Genistein Selectively Inhibits Platelet-Derived Growth Factor-Stimulated Versican Biosynthesis in Monkey Arterial Smooth Muscle Cells

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS

Vol. 339, No. 2, March 15, pp. 353–361, 1997 Article No. BB969854

Genistein Selectively Inhibits Platelet-Derived Growth Factor-Stimulated Versican Biosynthesis in Monkey Arterial Smooth Muscle Cells Elke Scho¨nherr,* Michael G. Kinsella,†,1 and Thomas N. Wight† †Department of Pathology, School of Medicine, University of Washington, Box 357470, Seattle, Washington 98195; and the *Institute for Physiological Chemistry and Pathobiochemistry, University of Mu¨nster, Mu¨nster, Germany

Received July 31, 1996, and in revised form December 3, 1996

Platelet-derived growth factor (PDGF) stimulates not only the proliferation and migration of arterial smooth muscle cells (ASMCs), but also the transcription, translation, and posttranslational processing of versican, a large chondroitin sulfate proteoglycan present in the extracellular matrix of blood vessels. PDGF receptor tyrosine kinase activity is required for signaling events associated with mitogenic and motogenic stimulation of cells by PDGF. Therefore, we have asked if inhibiton of tyrosine kinase activity by genistein also blocks the stimulation of both versican core protein synthesis and glycosaminoglycan (GAG) chain modifications induced by PDGF in ASMCs. The tyrosine kinase inhibitor, genistein, in a dose-dependent manner, reversibly inhibits PDGF-stimulated ASMC cell proliferation and RNA and core protein expression of versican, without affecting the expression of decorin and biglycan. In contrast, genistein does not affect the increase in GAG chain elongation that is induced by PDGF. This suggests that different aspects of the biosynthesis of versican are differentially regulated. To determine if such differential regulation involves downstream activation of protein kinase C, ASMCs were treated with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) to directly activate this kinase. In comparison to PDGF stimulation, TPA has little effect on expression of versican mRNA expression, nor does TPA stimulate ASMC cell proliferation. However, like PDGF, TPA increases [35S]sulfate incorporation into proteoglycans and GAG chain elongation. These results indicate that PDGF-induced GAG chain elongation, which is not inhibited by genistein treatment and is stimulated by protein kinase C activation, involves signaling pathways different from those that regulate PDGF-stimulated versican mRNA and protein expression. q 1997 Academic Press 1 To whom correspondence should be addressed. Fax: (206) 5433644.

Key Words: PDGF; tyrosine kinase; protein kinase C; proteoglycans; smooth muscle.

Platelet-derived growth factor (PDGF) is a mitogen for a number of different cell types, including arterial smooth muscle cells (ASMCs) (3, 4, 5 and references therein). In addition to inducing cell proliferation, PDGF stimulates the synthesis of components of the extracellular matrix by ASMCs, such as collagen (6, 7), fibronectin (8), thrombospondin (9), and proteoglycans (1, 2, 10). The effects of this growth regulatory peptide on its target cells are mediated by dimeric PDGF receptors, which are intercalated membrane glycoproteins with intrinsic tyrosine kinase activity (11, 12). It has been shown by site-directed mutagenesis (13) as well as with competitive tyrosine kinase inhibitors (14, 15) that tyrosine kinase activity is necessary for PDGFstimulated cell proliferation as well as stimulation of other intracellular signals, including the activation of phospholipase C and protein kinase C and the mobilization of Ca2/-ions and various ion fluxes (16). However, the relationship between these different signaling responses to PDGF and the initiation of DNA synthesis and extracellular matrix protein expression is not well understood. We have previously shown that PDGF influences the synthesis and structure of versican, which is a large chondroitin sulfate proteoglycan that is abundantly expressed by monkey ASMCs (1). The synthesis of this proteoglycan involves several different steps, including expression and transport of the protein core, and initiation and elongation of the glycosaminoglycan (GAG) side chains upon the core protein (17). In addition to stimulating ASMC proliferation, PDGF increases expression of versican mRNA and core protein and induces the synthesis of longer galactosaminoglycan 353

0003-9861/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.

