0 1990 WILEY-LISS. INC

TGF-P, a multifunctional peptide regulatory factor, is a 25 kDa dimer which acts as a potent growth inhibitor for most cells (epithelial and endothelial cells and lymphocytes), but can be mitogenic for cells of mesenchymal origin. Its effect on fibroblasts is both stimulatory and inhibitory depending on cell type, growth conditions, presence of serum and other growth factors, and the stage of differentiation. In addition, it has been shown that TGF-(3 antagonizes PDGF mitogenic effects in most fibroblastic cell lines, except for human embryonic fibroblasts (Roberts et al., 1988). Multiple membrane binding proteins (putative receptors) and multiple isoforms have been identified; their biologic implications and signal transduction mechanisms remain unknown (Massague et al., 1987). We have been interested in the effects of TGF-P on human dermal fibroblasts because this factor has been implicated in wound healing, as well as in the fibrosis of systemic sclerosis (scleroderma) (LeRoy et al., 1989). In this report we show that TGF-p acts synergistically with PDGF in inducing DNA synthesis in human dermal fibroblasts in vitro; the pattern of PDGF isoform responses and direct binding assays suggest that TGF-P functions via the increased functional expression of PDGF a receptors.

Received November 2, 1989; accepted June 20, 1990. *To whom reprint requests/correspondence should be addressed. This work was performed in the Division of Rheumatology and Immunology, Department of Medicine, Medical University of South Carolina, Charleston, SC 29425.

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MATERIALS AND METHODS Cells Human adult skin fibroblasts were obtained by biopsy of the dorsal forearm skin of healthy volunteers following informed consent and institutional approval. Primary ex lant cultures were established in 25 cm2 culture flas s (Costar, Cambridge, MA) in Dulbecco's modified Eagle's medium (DMEM) (Gibco, Grand Island, NY) supplemented with 10% fetal calf serum (FCS),2 mM L-glutamine, 50 pg/ml gentamicin sulfate (Quality Biological, Inc., Gaithersbur MD), and 5 pg/ ml amphotericin (Gibco, Grand Islan$ NY), the latter for 1 week. Monolayer cultures were maintained at 37°C in a humidified atmosphere of 10% C02/90% air and fed with DMEM containing 10% FCS, 2 m M L-glutamine, adn 50 pg/ml gentamicin.

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DNA synthesis Growth factor responses were measured by l3H1 thymidine incorporation. Primary cultured fibroblasts used in experiments were the third or fourth subpassage. Cells were seeded at the densities indicated in figure legends in DMEM supplemented with 10% FCS and synchronized with serum-free DMEM (SFM) containing 0.1% BSA. Control cultures received no growth factors and no serum after seeding. Cells were labeled with [3H] thymidine (sp. act. 3.22 Bq/mmol, final concentration 1 pCi/ml; NEN, Boston, MA), for 2 hours, washed three times with cold PBS and four times with 5% trichloroacetic acid. Five hundred microliters of 0.1% NaOH/O.l% SDS was added and the radioactivity of 250 pl aliquots was measured in 5 ml of PCS I1 (Amersham Corporation, Arlington Heights, IL) using a liquid scintillation system (Packard, Sterling, VA). TGF-PI and PDGF were purchased from R&D Systems Inc. (Minneapolis, MN). All experiments were done in triplicate.

then replaced with DMEM containing growth factors and, after varying times, total RNA was extracted using an acid guanidinium thiocyanate-phenol-chloroform method (Chomczynski and Sacchi, 1987). Ten micrograms of total RNA was electrophoresed on 1% agarose/formaldehyde gel and blotted onto nylon filters (ICN Biochemicals, Inc. Cleveland, OH). The filters were baked, prehybridized, and hybridized as follows: after a 2 hour incubation at 42°C in prehybridization buffer (50% deionized formamide, 5 x Denhardt's solution, 1%sodium dodecyl sulfate [SDSI, 5 x SSPE, and 100 pg/ml herring sperm DNA), filters were hybridized overnight with 5 x lo6 c p d m l of labeled probe at 42°C in the same buffer without herrin sperm DNA, followed by two times wash in 2X S PE for 15 min at room temperature, two times wash in 2 x SSPE/2%SDS for 45 min at 55"C, and two times wash in 0 . 1 SSPE ~ for 15 minutes at room temperature. Filters were exposed to Kodak X Omat AR film at -70°C overnight. Probe (PDGF-A chain, EcoRI fragment) was labeled with a random primed DNA labeling kit (Boehringer Mannheim, Indianapolis, IN). RESULTS TGF-P1 is mitogenic for human fibroblasts In experiments begun when the two laboratories were together, it was reported that in human foreskin fibroblasts TGF-fi induces PDGF-A chain mRNA levels and secretion of peptides that crossreact with antiPDGF antibody (Soma and Grotendorst, 1989). In human adult fibroblasts TGF-Pl also transiently induces PDGF-A mRNA (Fig. 1). Before examining the influence of TGF-PI on PDGF and its receptor, we asked if TGF-Pl was mitogenic for human fibroblasts in the absence of other added growth factors. To ensure that secreted autocrine PDGF-AA was present in sdficient amounts to exert its mitogenic effects, experiments

