JOURNAL OF CELLULAR PHYSIOLOGY 149:152-159 (1991)
Enhanced Sulfated-Proteoglycan Core Protein Synthesis by Incubation of Rabbit Chondrocytes With Recombinant Transforming Growth Factor$, CHARLES J. MALEMUD*, WENDY KILLEEN, THOMAS M. HERINC, AND ANTHONY F. PURCHIO Departments of Medicine (C.).M., W.K., J.M.H.) and Andtomy (C.I.M., T.M.H.), Case Western Reserve University, Cleveland, Ohio 44 106; Oncogen Corporation, Seattle, Washington 98 195 (A.F.P.) Rabbit articular chondrocytes were incubated with recombinant transforminggrowthiactor-p, (rhTGF-P,) and its effect on newly synthesized proteoglycan measured. rhTGF-P, stimulated proteoglycan synthesis at a concentration as low as 5 ngiml without further increases in radiosulfate incorporation up to 50 ngiml. The quantitative increase in radiosulfate incorporation in rh-TGF-P, -treated chondrocytes was greater in the cell-associated culture compartment than in the medium compartment. rhTGF-P, promoted an increased proteoglycan retention in the cell-associated compartment as evidenced by an increase in the t% of retention from 8 h to 11 h. Specific enhanced synthesis of [35S]-methioninelabeled core proteins was seen in rh-TGF-fi-treated chondrocytes. rh-TGF-P, increased the synthesis of the 2 core proteins derived from hydrodynamically large proteoglycans. They possessed apparent molecular weights of > 480 kD and 390 kD after 3-5% acrylamide gel electrophoresis. A compartmental analysis revealed that the cell-associated culture compartment contained only the larger of the 2 core proteins derived from large proteoglycans. Two other core proteins with apparent molecular weights 52 kD and 46 kD were also stimulated by rhTGF-P,. These results indicated that TGF-P probably plays a significant role in stimulating proteoglycan core protein synthesis in articular chondrocytes and therefore may be an important growth factor in the restoration of cartilage extracellular matrix after injury.
Recent studies have shown that TGF-P, belongs to a family of closely related growth-modulatory molecules including TGF-P2 (Seyedin et al., 1987; Cheifetz et al., 1987; Ikeda et al., 19871, TGF-P, (tenDijke et al., 1988; Derynck et al., 1988; Jakowlew et al., 1988a1, TGF-P, (Jakowlew et al., 1988b), Mullerian inhibitory substance (Cate et al., 1986), and the inhibins (Mason et al., 1985). TGF-P, is a molecule capable of evoking multiple pleiotypic responses when incubated with cells derived from embryonic mesenchyme. Numbered among these responses are inhibition of cell proliferation (Tucker et al., 1984; Roberts et al., 1985; Ranchalis et al., 1987; Coffey et al., 1988; Reiss and Dibble, 19881, stimulation of cell growth (Tucker et al., 1988; Roberts et al., 19811, and upregulation of expression of several extracellular matrix macromolecules, namely fibronectin and collagen (Ignotz and Massague, 1986; Centrella et al., 1987). The effects of TGF-P, on proteoglycan synthesis in organ culture and in cell cultures appears partially dependent on whether or not cells are in a developmental stage or are fully differentiated. For example, in articular chondrocytes isolated from fetal rat bones, purified TGF-P suppressed proteoglycan and Type I1 collagen synthesis, which could be modulated to normal 0 1991 WILEY-LISS. INC.
levels by the addition of fibronectin and agents that interact with the cytoskeleton (Rosen et al., 1988). The marked change in cell shape induced by TGF-P, suggested that, at least in part, the effects of TGF-P, were mediated through its interaction with the cytoskeleton whose structure significantly alters the surrounding pericellular matrix. Furthermore, the effects of TGF-P, on Type I1 collagen synthesis appears to involve alteration in Type I1 collagen gene promoter andior enhancer region activity (Horton et al., 1989). In studies using whole chick limb bud micromass cultures (Gay and Kosher, 19841, different results were obtained (Kulyk e t al., 1989). In this study, TGF-P, (1-10 ngiml) elicited a n increase in Alcian blue staining, radionuclide incorporation into glycosaminoglycans (GAG), and mRNA transcription as measured by hybridization of mRNA to cDNAs encompassing the Type I1 collagen gene and chick proteoglycan core protein gene. By contrast with the variable results obtained with embryonal chondrocytes, in mature chondrocyte culReceived February 5, 1991; accepted June 3, 1991.