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chains on both versican and the smaller secreted proteoglycans, decorin and biglycan (1, 2). We have determined that, in addition to blocking PDGF receptor tyrosine kinase activity and subsequent proliferation of ASMC, genistein also affects some, but not all, signaling pathways involved in PDGF-inducible versican biosynthesis. In addition, we have examined if protein kinase C, which is stimulated downstream from PDGF receptor activation, affects particular steps in versican biosynthesis that are not inhibited by genistein. Our results indicate that PDGF-mediated changes in versican mRNA and core protein expression require genistein-inhibitable tyrosine kinase activity and are not mimicked by protein kinase C activation alone. These effects on the regulation of versican expression correlate with the stimulation of ASMC proliferation by PDGF, which is also inhibited by genistein and not induced by phorbol esters. Unlike other PDGF-dependent effects on versican synthesis, the stimulation of GAG chain elongation is not inhibited by genistein and can be induced independently by the stimulation of protein kinase C. Thus, we conclude that different steps in versican proteoglycan synthesis are differentially regulated in ASMCs by different signaling pathways induced by PDGF. EXPERIMENTAL PROCEDURES

Materials GdnHCl (grade 1), Tris base, cetyl pyridinium chloride, urea, Nethylmaleimide, phenylmethylsulfonyl fluoride, bovine serum albumin, and blue dextran were purchased from Sigma (St. Louis, MO); 6-aminohexanoic acid, benzamidine HCl, NTB-2 autoradiography emulsion, and XAR-2 film were from Eastman Kodak Co. (Rochester, NY); chondroitin ABC lyase was from Seikagaku Kogyo Co., Ltd., through ICN Biomedicals (Costa Mesa, CA); Triton X-100 was from Boehringer-Mannheim (Mannheim, FRG); Sepharose CL-2B and Sepharose CL-6B were from Pharmacia LKB Biotechnology Inc. (Mechanicsburg, PA); electrophoresis chemicals were bought from BioRad (Richmond, CA); Na[35S]SO4 [43 Ci/mg S] (carrier free), [methyl3 H]thymidine [60–90 Ci/mM] and Universol scintillation fluid were from ICN Biomedicals; L-[4,5-3H]leucine (74 Ci/mM) was from Amersham Corp. (Arlington Heights, IL); and PDGF AB purified from human platelets was a gift of Elaine W. Raines (University of Washington). All other chemicals were reagent grade.

Cell Culture General procedure. Arterial smooth muscle cell cultures were established from strips of intimal–medial tissue from thoracic aortae of 3- to 4-year old pigtail monkeys (Macaca nemestrina). The cells were cultured as previously described (18–20). Cells between the 4th and the 12th passage were plated into 35-mm-diameter dishes (1.3 1 105 cells/dish) or 24-well trays (25 1 104 cells/well) and maintained in Dulbecco–Vogt modified Eagles minimal medium with high glucose, pyruvate, and nonessential amino acids supplemented with 105 units/liter penicillin, 105 mg/liter streptomycin (Life Technologies, Inc.), and 5% new born calf serum (NBCS, Biolabs), which was changed every second day. Cultures reaching visual confluence were made quiescent by lowering the serum concentration to 0.1% NBCS for 2 days.

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Stimulation and metabolic labeling. The medium was replaced by fresh medium containing 0.1% NBCS, with or without additional factors, 24 h prior to harvest. The dosages of PDGF-AB (10 ng/ml) and 12-O-tetradecanoylphorbol-13-acetate (TPA, 5 nM; Sigma, St. Louis, MO) were determined empirically by assessing the increase in incorporation of [35S]sulfate into proteoglycans by ASMCs exposed to these factors. Genistein (Calbiochem, San Diego, CA) was used at a concentration of 74 mM, as determined by dose–response studies (see Fig. 1). The cells were labeled with 50, 100, or 200 mCi/ml [35S]sulfate, 10 mCi/ml [3H]leucine, or 2 mCi [3H]thymidine in complete medium as indicated using labeling times up to 24 h. For each experiment, the number of cells was determined with a particle counter after trypsinization of parallel dishes. Measurement of thymidine incorporation into nuclei. The medium was removed and the cells were washed five times with icecold PBS and fixed with Holley’s fixative (21). The fixed cells were coated with NTB-2 photoemulsion (Eastman Kodak Co., Rochester, NY) and developed after 3 weeks. Labeled and unlabeled nuclei were counted under the microscope.

Measurement of Sulfate Incorporation into Proteoglycans Proteoglycans were extracted from the medium and the cell layer as described (19, 20). The medium was removed, and the cell layer was washed once with PBS, extracted for 15 min with cold 4 M guanidine-HCl, pH 5.8, containing 2.5 mM ethylenediaminetetraacetic acid (EDTA), 0.1 M 6-aminohexanoic acid, 5 mM benzamidineHCl, 10 mM N-ethylmaleimide, and harvested by scraping. The incorporation of [35S]sulfate into proteoglycan was measured by cetyl pyridinium chloride precipitation (22). Briefly, aliquots of the cell layer fraction and the medium were spotted on filter paper and washed five times for 1 h in 1% cetyl pyridinium chloride with 0.05 M NaCl. The amount of precipitate on the dried filter paper was determined by liquid scintillation counting.