lZ5IPDGF binding assay Saturation binding assays were done according to the method of Bowen-Pope and Ross (1982). Cells were plated in 24-well tra s in DMEM medium supplemented with 10% FC and grown to confluence, followed by 48 hours incubation in DMEM with 0.1% BSA. Varying concentrations (0.125-8 ng/ml) of 9labeled PDGF-AA (sp. act 70 pCi/pg, purchased from UBI, Lake Placid, NY, custom iodinated by BTI, Stou hton, MA) or lZ6I-labeled PDGF-BB (sp. act. 45 p i/p.g; Biomedical Technologies Inc., Stoughton, MA) were added in binding buffer for 2 hours at 22°C. Cells were then washed with PBS/O.l% BSA and harvested with solubilization buffer. Total cell-associated 1251was determined. Nonspecific binding was determined by the addition of 100-fold excess of unlabeled PDGF-AA or PDGF-BB (Upstate Biotechnology Inc., Lake Placid, NY).

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RNA preparation and northern blot analysis Cells were grown to confluence in DMEM supplemented with 10% FCS in 15 cm-diameter dishes (Falcon Products, Cockeyville, MD), washed three times with DMEM with 0.1% BSA, followed by a 24-hour preincubation in this medium. Medium was

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4

Fig. 1. Northern blot analysis of expression of PDGF-A chain at 3 hours (lanes 2-4) and 6 hours (lanes 5-71; after growth factors stimulation (lane l), control; lanes 2 and 5 after stimulation with PDGF (LO ng/ml); lanes 3 and 6 with PDGF + TGF-Pl; lanes 4 and 7 with TGF-pl(1 ng/ml). Arrowheads indicate 2.8 kb, 2.3 kb, and 1.9 kb PDGF-A chain mRNA. Top: Represents ethidium bromide staining of the gel.

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TGF-P AND PDGF Q RECEPTOR EXPRESSION

were performed using dense cell cultures and minimal medium volume after addition of growth factors (0.3 ml/per well). As shown in Table 1, under these conditions TGF-P1 was weakly mitogenic. Kinetics of mitogenic responses to TGF-P1 alone were delayed and more persistent when compared to PDGF (vide infra). TGF-P1enhances mitogenic response to PDGF We measured proliferative responses of human adult dermal fibroblasts to PDGF (10 ng/ml), TGF-P1 (1 ngl ml), or PDGF and TGF-PI in combination. To eliminate the effect of other serum-derived factors, including variable and unknown concentrations of PDGF and TGF-P, cells were exposed to 10% FCS only for 3 hours after plating to allow adherence, then medium was changed to serum free for 24 hours prior to growth factor additions. The kinetics of i3H1 th midine incorporation were studied for 48 hours and [3HI thymidine incorporation was measured every 6 hours. PDGF alone showed potent mitogenic effect with peaks at 18 and 36 hours, while TGF-P was not mitogenic under these conditions. The combination of PDGF and TGF-P, consistently enhanced L3H] thymidine incorporation compared to PDGF alone (Fig. 2). Maximum enhancement of DNA synthesis by TGF-P1 occurred 30 hours after growth factor addition (Fig. 2b). With PDGF alone, responses were linear between concentrations of 0.008 and 0.13 ng/ml and maximal between 0.13 and 20ng/ml (data not shown). The ability of TGF-P1 to enhance i3H] thymidine incorporation in PDGF-treated fibroblasts was dose dependent; responses were linear between TGF-P1 concentrations of 0.15 and 0.62 ng/ml with a plateau between 0.62 ng/ml and 5.0 ng/ml (data not shown). Differences in PDGF kinetics and TGF-P, mitogenic potency in comparing Table 1 and Figure 2 are due to cell density and experimental conditions. To explore further this delayed effect of TGF-P1, we preincubated human fibroblasts with TGF P1 for varying times before PDGF stimulation. As depicted in Figure 3, preincubation of cells with TGF-P1resulted in significantly greater responses to PDGF, maximal after 8 hours at all TGF-P1 concentrations studied. Dose differences with shorter preincubation times were observed between 0.2 ngiml and 5.0 ng/ml. In addition, we compared mitogenic responses to individual PDGF isoforms after TGF-P, preincubation (recombinant PDGF-AA and BB, and platelet-derived PDGF-AB). As shown in Table 2, increased mitogenic effects were seen with all three PDGF isoforms, consistent with increased a receptor expression.