*To whom reprint requests should be addressed.
TGF-p STIMULATION OF CHONDROCYTE PROTEOGLY CANS
tures and cartilage explants, TGF-P, has been reported to stimulate proteoglycan synthesis (Redini et al., 1988; Morales and Roberts, 19881, while a t the same time decreasing proteoglycan turnover and degradation (Chandrasekhar and Harvey, 1988). In this regard, TGF-P, may inhibit stromelysin synthesis (Kerr et al., 1990) by causing a reduction in c-fos interaction with the AP-1 site of the stromelysin gene, which regulates stromelysin gene transcription (Kerr et al., 1988). An increase in proteoglycan synthesis after exposure of chondrocytes to TGF-PI as measured by 35S04 or [3H1-glucosamine incorporation could reflect increased GAG chain length or stimulated GAG chain initiation caused by indirect effects of TGF-P, on galactosyltransferase, xylosyltransferase, or other GAG chain initiation recognition signals. In this context, Rapreager (1989) showed that TGF-P, causes the addition of increased GAG to the cell surface proteoglycan (i.e., syndecan) of mouse mammary epithelial cells. In the present study, we have used recombinant TGF-P, (rhTGF) to assess whether or not increased proteoglycan synthesis by chondrocytes results from stimulating production of more core proteins for eventual GAG chain addition or by a stimulation of GAG chain initiation or elongation.
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from 1 h to 24 h. In most experiments, proteoglycan synthesis was measured after 24 h since we had previously shown that all the isoforms in the proteoglycan repertoire synthesized by rabbit chondrocytes are found in the cell cultures a t that time (Srivastava e t al., 1974; Malemud and Papay, 1984). Newly synthesized proteoglycans deposited in the pericellular matrix (cell-associated fraction, CAF) and secreted into the medium (M) were measured separately in 3 experiments. In 3 additional experiments, radiosulfate incorporation was measured on the combined CAF and M compartments. Analysis of newly synthesized proteoglycans The CAF and M compartment were extracted with 4M GuHCl/O.lM sodium acetate buffer, pH 5.8, for 24 h at 4°C. The extraction buffer contained a mixture of protease inhibitors (Oegema et al., 1975).The extracted material was dialyzed exhaustively against distilled deionized water (Q = 17 ohms) and the retained radioactivity counted. In most cases, each group contained 6 culture wells. After obtaining quantitative data on radiosulfate incorporation, the CAF or M fractions were pooled for each group and subjected to density gradient equilibrium centrifugation in CsCl under dissociative conditions (Malemud and Papay, 1984).The starting density was 1.5 gCsClim1. The most dense fraction (p = 1.6 gCsCl/ml) termed D1 was analyzed further.