Proteoglycan Isolation and Analysis Ion-exchange chromatography. Extracted proteoglycans were dialyzed against 8 M urea, 2 mM EDTA, 0.5% Triton X-100, 50 mM Tris/HCl, pH 7.5 prior to purification on DEAE-Trisacryl (IBF Biotechnics, Columbia, MD) minicolumns (1 ml resin) equilibrated with the dialysis buffer. Medium samples were directly applied. The columns were washed extensively with the same buffer containing 0.25 M NaCl to remove glycoproteins, and the proteoglycans were eluted with the same buffer containing 3 M NaCl. The isolated material was further concentrated by contact desiccation (Aquacide II; Calbiochem, San Diego, CA) and dialyzed extensively against distilled water. Aliquots of the dialysate were counted and lyophilized for analysis. Gel electrophoresis. SDS–PAGE was performed according to the procedure of Laemmli (23) on 3–12% gradient slab gels with a 3% stacking gel. The labeled proteoglycans and proteins were visualized by fluorography of dried gels previously treated with Enlightening enhancer (New England Nuclear, Boston, MA) and exposed to XAR2 film (Eastman Kodak Co.) at 0707C. For Western blotting of chondroitin ABC lyase-generated versican core protein, SDS–PAGE gels were equilibrated in 50 mM Tris, 40 mM glycine, pH 9.2, transfer buffer with 20% methanol, and 0.0375% SDS and transferred to nitrocellulose (BA83; Schleicher and Schuell, Inc., Keene, NH) for 1.5 h with a semidry transfer apparatus (Transblot SD; Bio-Rad Laboratories, Hercules, CA). Nitrocellulose membranes were blocked with 2% bovine serum albumin (Fraction V, Boehringer-Mannheim Corp.) in Tris buffered saline with 0.05% Tween 20 and exposed to 1:1000 rabbit anti-recombinant human versican antibody (kindly provided by Dr. R. Le Baron, University of Texas at San Antonio) overnight at 47C. After incubation of the

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blot with alkaline phosphatase-conjugated secondary antibodies, bands that bound primary antibodies were visualized by a enzymelinked chemiluminescence procedure (Tropix, Bedford, MA). Chemical and enzymatic degradation. Proteoglycans were isolated by gel filtration under dissociative conditions (1) and GAGs were chemically released by reductive b-elimination (24), using 1 M sodium borohydride in 50 mM NaOH for 24 h at 457C. The reaction was terminated by neutralizing the sample with glacial acetic acid. Glucuronic acid and iduronic acid containing GAGs were degraded with 0.03 units/ml chondroitin ABC lyase in 0.3 M Tris/HCl, pH 8.0, 0.6 mg/ml bovine serum albumin, 18 mM sodium acetate for 3 h at 377C. Core proteins were prepared from [3H]leucine-labeled proteoglycans by digestion with chondroitin ABC lyase for 2 h, as described above, except for the presence of proteinase inhibitors (5 mM benzamidine-HCl, 1 mM phenylmethylsulfonyl fluoride, 10 mM N-ethylmaleimide, and 0.1 M 6-aminohexanoic acid). Analytical gel filtration. Glycosaminoglycans, prepared by reductive b-elimination (see above) were chromatographed on a Sepharose CL-6B column (0.7 1 63 cm) in 0.2 M Tris/HCl, pH 7.0, 0.2 M NaCl (25).

dent fashion (Fig. 1A). The effect of PDGF on proteoglycan synthesis was completely blocked by the inhibitor at a concentration of 74 mM, while 18 mM genistein was sufficient to block PDGF-stimulated cell proliferation. These results indicate that PDGF stimulation of both proteoglycan synthesis and cell proliferation of ASMCs is dependent on tyrosine kinase activity. Quiescent ASMCs were then pretreated with PDGF and genistein for 24 h before the incubation was continued with PDGF alone to test if the effects of genistein are reversible (Fig. 1B). [35S]Sulfate incorporation into proteoglycans of cells pretreated with genistein, compared to cells stimulated with PDGF in the absence of genistein over 48 h, showed a slower, but comparable, increase in proteoglycan synthesis, indicating that the cells are viable and that proteoglycan synthesis is not irreversibly inhibited by the treatment.