TABLE 1. Kinetics of growth factor-stimulatedfibroblast proliferation' 30 hours

Control TGF-Pl PDGF

+

Time after addition (cprn/well) 36 hours 48 hours

298 38 873 f 40 15,736 369

*

288 6 613 38 4,765 f 305

*

339 z t 29 747 f 72 421 f 84

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INCUBATION TIME (HOURS)

Fig. 2. Kinetics of stimulation of DNA synthesis by PDGF, TGF-p,, or by PDGFITGF-P, in combination. Cells were seeded at a density of 5,000 cellsicm' and preincubated in DMEM + 0.1%BSA for 24 hours. Cells were stimulated by growth factors for the indicated times followed by a 2 hour labeling period with t3H] thymidine. Four different fibroblast strains were studied and one representative is depicted in a: PDGF (10 ngiml) (HI;PDGF (10 ngiml) and TGF-P, (1 ng/ml) (a), TGF-P, (1 ng/ml) (a); (control (0).bi The E3H1 thymidine incorporation was compared to that of PDGF-stimulated fibroblasts which was set at 100%.Represents mean value + SE for 4 fibroblast strains: PDGF (10 ng/ml) and TGF-p, (1 ngiml) ( 0 ) ;TGF-P, (1 ngiml) ( A ) : control culture (0).

TGF-P1 increases PDGF a receptor number To explain the enhanced mitogenic effects of PDGFAA (Table 21, we asked directly if it was due to the increase in PDGF a receptor number or affinity. Since only a PDGF receptors bind to PDGF-AA, we performed saturation binding assays using lZ5IPDGF-AA (Fig. 4A). Scatchard analysis of binding isotherms show increased PDGF-a receptor number with no apparent change in bindin affinity (Fig. 4C). Saturation binding assay using "1 PDGF-BB also showed increased binding of this ligand after TGF-P1 pretreatment (Fig.

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ISHIKAWA ET AL. zrp

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Fig. 3. Effect of TGF-P preincubation time on DNA synthesis in PDGF-stimulated fibroblasts. Cells (13,000/cm2) were seeded in DMEM/10% FCS in 24 well plates and growth for 2 days followed by extensive washes with DMEM containing 0.1% BSA and overnight incubation in this media (serum-free). TGF-p (0.2,1, and 5 nglml) was added for 1 , 4 , or 8 hours, then washed extensively. PDGF (10ng/ml)

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Concentration of TGFI, (ns/ml) Preincubotion time (hr)

was subsequently added for 18 hours, followed by labeling with 3H-thymidine (1 pCilml) for 2 hours. Each value represents the mean of two separate triplicate well experiments. Duplicates agreed within 5%.Values represent increases over PDGF stimulation which was set at 100%.