MATERIALS AND METHODS rhTGF rhTGF (rhTGF-P,) was purified from serum-free medium conditioned by clone 17 cells (a clone of ChiHydrodynamic size of D 1 proteoglycans nese hamster ovary cells) transfected with a cDNA The size of the proteoglycan i n the D1 fraction was clone encoding TGF-PI (Gentry e t al., 1987; Purchio by chromatography on Sepharose CL-2B et al., 1988) as previously described (Gentry et al., assessed (Malemud and Papay, 1984). 1988). The final preparation was at a concentration of 170 pgiml in a solution containing 30% acetonitrile/ GAG chain size 0.05% trifluoracetate and stored a t 4°C. Dilute stock The size of GAG chains was assessed by first removsolutions of rhTGF-PI were prepared in Dulbecco’s modified Eagle’s medium (DME) on the day of its use ing the newly synthesized GAG chains from the core and further dilutions made to arrive a t various concen- protein via alkaline borohydride reduction (Yanagishita and Hascall, 1979) followed by Sepharose CL-6B trations used in the study. chromatography (Malemud and Papay, 1984). C h o n d r o c y t e cultures Assessment of proteoglycan retention Primary cultures of young immature rabbit articular in the C A F chondrocytes were prepared by enzymatic dissociation Chondrocytes were radiolabeled with 35S0, for 24 h of pooled shoulder, hip, and knee cartilage, as previously described (Sokoloff et al., 1970). The cultures in DME containing fetal bovine serum (10%). The cells were initiated in Ham’s F12 medium supplemented were washed free of unincorporated radioactivity and with fetal bovine serum (10%; Hazelton Biologics Inc., rhTGF-P, (50 ngiml) added without serum. Controls did Hazelton, PA), antibiotics, and antimycotics (Gibco, not receive rhTGF-pl. At 1, 6, 12, 24, and 48 h, the Grand Island, NY). The cultures were maintained a t amount of incorporated radiosulfate retained in the 37°C in a n atmosphere of 10% C02/90% air) subpas- CAF was measured after exhaustive dialysis of 4 M saged once and replated at high-density (2 x lo5 GuHCl extracts of the CAF. cellsil.2 cm diameter culture well) in DME suppleEffect of r h T G F on proteoglycan core protein mented with fetal bovine serum (10%) and maintained We compared 35S04-labeled D1 fractions with D1 a t 37°C for 2-3 days, at which time rhTGF-pl was fractions radiolabeled with [35S]-methionine (sp. act. added for 24 h. 1333 Ciimmol; Amersham) in minimal essential meRadiosulfate incorporation dium without methionine. D1 fractions were digested The incorporation of Na,35S0, (sp. act. Carrier Free; with chondroitin lyase ABC and keratanase (0.125 43 Ciimg at loo%, isotopic enrichment; ICN) was used units ABCiO.l unit keratanase; Seikagaku America to assess the effect of rhTGF-P, on proteoglycan syn- Inc.) to remove GAG chains, subjected to 3-16% gradithesis (Srivastava et al., 1974). Cultures were incu- ent slab gel electrophoresis containing 0.1% SDS (SDSI bated with rhTGF-PI (1-50 ngiml) in the presence of PAGE) (Laemmli, 19701, and visualized by autoradiogfetal bovine serum (10% viv) for periods of time ranging raphy.
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Resolution of isoforms of the large proteoglycan Chondroitin lyase ABClkeratanase-digested D1 fractions were electrophoresed on 3-5% acrylamide gels as previously described (King and Witman, 1987). The a and p forms of the heavy chain of dynein (480 kD and 440 kD, respectively) were used to mark the migration rate of a known protein from the top of the running gel to the 200 kD marker protein.