Isolation and Northern Blotting of RNA

Genistein Selectively Blocks PDGF-Stimulated [35S]Sulfate Incorporation into Versican, but Has No Effect on Increases in Proteoglycan Hydrodynamic Size

Total RNA was prepared by the single-step method as described by Chomczynski and Sacchi (26). Ribonucleic acids (10–15 mg/lane) were electrophoresed overnight in 1% (w/v) agarose gels containing formaldehyde (27) and transferred to Zeta-Probe GT blotting membranes (Bio-Rad) and crosslinked with uv light, as previously described (28). Filters were prehybridized at least 2 h at 427C in a solution containing 50% (v/v) formamide (Life Technologies, Inc.), 61 SSPE (11 SSPE Å 0.15 M NaCl, 0.2 M NaH2PO4 , and 0.02 M tetrasodium EDTA), 51 Denhardt’s solution (11 Denhardt’s Å 0.02% Ficoll, 0.02% polyvinylpyrrolidone, and 0.02% bovine serum albumin), 0.5% SDS, 5% dextran sulfate (5 prime r 3 prime, Inc.), and 100 mg/ml salmon sperm DNA (Sigma). Hybridizations with 32P-labeled cDNA probes (prepared as described below) were carried out at 427C in the same solution for at least 16 h, after which the filters were washed thrice with 21 SSPE/0.1% SDS at 427C and twice with 0.31 SSPE/0.1% SDS at 657C. Several cDNAs were used as probes for PG RNA on Northern blots, including full-length human biglycan cDNA (P16) and full-length bovine decorin cDNA, provided by Dr. L. Fisher (29) and Dr. M. Young (30), respectively, of the National Institute of Dental Research (Bethesda, MD) and human versican cDNA (C7) from Dr. E. Ruoslahti (31). Northern blots were normalized for loading by comparison to hybridization of bovine 28S rRNA cDNA that was kindly provided by Dr. E. H. Sage (University of Washington, Seattle, WA). Probes were 32P-labeled by random priming, using 5*-[a-32P]dCTP (Amersham Corp.), and used to hybridize with RNA on Northern blots prepared as described above. Autoradiographs were prepared by exposure on Kodak XAR2 film at 0707C and then developed.

RESULTS

Stimulation of ASMC Cell Proliferation and [35S]Sulfate Incorporation into Proteoglycans by PDGF Is Inhibited by Genistein The stimulation of quiescent monkey arterial smooth muscle cells with PDGF (10 ng/ml) increased the incorporation of [35S]sulfate into proteoglycans and cell proliferation by 48 h when compared to controls cultured with 0.1% NBCS (Fig. 1A). The tyrosine kinase inhibitor, genistein, decreased PDGF-stimulated incorporation of [35S]sulfate into proteoglycans in a dose-depen-

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[35S]Sulfate-labeled proteoglycans synthesized by quiescent ASMC, or cells treated with PDGF in the absence or presence of genistein, were separated according to their hydrodynamic size by molecular sieve chromatography on a Sepharose CL-2B column under dissociative conditions (Fig. 2A) to determine which proteoglycan population(s) are most affected by genistein inhibition of PDGF stimulation. Both secreted and cell layer-associated ASMC proteoglycans are separated into two major size classes by Sepharose CL-2B chromatography, in agreement with previous studies (1). In addition, for cell layer samples, 10–20% of the applied radioactivity eluted with the void volume of the column for all treatments. This peak contains aggregates of heparan sulfate proteoglycans (19), and was not examined further. The primary effects of both PDGF stimulation and genistein inhibition on proteoglycan synthesis were observed in the first major peak (Fig. 2A, I), which contains primarily versican (1, 32). This peak showed the largest changes in relative [35S]sulfate incorporation, which averaged about a threefold increase over quiescent controls in cell layer samples extracted from PDGF-stimulated cultures and doubled in medium samples. When cells were stimulated with PDGF in the presence of genistein, however, incorporation of [35S]sulfate into this peak was reduced below the level of quiescent cells. In addition, the apparent hydrodynamic size of the proteoglycans that eluted in this peak increased after PDGF treatment, with the Kav changing from 0.36 in proteoglycans secreted by quiescent cells to 0.30 for proteoglycans harvested after PDGF stimulation. The increase in hydrodynamic size of the proteoglycans could not be abolished by genistein

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FIG. 1. Effect of genistein on PDGF-stimulated [35S]sulfate incorporation and proliferation of ASMCs in culture. (A) ASMCs were made quiescent by serum starvation and treated 24 h prior to harvest with or without PDGF and the indicated concentration of genistein. Proteoglycan synthesis was determined by [35S]sulfate incorporation into CPC precipitates of the combined medium and cell layer (circles), and cell proliferation was analyzed by cell counting 48 h after stimulation (triangles). The determinations were done in duplicate from two different wells and compared to untreated controls (open symbols). The error was always lower than 10% of each value. (B) Reversibility of the effect of genistein on PDGF-stimulated proteoglycan synthesis. ASMC were made quiescent by serum starvation and treated with PDGF (black circles) or PDGF and genistein (white circles) for 24 h, at which time genistein was washed out and fresh PDGF-containing medium was added (arrow). [35S]Sulfate was added at 6-h intervals prior to the harvest of the cells. Incorporation of [35S]sulfate was determined by CPC precipitation. The determinations were done in duplicate from two different wells. The error was always lower than 10% of each value.