PDGF-AA binds only to aa receptors, these data, although indirect, suggest that aa receptor number TGF-8 1 pretreatment (cpm/well) was increased by TGF-P1. Elevated mitogenic re+ PDGF isoform (ng/ml) sponses to PDGF-AB and PDGF-BB, which bind to a AA and P receptor subunits, can be accounted for by 10 892 k 42 2,358 f 195 increased a receptor number. If increased OLPor PP 1.0 372 i 26 1,211 f 41 rece tor levels were present, we would expect relaAB tive y greater responses to PDGF-AB and BB than to 10 2,958 f 200 4,766 f 90 1.0 636 f 45 1,708 f 42 PDGF-AA. As observed, responses of the three isoforms BB were similar. Direct measurements of individual recep10 3,219 t 30 6,165 f 660 tor levels with specific binding assays confirm that OL 1.0 794 f 95 2.529 f 182 receptor number, but not affinity, is increased after ‘Cells were grown as describedin Figure 3 and preincubatedwith TGF-Bl(1 ng/ml) TGF-P exposure. Future studies with specific receptor for 8 hours. Control cells were incubated in DMEM with 0.1%BSA. PDGF isoforms (recombinant PDGF-AA and PDGF-BB. Upstate Biotechnology, Inc., Lake Placid, antibodies are planned. NY; human platelet PDGF, R & D Systems, Inc., Minneapolis, MN) at indicated Transmodulation of PDGF receptors has been previConcentrations were added for 18 hours, followed by 2 hours of [3H]-thymidine labeling. The Nos represent mean value from two separate triplicate experiments. ously investigated in NIH 3T3 cells (Gronwald et al., 1989).In these cells, unlike human fibroblasts, TGF-P1 treatment decreases responsiveness to PDGF-AA, probably by decreasing a subunit receptor number, 4B) and Scatchard analysis was consistent with in- while slightly inducing P subunit receptor number. The basis for the differences between these two cell types is creased receptor number (Fig. 4D). unclear; it may be relevant that in NIH 3T3 cells, as DISCUSSION well as in human osteogenic sarcoma cell line MG-63, This study analyzes the effects of TGF-P1 on fibro- the number of a and P receptors are roughly equal. It is blasts singly and in concert with the other major possible that increased a receptor number and down growth factor for anchorage-dependent, mesenchymal re lation by TGF-P may be characteristic of immorcells, PDGF. In the absence of serum, PDGF alone ta ized or transformed cells, contributing to the wellexhibited potent mitogenic effects, while TGF-P1 alone known plieotrophic proliferative effects of TGF-P. was weakly mitogenic. The combination of PDGF and In human fibroblasts TGF-P induces PDGF-A mRNA TGF-P, appeared to be synergistic. Kinetic experi- level and secretion of peptides that crossreact with ments demonstrated that DNA synthesis stimulated by PDGF antibody (Paulsson et al., 1988; Soma and GroTGF-P, in PDGF-treated cells follows a lag period (as tendorst, 1989). In this study we showed that TGF-P, compared to PDGF), indicating an indirect mechanism. acts as a weak mitogen for human fibroblasts in vitro, Comparison of mitogenic responses to individual presumably via coordinate induction of autocrine PDGF isoforms after TGF-P1 preincubation showed PDGF-AA and its a receptor. It remains to be determitogenic effects with all three PDGF isoforms. Since mined whether, analogous to PDGF-A chain regulaTABLE 2. TGF-8 1 effect on mitogenic potency of PDGF isoforms’

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TGF-P AND PDGF a RECEFTOR EXPRESSION

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Fig. 4. Saturation binding of 1261-labeledPDGF-AA (A) and lz5Ilabeled PDGF-BB (B) in TGF-pl-treated and control fibroblasts. Cells were grown as described in “Materials and Methods” and incubated with 1 ngiml of TGF-p ( 0 ) for 8 hours; control cells were incubated in

DMEMO.1BSA (0)before addition of lZ5IPDGF AA or BB. Scatchard analysis of PDGF-AA binding (C) and PDGF-BB binding (D) with ( 0 ) and without (0) TGF-p premncubation.

tion, TGF-P induction of a receptor subunit is at the transcriptional level. Recent cloning of PDGF a receptor subunit cDNA should facilitate further study. The distribution of PDGF isoforms varies in different species and cell types. Human serum contains only a small fraction (15%) of total PDGF-mitogenic activity as PDGF-BB, which is the predominant isoform in serum from many other species. Conditioned medium from cultured cells, with the exception of vascular endothelial cells and v-sis transformed cells that secrete predominantly PDGF-BB, contain PDGF-AA or PDGF-AB (Bowen-Pope et al., 1989). On the other hand, a receptors, essential for mitogenic responses to PDGF-AA homodimer and PDGF-AB heterodimer, constitute less than 10% of the a/p receptor total in diploid human fibroblasts. Low levels of a receptor

subunits may therefore be rate limiting for fibroblast mitogenic responses to PDGF and subject to regulation in certain physiological conditions. For exam le, simultaneous release and action of PDGF and T F-P from platelets or activated macrophages can, by upregulating a receptor subunits, greatly potentiate the proliferative potential of fibroblasts during wound healing or other pathological conditions (e.g., atherosclerosis or scleroderma). TGF-p is a potent inducer of extracellular matrix synthesis and may participate in tissue formation during development, tissue maintenance, and repair of injury. A proliferative effect of TGF-P1 on matrixproducing cells may play an additional stimulatory role in these settings. Conversely, the demonstrated proliferative effects of TGF-P through PDGF and PDGF