RESULTS Effect of rhTGF-Pl on 35S04 incorporation Incubation of rabbit chondrocytes a t high-density with rhTGF-P, stimulated proteoglycan synthesis (Fig. 1).The effect was demonstrable at a concentration of 5 nglml and was apparently saturable since no further increase was apparent a t concentrations above 5 ng/ml. The stimulation of proteoglycan synthesis was characterized predominantly by increased deposition of newly synthesized proteoglycan into the CAF. At a rhTGF-p, concentration of 50 nglml, the increase over control values for CAF 35S04incorporation was 32.9%, 55.9%, and 210% in 3 separate experiments. Furthermore, in label-chase experiments, rhTGF-P, treatment resulted in a n increase in the retention of the newly synthesized proteoglycan in the CAF (Fig. 2). These differences were statistically significant a t 12 h and 24 h after addition of rhTGF-p, but not so at 48 h. In this regard, rhTGF-p, increased the tb of retention of newly synthesized proteoglycan from 8 h to 11h. Effect of rhTGF-p, on D1 hydrodynamic size and GAG chain length After 24 h incubation with rhTGF-p, (50 ng/ml) the K,, of newly synthesized proteoglycan in control cultures was 0.34 compared to 0.37 for rhTGF-&-treated cultures as measured by Sepharose CL-2B chromatography. In order to examine whether or not rhTGF-P, altered the size of newly synthesized proteoglycans, D1 fractions were examined in label-chase studies. The K,, of the D1 fraction recovered from the medium compartment of control cultures after 24 h of labeling with 35s04 during rhTGF-P, treatment was 0.34. After 6 h of chase in control medium or medium with rhTGF-p,, the K,, of the D1 fraction was 0.32 for control cultures and 0.34 for rhTGF-P,-treated cultures. No changes were seen up to 48 h. The results show that rhTGF-6, did not modify the glycosylated intracellular newly synthesized proteoglycan. Treatment with rhTGF-p, did not alter the GAG chain length of newly synthesized proteoglycan. The sodium borohydride reduced product of both control and rhTGF-p, cultures eluted on Sepharose CL-6B at a K,, of 0.54, corresponding to a M, of 16,500, according to the data of Wasteson (1971).
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Fig. 1. rhTGF-p, stimulates 3sS04incorporation into newly synthesized chondrocyte proteoglycans. First passage chondrocytes were incubated with 0-50 ngiml rhTGF-p, for 24 h and the CAF and M compartment analyzed for retained 35S04in the combined CAF and M. Mean i S.D. n = 6. *PI< 0.051; Student's t-test.
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Chase (hr.) Fig. 2. rhTGF-p, increases the retention of newly synthesized proteoglycan in the CAF compartment. First passage chondrocytes were radiolabeled with "SO4 for 24 h, in the presence of fetal bovine serum (10%viv) washed and incubated with or without rhTGF-p, (50 ngiml) for up to 48 h without serum. The amount of retained radioactivity in the CAF was measured after 4 M GuHCl extraction and dialysis. Mean i S.D. n = 6; "PI< 0.051.0, without rhTGF-PI; with rhTGF-P,. t%, time in which 50% of "SO4 is retained in CAF.
be discerned. The proteoglycan subpopulation a t the top of the running gel is the hydrodynamically large proteoglycan (PG,), whereas the two other proteoglycan subpopulations migrated a s a broad band; one SDWPAGE of newly synthesized proteoglycan subpopulation migrated slower and the other migrated Newly synthesized proteoglycan transported into the to a similar position as the 200 kD marker protein. M compartment, radiolabeled with 35S0, or [35Sl-me- After enzyme digestion, a faint radiosulfate signal was thionine was displayed on 3-16% gradient slab gels seen, corresponding to the core proteins of the large (Fig. 3). The separation of 35S04-labeledproteoglycans proteoglycans containing sulfated-oligosaccharide before and after digestion with chondroitin lyase ABCI stubs. keratanase is shown (Fig. 3A; lanes 1-4; lanes 5-8, The migration of partially deglycosylated core prorespectively). Three proteoglycan subpopulations can tein was visualized by autoradiographs of [35Sl-methio-
TGF-p STIMULATION OF CHONDROCYTE PROTEOGLYCANS
A
155
6 DI - 3 5 ~ 0 ~ 1 2 3 4
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5 6 7 8
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M, x 200 92.5 66 46 30 21.5 -
Fig. 3. Three-16% gradient SDS/PAGE slab gel of newly synthesized proteoglycan. Chondrocytes were radiolabeled with either 3 s S 0 , or LJ5S1-methioninefor 24 h in the presence of 0-50 ng/ml rhTGF-p,. The CAF and M compartments were extracted with 4 M GuHCl and dense proteoglycan fraction D1 generated by equilibrium density centrifugation under dissociative conditions. The D1 fractions were digested or not with chondroitin lyase ABCikeratanase prior to SDSIPAGE. (A)
lanes 1-4, M compartment, 0,5, 10,50 ngiml rhTGF-PI,no digestion; lanes 5-8, after enzyme digestion; (B)same conditions as in (A)but D1 radiolabeled with P5SJ-methionine. Molecular weight markers are, myosin (200kD), phosphorylase B (92.5kD), albumin (66kD), ovalbumin (46kD),carbonic anhydrase (30kD), and soybean trypsin inhibitor (21.5kD).