FIG. 2. Genistein selectively inhibits PDGF-stimulated incorporation of [35S]sulfate incorporation into specific proteoglycans. (A) Sepharose CL-2B dissociative gel filtration chromatography of [35S]sulfate-labeled proteoglycans extracted from the medium (above) and cell layer (below) of ASMC cultures 24 h after treatment. Quiescent, unstimulated (no symbol), PDGF (filled circle), PDGF / genistein (open circle). (B) SDS–PAGE. Samples of [35S]sulfate-labeled proteoglycans extracted from the medium of ASMC cultures 24 h after treatment were run on a reducing 3–12% gradient SDS–PAGE gel and fluorography of the dried gel was performed. Note that genistein treatment, while inhibiting PDGF-stimulated incorporation into the versican band, cannot reverse PDGF-stimulated increases in decorin and biglycan apparent Mr . The first lane contains 14C-labeled protein molecular weight standards (Mr 1 1003).

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treatment. The second (Kav Ç0.65) major Sepharose CL-2B peak (Fig. 2A, II), which contains decorin and biglycan (2), showed little change in [35S]sulfate incorporation in either medium or cell layer samples after any treatment. Aliquots of the [35S]sulfate-labeled proteoglycans from the medium were also separated by SDS–PAGE gradient gels under reducing conditions (Fig. 2B) to characterize further the apparent sizes and relative incorporation of [35S]sulfate into different proteoglycan bands. In previous experiments under similar conditions (1, 2), versican was found as a broad band in the 3% polyacrylamide stacking gel, and biglycan and decorin ran as bands larger than 200 kDa and smaller than 200 kDa relative to protein standard markers, respectively. A small amount of a heparan sulfate proteoglycan was found at the interface between stacking and running gels. Gel electrophoresis of secreted proteoglycans indicates that, compared to proteoglycans secreted by quiescent cells, PDGF stimulates [35S]sulfate incorporation into the GAG chains of versican and biglycan by ASMCs and increased the apparent size of both biglycan and decorin (versican size is also increased (Fig. 2A)). Genistein selectively inhibited the incorporation of [35S]sulfate into versican by PDGFstimulated cells. However, genistein did not affect the PDGF-stimulated incorporation of [35S]sulfate into biglycan, nor does genistein treatment abolish increased proteoglycan size due to increases in GAG chain length (see below, Fig. 5). PDGF-Stimulated [35S]Sulfate Incorporation Is Mimicked by Phorbol Ester Treatment ASMCs were treated with PDGF or the protein kinase C activator TPA and compared to unstimulated cells to address whether stimulation of protein kinase C, which is activated downstream in signal transduction by PDGF (16, 33), has effects similar to PDGF stimulation of proteoglycan synthesis and cell proliferation. TPA treatment, unlike PDGF, did not increase DNA synthesis by ASMCs, as measured by the incorporation of [3H]thymidine into nuclei of cells over 48 h after stimulation (Fig. 3A). TPA directly stimulates protein kinase C (34) and increases [35S]sulfate incorporation into proteoglycans in a dose-dependent manner, with 5 nM giving maximal incorporation (data not shown). PDGF or TPA treatment both doubled total [35S]sulfate incorporation into proteoglycans (Fig. 3B), suggesting that some of the effects of PDGF on proteoglycan synthesis may be mediated by the activation of protein kinase C alone. However, because protein kinase C does not stimulate ASMC proliferation, it is clear that not all signaling through the PDGF receptor is mediated by subsequent protein kinase C activation. [35S]Sulfate-labeled proteoglycans were harvested

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FIG. 3. Comparison of DNA synthesis (A) and proteoglycan synthesis (B) in ASMCs. Quiescent ASMCs were treated in the presence of 0.1% newborn calf serum without or with PDGF, PDGF and genistein, or TPA alone. (A) Percentage of [3H]thymidine-labeled nuclei over 48 h in comparison to unlabeled nuclei under the different conditions. The determinations were done in duplicate from two different wells. The error was always lower than 10% of each value. (B) [35S]Sulfate incorporation into total (cell layer / media) proteoglycans, normalized to cell number, over a 24-h period after treatment of control, quiescent cultures. NBS, 0.1% newborn calf serum.