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Escobedo, J.A., Navankasatussas, S., Cousens, L.S., Coughlin, S.R., Bell, G.I., and Williams, L.T. (1988) A common PDGF receptor is activated by homodimeric A and B forms of PDGF. Science, 240~1532-1534. Gronwald, R.G.K., Seifert, R.A., and Bowen-Pope, D.F. (1989) Differential regulation of expression of two platelet-derived growth factor receptor subunits by transforming growth factor-p. J. Biol. Chem., 264:8120-8125. Hammacher, A., Hellman, U., Johnsson, A,, Ostman, A,, Gunnarsson, K., Westermark, B., Wasteson, A,, and Heldin, C.-H. (1988)A major part of platelet-derived growth factor purified from human platelets is a heterodimer of one A and one B chain. J. Biol. Chem., 263:16493-16498. Johnsson, A,, Heldin, C.-H., Westermark, B., and Wasteson, A. (1982) Platelet-derived growth factor: Identification of constituent polypeptide chains. Biochem. Biophys. Res. Commun., 104:6&74. LeRoy, E.C., Smith, E.A., Kahaleh, M.B., Trojanowska, M., and Silver, R.M. (1989) A strategy for determining the pathogenesis of systemic sclerosis. Arthritis Rheum., 32817425. Massague, J.,Cheifetz, S., Ignotz, R.A., and Boyd, F.T. (1987) Multiple type+ transforming growth factors and their receptors. J. Cell. Physiol., 543-47. Matsui, T., Heidaran, M., Miki, T., Popescu, N., La Rochelle, W., Kraus, M., Pierce, J., and Aaronson, S. (1989) Isolation of a novel Betsholtz, C., Johnsson, A., Heldin, C., Westermark, B., Lind, P., receptor cDNA establishes the existence of two PDGF receptor Urdea, M., Eddy, R., Shows, T., Philpott, K., Mellor, A,, Knott, T., genes. Science, 243:800-804. and Scott, J. (1986) DNA sequence and chromosomal localization of Paulsson, Y., Beckmann, M.P., Westermark, B., and Heldin, C.-H. human platelet-derived growth factor A-chain and its expression in (1988) Density-dependent inhibition of cell growth by transforming tumour cell lines. Nature, 320:695-699. growth factor-pl in normal human fibroblasts. Growth Factors., Bowen-Pope, D.F., and Ross, R. (1982) Platelet-derived growth factor. 1:19-27. J. Biol. Chem., 2575161-5171. Roberts, A.B., Thompson, N.L., Heine, U., Flanders, C., and Sporn, Bowen-Pope, D.F., Hart, C.E., and Seifert, R.A. (1989) Sera and M.B. (1988) Transforming growth factor-p: possible roles in carcinoconditioned media contain different isoforms of platelet-derived genesis. Br. J. Cancer, 57594-600. growth factor (PDGF) which bind to different classes of PDGF Ross, R., Raines, E.W., and Bowe-Pope, D.F. (1986) The biology of receptor. J . Biol. Chem., 264:2502-2508. platelet-derived growth factor. Cell, 46155159. Chomczynski, P., and Sacchi, N. (1987) Single-step method RNA Seifert, R.A., Hart, C.E., Phillips, P.E., Forstrom, J.W., Ross, R., isolation by acid guanidinium thiocyanate-phenol-chloroformexMurray, M.J., and Bowen-Pope, D.F. (1989) Two different subunits traction. Anal. Biochem., 162:156-158. associate to create isoform-specific platelet-derived growth factor Claesson-Welsh, L., Eriksson, A,, Moren, A., Severinsson, L., Ek, B., receptors. J. Biol. Chem. 264:8771-8778. Ostman, A., Betsholtz, C., Heldin, C.-H. (1988) cDNA cloning and Soma, Y., and Grotendorst, G.R. (1989) TGF-P stimulates primary expression of a human platelet-derived growth factor (PDGF) human skin fibroblast DNA synthesis via an autocrine production receptor specific for B-chain-containing PDGF molecules. Mol. Cell. of PDGF-related peptides. J. Cell. Physiol. 140:246-253. Biol., 8:3476-3486.

receptor expression could be coordinately linked to the transcriptional control of several extracellular matrix genes; these intriguing ideas are readily amenable to a variety of experimental approaches. Coordinate initiation of both proliferation and matrix synthesis by TGF-P1 provides a new approach in our understanding of the role of this polypeptide in vivo. ACKNOWLEDGMENTS The authors acknowledge the support of the National Institutes of Health, the RGK Foundation, the Phillip and Jane Williams Fellowship, the Harwood Institute, the Scleroderma Research Foundation, the Health Sciences Foundation, and the Biomedical Support Fund of the Medical University of South Carolina, and thank Christine Kjelgaard for manuscript preparation and Ann Donaldson for figure preparation. LITERATURE CITED

Mitogenic effect of transforming growth factor beta 1 on human fibroblasts involves the induction of platelet-derived growth factor alpha receptors.

Platelet-derived growth factor (PDGF) and transforming growth factor beta (TGF-beta), potent modulators of mesenchymal cell growth and differentiation...
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