used, the LIIiLI ratio fluctuated somewhat (range, 0.80-0.92). Two additional core proteins generated from the PG Subuouulation 5 50 small proteoglycan subpopulations (Fig. 3B; lanes 1 4 , LT +8.8 +13.6 +5R.F, .. ~. arrowheads) are seen migrating somewhat slower than t42.0 +55.1 f87.3 the 46 kD marker. Both small proteoglycans core “PG-S,” t24.0 +41.2 +77.0 proteins were stimulated equally by rhTGF-p, “PG-So” f14.1 +31.9 +66.0 (Table 1). IThe results in Figure 3 8 (lanes 5-8) were analyzed by scanning laser densitometry To determine more accurately the apparent size of (Shuckett and Malemud, 1988). “PG-S,” and “PG-S,” refer to the 2 cure proteins shown by arrowheads in Figure 3. the large proteoglycan core proteins LI and LII, the D1 fraction was electrophoresed in 3-5% acrylamide. The a and p chains of dynein (480 kD and 440 kD, respectively) were used to mark protein migration between the beginning of the running gel and the 200 kD nine-labeled D1 fractions. These results are shown in marker protein. The results shown in Figure 4 display methionine-labeled core proteins genFigure 3B. There was a n substantial increase in the the 2 large [35S] radioactive signal in both core proteins (LI, LII) gener- erated after chondroitin lyase/keratanase digestion of ated from the large proteoglycan (PG,) in rhTGF- D1 fractions derived from chondrocytes in the absence p,-treated cultures (Fig. 3B). The laser densitometric and presence of (5 ng/ml) rhTGF-p,. The apparent size scanning (Shuckett and Malemud, 1988) results are of the core protein L, was in excess of 480 kD, since i t summarized in Table 1. Increased radioactivity in L, migrated slower than the dynein markers. The size of was seen almost exclusively at higher concentrations of L,, was about 390 kD. Laser densitometric scanning rhTGF-p,, (50 ngiml). However, lower concentrations showed that rhTGF-P1 did not alter the migration rate of rhTGF-p, (5 ng/ml) resulted in augmented LII syn- of either LI or LII. In the CAF, only L, was found (Fig. 5). Treatment thesis. Over the range of rhTGF-p, concentrations TABLE 1. rhTGF-pl Stimulates proteoglycan core protein synthesis’ [rhTGF-Pl] ng/ml A% 10
MALEMUD ET AL.
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-
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Fig. 4. Three-5% acrylamide gel electrophoresis of [3sSl-methioninelabeled D1 derived from chondrocytes treated with rhTGF-P, (5 ng/ml). D1 was digested with chondroitinase ABCikeratanase. The w and p chains of dynein (generously supplied by Dr. Stephen King, Worcester Foundation) was used as protein markers between the top of the running gel and the 200 kD protein marker. The gels were exposed to BetaMax film and then scanned by laser densitometry (Shuckett and Malemud, 1988).(- 1, no additions, (+)-rhTGF-p, (5 ngiml). L, and L,, are the two core proteins generated from PG,.
with rhTGF-p, (50 ngiml) augmented the synthesis of this core protein.