from the media of PDGF and TPA-treated ASMCs and compared with samples from quiescent cells by SDS– PAGE (Fig. 4), to determine which aspects of proteoglycan synthesis are affected by phorbol ester treatment. TPA increased [35S]sulfate incorporation into versican, in addition to causing the synthesis of versican, biglycan, and decorin (Fig. 4) of higher Mr , similar to the results of PDGF stimulation. The PDGF-Mediated Effect on Versican Glycosaminoglycan Chain Elongation Is Not Inhibited by Genistein To analyze whether the regulation of GAG chain length by PDGF is inhibited by genistein, [35S]sulfatelabeled versican secreted into the medium after the different treatments was isolated by gel filtration chromatography. GAG chains from isolated versican were released by reductive b-elimination and applied to Sepharose CL-6B columns (Fig. 5). PDGF in the presence or absence of genistein caused significant shifts in the elution position of the isolated GAG chains when compared to controls. PDGF and all other treatments caused shifts from Kav Å 0.33 for quiescent cells to Kav Å 0.26–0.23. A shift in Kav from 0.34 to 0.24 accounts for a change in the Mr from approximately 45,000 in

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FIG. 4. Comparison of [35S]sulfate-labeled proteoglycans secreted into the medium. Quiescent cells were treated for 24 h with [35S]sulfate in the presence of 0.1% serum with or without either PDGF (10 ng/ml) or TPA (5 nM). [35S]Sulfate-labeled proteoglycans were isolated from the medium by ion-exchange chromatography and separated on 3–12% gradient SDS–PAGE with a 3% stacking gel under reducing conditions. NBS, 0.1% newborn calf serum.

densitometry of this band in fluorographs. A similar stimulation of versican core protein synthesis by PDGF occurred in the cell layer samples (result not shown). In contrast, the incorporation of [3H]leucine into decorin and biglycan core proteins (Ç43–45 kDa) (2) was not affected (not shown). These results indicate that the PDGF-mediated increase in versican synthesis is apparent at the level of increased core protein synthesis and is inhibited by genistein. Northern blot analyses were performed to determine if regulation of versican core protein synthesis by PDGF and its inhibition by genistein are mediated at the mRNA level. Thus, total RNA was harvested at various times after treatment of cells, and blots were prepared and hybridized with 32P-labeled cDNA probe for versican, decorin, and biglycan (Fig. 7). PDGF induced a significant increase in versican transcript levels over unstimulated controls, in agreement with previous experiments that indicated that versican mRNA transcripts reach maximal levels 12 h after PDGF stimulation (K. Braun and T. N. Wight, unpublished observations). Genistein blocked the PDGF-induced increase

versican from unstimulated cultures to 70,000 after treatment (25). These results indicate that the stimulation of chondroitin sulfate chain elongation that is induced by PDGF is not inhibited by genistein. The effects of protein kinase C activation on the posttranslational assembly of GAG chains on versican core proteins was examined by TPA treatment. TPA also induced the assembly of GAG chains of larger size, as assessed by Sepharose CL-6B gel filtration chromatography (Fig. 5). Similar changes in the posttranslational modifications of the GAG chains of the small leucinerich proteoglycans, biglycan, and decorin, were also observed (not shown). Genistein Selectively Inhibits PDGF-Stimulated Expression of Versican mRNA and Core Protein Proteoglycans from quiescent cells, cells treated with PDGF, or cells treated with PDGF / genistein were metabolically labeled for 24 h with [3H]leucine, to determine whether the tyrosine kinase-dependent effects of PDGF stimulation on proteoglycan synthesis are evident at the level of versican core protein expression. SDS–PAGE analysis of the chondroitin ABC lyase-generated core proteins of [3H]leucine-labeled proteoglycans showed a single band at Ç450 kDa for versican (1) (Fig. 6A), and Western blotting using an anti-versican antibody detected a single major band of the same size (Fig. 6B). PDGF doubled the versican core 3H radioactivity, and genistein reduced incorporation to 30% of the level of quiescent cells, as determined by scanning

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FIG. 5. Gel filtration analysis of GAG chain lengths of versican synthesized by quiescent and stimulated ASMC. Versican was isolated by Sepharose CL-2B and ion-exchange chromatography from the medium of ASMC cultures after treatment with various agents and continuous labeling for 24 h with [35S]sulfate. Chondroitin sulfate chains were released by reductive b-elimination from purified versican. The profiles represent Sepharose CL-6B gel filtration chromatography, in 0.2 M Tris/HCl, pH 7.0, 0.2 M NaCl, of isolated versican chondroitin sulfate chains. The dashed vertical line indicates the Kav of the GAG chains of proteoglycans from unstimulated cells. NBS, 0.1% newborn calf serum.

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FIG. 6. Comparison of [3H]leucine-labeled proteoglycans secreted into the medium of ASMCs and their core proteins after treatment with and without PDGF and genistein. (A) [3H]Leucine-labeled proteoglycans were isolated from the medium of ASMC cultures by ionexchange chromatography after treatment of cultures with PDGF with or without genistein and continuous labeling for 24 h. [3H]Leucine-labeled core proteins were prepared by digestion with chondroitin ABC lyase in the presence of proteinase inhibitors and separated under reducing conditions by electrophoresis on a 3–12% gradient SDS–PAGE gel with a 3% stacking gel. Versican core protein is indicated by an arrow. (B) Proteoglycans were prepared from similarly treated media samples (18 h after treatment) from unlabeled cultures and Western blots performed. A major band at Ç450 kDa (arrow) was recognized by an anti-human versican antibody. An aliquot of chondroitin ABC enzyme and buffer (Chond’ase ABC) and a medium aliquot the had not been exposed to cells (Fresh Medium) were processed with the experimental samples as negative controls.

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to assume that the signal transduction pathways for these two factors must have some common intermediates, as well as differences, considering the divergence of effects on ASMC proliferation. Because previous studies have demonstrated that PDGF stimulation modifies several aspects of versican synthesis, including processes necessary for both core protein expression and synthesis and chondroitin sulfate chain elongation, we chose to examine tyrosine kinase and protein kinase C-dependent pathways that are induced by PDGF treatment for points of divergence among the effects on proteoglycan expression and structure and cell proliferation. Protein kinases mediate a large variety of intracellular signals leading to the control of the cell cycle, cell differentiation, and protein expression (35). Two major groups of protein kinases, tyrosine kinases (36, 37) and serine/threonine kinases (38), are known. Signal transduction through the PDGF receptor involves both types of kinase activity (12). In this study, the regulation of versican synthesis was examined to determine whether there are aspects of that synthesis that require stimulation of both tyrosine kinases and/or the serine/threonine kinase, protein kinase C. We demonstrate that tyrosine kinase activity stimulated by PDGF, and inhibited by genistein, affects core protein synthesis and mRNA expression of versican, but is not required for the stimulation of GAG chain elongation. We used the isoflavinoid, genistein, which competes with adenosine

in versican mRNA levels, but had no effect on mRNA levels for decorin or biglycan in PDGF-treated cultures. TPA did not increase versican transcripts to the level observed for PDGF treatment. Nor was there an apparent increase in biglycan transcript levels after protein kinase C activation. Therefore, while TPA clearly affects the posttranslational modifications of both leucine-rich proteoglycans and versican (see Figs. 4 and 5), as evidenced by increased size and incorporation of [35S]sulfate into these proteoglycans, protein kinase C activation alone may not be sufficient cause of the stimulation of versican RNA expression by PDGF. DISCUSSION

Previous studies have shown that PDGF not only stimulates cell proliferation by ASMCs, but also increases synthesis of extracellular matrix proteins such as versican, a large chondroitin sulfate proteoglycan (1). However, it is not known whether similar or different pathways of PDGF signal transduction are involved in these diverse effects. For example, both PDGF and TGF-b1 stimulate versican synthesis, despite differences in the effects of these growth modulatory factors on ASMC proliferation (1). Therefore, it is reasonable

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FIG. 7. Northern blot analysis of the effect of genistein and TPA on proteoglycan expression by quiescent and PDGF-stimulated ASMC. Total RNA was extracted from control, quiescent cultures of ASMC and cultures treated for 12 h with either PDGF or TPA, with or without genistein. RNA was separated by denaturing agarose gel electrophoresis, blotted to membranes, and probed with 32P-labeled cDNAs for versican, biglycan, or decorin and autoradiography performed. Relative RNA loading is demonstrated by photographs of the ethidium bromide-stained rRNAs (EtBr).