DISCUSSION Stimulation of chondrocyte proteoglycan synthesis would be advantageous to cartilage after injury or other traumatic insult. In the early stages of osteoarthritis, abortive cellular proliferation and increased synthesis of proteoglycans and collagen has been reported (Malemud, 1985). However, these early events are not sustained. The possible failure of available growth factors to affect the outcome of the osteoarthritic process should be considered. It seems plausible that TGF-P is involved in cartilage responses to injury, since it has been extracted and purified from articular cartilage (Seyedin et al., 1986, 19881, and recent studies suggest a n autocrine feedback loop, whereby TGF-P upregulates the transcription of mRNA for TGF-P (Joyce et al., 1990; Malemud and Purchio, unpublished data). The mechanism for activation of the pre-ipro-form of TGF-P in cartilage remains to be elucidated but may involve proteolytic cleavage which has been shown to activate the pre-/pro-form of TGF-P (Gentry et al., 1988).A pH in the range 2.5-3.5, which can result in test-tube
activation of the precursor form of TGF-61 (Gelb e t al., 19901, seems less likely to occur in cell culture. This suggests that the only active form of TGF-PI came from rhTGF-P, and not a combined effect of TGF-p, activated in fetal bovine serum and rhTGF-6,. Gelb et al. (1990)could not find any detectable active TGF-P in the conditioned medium of growth plate chondrocytes maintained serum free for 24 h. In adult rabbit articular chondrocytes, purified TGF-P was previously shown to stimulate proteoglycan synthesis (Redini et al., 1988). The present studies revealed that the rhTGF-p, had a similar effect. The studies indicated that, in all likelihood, rhTGF-P, caused a n upregulation of core protein synthesis, which then became available for GAG chain initiation. It was noteworthy that rhTGF-P, stimulation of core protein synthesis was concentration-dependent. The L, form of core protein required a higher concentration of rhTGF-p, than did L,, to stimulate its synthesis. The apparent M, of 390 kD for L,, was in good agreement with the cell-free translation product from chick sternal cartilage mRNA immunoprecipitated with antisera to core protein (Vertel and Hitti, 1987) and the hydrogen fluoride-treated newly synthesized chick proteo-
TGF-6 STIMULATION OF CHONDROCYTE PROTEOGLYCANS
M,X
480,440 (dynein)
200 -
93 69 Fig. 5. Three -5% acrylamide gel electrophoresis of 135S1-methionine labeled D1 from the CAF of chondrocytes treated with rhTGF-p, (50 ngiml). D1 was digested with chondroitinase ABCikeratanase prior to electrophoresis. The L, form is the only core protein seen in the cell-associated (C) compartment. L,, is the expected migration position of this core protein.
157
drocytes and other cell types (Fisher et al., 1989; Melching and Roughley, 1989; Pulkkinen et al., 1990). We have, a t present, been unable to unequivocally characterize either of these molecules. Neither antidecorin antibody (Sawhney et al., 1991) nor antisera made against synthetic peptides derived from the amino acid sequences of DS-PG-I or DS-PG-I1 core protein (Fisher et al., 1989) immunoprecipitated the chondroitinase ABC-digested “small” rabbit proteoglycans. Significant differences may exist between the rabbit form of these “small” proteoglycans and “small” proteoglycans from other species. Although the molecular mechanism for the rhTGFP1 effect on rabbit chondrocyte proteoglycan remains unknown, a recent report sheds some light on how the cell cycle may play a role in this stimulation. TGF-6, increases the time chondrocytes spend in the interface between the S phase and mitosis (Vivien et al., 1990). We have previously shown that proteoglycans are synthesized by rabbit chondrocytes in both the G, and G, phase of the cell cycle (Malemud and Sokoloff, 1974). These results are noteworthy since the proliferative capacity of chondrocytes participating in repair of cartilage lesions may be directly related to their capacity to also synthesize extracellular matrix macromolecules. Thus, TGF-P, may act to stimulate proteoglycan synthesis only when early proliferative events consonant with the initial stages of attempts at cartilage repair after trauma cease or become less prominent.