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triphosphate for its binding site on tyrosine kinases (39), to inhibit this activity. This compound is a normal product of vegetarian diet and inhibits endothelial cell growth and neovascularization (40) at concentrations similar to that used in this study. At concentrations that effectively inhibit tyrosine kinases, genistein has little effect on most other kinases, such protein kinase C or cyclic adenosine monophosphate-dependent kinases (39). Because genistein does not interfere with receptor internalization (14), control of versican expression by PDGF is unlikely to require this regulatory mechanism. However, genistein does inhibit the serine/ threonine kinase, S6-kinase (41), which is activated by both PDGF and TPA (42, unpublished observations). Inhibition of S6-kinase may therefore contribute to the inhibition by genistein of versican core protein synthesis and RNA expression. However, S6-kinase inhibition is unlikely to be a sufficient cause, since TPA had little effect on versican RNA expression in this study. The irreversible inhibition of topoisomerases II and III by genistein (43, 44) can be ruled out as affecting proteoglycan synthesis, because proteoglycan synthesis resumes after removal of genistein even after 24 h. However, this effect of genistein may be responsible for the inhibition of cell proliferation after prolonged (24 h) genistein treatment. Finally, while genistein clearly inhibits tyrosine kinase activity directly stimulated by PDGF, including the receptor kinase, it should be noted that other tyrosine kinase activities not specifically induced by PDGF may also be inhibted by genistein. For example, the observation that genistein treatment alone reduces versican mRNA below control levels (Fig. 7) may be explained by the inhibition by genistein of tyrosine kinases necessary for other general cellular processes. Although genistein effectively blocked versican RNA expression and core protein synthesis, which are stimulated by PDGF, inhibition of tyrosine kinase activity by genistein did not inhibit the attendant increase in GAG chain elongation. These results indicate that some signaling involved in PDGF stimulation of versican biosynthesis can be initiated in the absence of, and independently from, genistein-inhibitable autophosphorylation of the PDGF receptor tyrosine kinase. Similar apparently tyrosine kinase-independent activities induced by PDGF have been reported recently by Mundschau et al. (45), who demonstrated that the induction of erg-1 by PDGF occurs in the absence of measurable tyrosine kinase activity. Such results suggest the existence of PDGF signaling pathways that operate independently from PDGF receptor tyrosine kinase activity. Our finding that agonists of protein kinase C also stimulate pathways that are activated by PDGF, but are not inhibited by genistein, suggest that protein kinase C may be involved in genistein-insensitive PDGF signaling. Additional evidence that GAG chain

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elongation is dependent on protein kinase C activity comes from a recent work that indicates that the protein kinase C inhibitor, H7, blocks GAG chain elongation induced by PDGF treatment (L. Cardoso and T. N. Wight, unpublished observations). Taken together, these results indicate that different aspects of the synthesis of a complex proteoglycan, such as versican, are controlled independently through overlapping, but distinct, signaling pathways. This conclusion implies that the number of versican molecules can be regulated independently from the length of GAG chains. Altered GAG chain length may influence the binding of number of ligands that interact with versican chondroitin sulfate chains (10) and overall proteoglycan hydrodynamic size, while interaction of ligands that bind versican core protein domains, such as hyaluronan and tenascin, may be influenced only by changes in the number or distribution of versican molecules. The results presented in this study show that sulfate incorporation into versican GAG chains and increased GAG chain elongation can occur in the absence of cell proliferation, but an increase in versican expression and synthesis is linked to increased cellular proliferation by ASMCs, in agreement with previous observations (1). It is unknown whether the observed increase in versican synthesis is necessary for cell proliferation, or only occurs concomitant to it. Recently, however, versican synthesis has been reported by proliferating cells both in vitro and in tissues (46), suggesting that this proteoglycan may be necessary as cells modulate their pericellular matrix to achieve favorable conditions for cell proliferation. In summary, the results of these studies suggest that different aspects of versican synthesis may be differentially regulated. Thus, like ASMC cell proliferation, PDGF stimulation of versican RNA and core protein expression may not require protein kinase C activation, since these effects are blocked by genistein and are not independently stimulated by TPA. However, the increase of [35S]sulfate incorporation into GAG chains as well as GAG chain elongation may be protein kinase C-dependent, since they are induced directly by TPA stimulation. In more general terms, this work demonstrates that the stimulation of different signal transduction pathways by growth factors can differentially regulate the types, amounts, and structures of proteoglycans synthesized by ASMCs. Such independent regulation of different aspects of versican biosynthesis by kinase activities induced by growth factors may play a role in the secretion of structurally, and thus functionally, different forms of versican as different growth factors activate cells at different tissue sites. ACKNOWLEDGMENTS We are grateful to Elaine W. Raines, Department of Pathology, University of Washington, for the determination of PDGF receptor

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REGULATION OF VERSICAN SYNTHESIS BY PDGF numbers and for providing PDGF for these experiments; and to Dr. Lee Graves, of the Howard Hughes Institute, University of Washington, for protein kinase assays and many helpful discussions. We also thank Dr. Hans Kresse, University of Mu¨nster, and Dr. Eva Qwarnstro¨m, University of Washington, for critical reading of the manuscript. This work was supported by grants from the Deutsche Forschungsgemeinschaft (E.S.) and the National Institutes of Health (HL 18645, T.N.W.).

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