ACKNOWLEDGMENTS This work was supported in part by grant AG-02205 from the National Institute on Aging (USPHS). LITERATURE CITED
glycan core protein (Campbell et al., 1990). Recent studies using rotary shadowing electron microscopy of chick core protein (Dennis et al., 1990) revealed a core protein size larger than predicted by a cDNA clone for the rat chondrosarcoma core protein (Doege et al., 1987). The larger form of core protein we have observed (> 480 kD) has not been previously described in chondrocytes, but large chondroitin sulfate-containing core proteins of this size or somewhat smaller (> 400 kD) have been reported in brain tissue (Gowda et al., 1989). It is not known at present whether or not the 480 kD core protein represents a n alternative spliced form of the large proteoglycan gene or a unique gene product. Other studies have reported that TGF-6, stimulates decorin synthesis in rat mesangial cells (Border et al., 1990). A recent report by Breuer et al. (1990) found no effect of TGF-P on decorin in human skin fibroblasts and a human osteosarcoma cell line. Using laser densitometric scanning, a distinct increase in the radioactive signal was seen in both of the 2 “small” proteoglycans. The larger of these 2 core proteins (PG-S,) migrated slower than PG-S, presumably due to more glycosaminoglycan stubs. It may be structurally similar to DS-PG-I (biglycan) synthesized by bovine chon-
Border, W.A., Okuda, S., Languino, L.R., and Ruoslahti, E. (1990) Transforming growth factor p regulates production of proteoglycans by mesangial cells. Kidney Int., 37:689-695. Breuer, B., Schmidt, G., and Kresse, H. (1990) Non-uniform influence of transforming growth factor-p on the biosynthesis of different forms of small chondroitin sulfateidermatan sulfate proteoglycans. Biochem. J.,269551-554, Campbell. S.C.. Kruerrer. R.C., and Schwartz. N.B. (1990) Dedvcosvlation of chondroitb sulfate Droteodvcan and derived Diutides. Biochemistry, 29t907-914. Cate, R.L., Mattaliano, R.J., Hession, C., Tizard, R., Farber, N.M., Cheune. A,, Ninfa. E.G.. Frev. A.Z.. Gash, D.J., Chow, E.P.. Fisher, R.A., Bertonis, J.M., Torres,’ G., Wallner, B.P., Ramachandran, K.L., Ragin, R.C., Manganaro, T.F., MacLaughlin, D.T., and Donahoe, P.K. (1986) Isolation of the bovine and human genes for Mullerian inhibiting substance and expression of the human gene in animal cells. Cell, 45:685-698. Centrella, M., McCarthy, T.L., and Canalis, E. (1987) Transforming growth factor p is a bifunctional regulator of replication and collagen synthesis in osteoblast-enriched cell cultures from fetal rat bone. J . Biol. Chem., 2622869-2894. Chandrasekhar S., and Harvey, A. (1988) Transforming growth factdor-p is a potent inhibitor of IL-I induced protease activity and cartilage proteoglycan degradation. Biochem. Biophys. Res. Commun., 157:1352-1359. Cheifetz, S., Weatherbee, J.A., Tsang, M.L.-S., Anderson, J.K., Mole, J.E., Lucas, R., and Massague, J . (1987) The transforming growth factor-p system, a complex pattern of cross-reactive ligands and receptors. Cell, 48:409-415. Coffey, R.J., Sipes, N.J., Bascum, C.C., Graves-Deal, R., Pennington, C.Y., Weissman, B.E., and Moses, H.L. (1988)Growth modulation of mouse keratinocytes by transforming growth factors. Cancer Res., 48t159-1602. Dennis, J.E., Carrino, D.A., Schwartz, N.B., and Caplan, A.I. (1990) Ultrastructural characterization of embryonic chick cartilage proU “
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teoglycan core protein and the mapping of a monoclonal antibody epitope. J . Biol. Chem., 265t12098-12103. Derynck, R., Lindquist, P.B., Lee, A., Wen, D., Tamm, J.L., Graycar, J.L., Rhee, L., Mason, A.J., Miller, D.A., Coffey, R.J., Moses, H.L., and Chen, E.Y. (1988) A new type of transforming growth factor-p, TGF- p3. EMBO J., 7:3737-3743. Doege, K., Sasaki, M., Horigan, E., Hassell, J . Jr., Yamada, Y. (1987) Complete primary structure of rat cartilage proteoglycan core protein deduced from cDNA clones. J . Biol. Chem., 262:1775717767. Fisher, L.W., Termine, J.D., and Young, M.F. (1989) Deduced protein sequence of bone small proteoglycan (biglycan) shows homology with proteoglycan I1 (decorin) and several nonconnective tissue proteins in a variety of species. J. Biol. Chem., 264:4571-4576. Gay, S.W., and Kosher, R.A. (1984) Uniform cartilage differentiation in micromass cultures prepared from a relatively homogeneous population of chondrogenic progenitor cells of the chick limb bud: Effect of prostaglandins. J . Exp. Zool., 232t317-326. Gelb, D.E., Rosier, R.N., and Puzas, J.E. (1990) The production of transforming growth factor-p by chick growth plate chondrocytes in short term monolayer culture. Endocrinology, 127t1941-1947. Gentry, L.E., Lioubin, M.N., Purchio, A.F., and Marquardt, H. (1988) Molecular events in the processing of recombinant Type 1 prepro-transforming growth factor p to the mature polypeptide. Mol. Cell. Biol., 8t4162-4168. Gentry, L.E., Webb, N.R., Lim, G.J., Brunner, A.M., Ranchalis, J.E., Twardzik, D.R., Lioubin, M.N., Marquardt, H., and Purchio, A.F. (1987) Type 1 transforming growth factor beta: Amplified expression and secretion of mature and precursor polypeptides in Chinese hamster ovary cells. Mol. Cell. Biol., 7t3418-3427. Gowda, D.C., Margolis, R.U., and Margolis, R.K. (1989) Presence of the HNK-1 epitope on poly (N-acetyllactosaminyl) oligosaccharides and identification of multiple core proteins in the chondroitin sulfate proteoglycans of brain. Biochemistry, 28r446W474. Horton, W.E., Higginbotham, J.D.. and Chandrasekhar. S. (1989) Transforming growth factor-p and fibroblast growth factor act synergistically to inhibit collagen 11 synthesis through a mechanism involving regulatory DNA sequences. J . Cell. Physiol., 141;&15. Ignotz, R.A., and Massague, J . (1986) Transforming growth factor-p stimulates the expression of fibronectin and collagen and their incorporation into the extracellular matrix. J . Biol. Chem., 261t4337-4345. Ikeda, T., Lioubin, M.N., and Marquardt,, H. (1987) Human transforming growth factor type p2: Production by a prostatic adenocarcinoma cell line, purification and initial characterization. Biochemistry, 26t24062410. Jakowlew, S.B., Dillard, P..J., Kondaiah, P., Sporn, M.B. and Roberts, A.B. (1988a) Complementary deoxyribonucleic acid cloning of a novel transforming growth factor-p messenger ribonucleic acid from chick embryo chondrocytes. Molec. Endocrinology, 2:747-755. Jakowlew, S.B., Dillard, P.J., Sporn, M.B., and Roberts, A.B., (198813) Complementary deoxyribonucleic acid cloning of a messenger ribonucleic acid encoding transforming growth factor p4 from chick embryo chondrocytes. Molec. Endocrinology, 2t11861195. Joyce, M.E., Roberts, A.B., Sporn, M.B., and Bolander, M.E. (1990) Transforming growth factor-p and the initiation of chondrogenesis and osteogenesis in the rat femur. J . Cell Biol., 110t2195-2207. Kerr, L.D., Holt, J.T., and Matrisian, L. (1988) Growth factors regulate transin gene expression by c-fos-dependent and c-fosindependent pathways. Science, 242t142P1427. Kerr, L.D., Miller, D.B., and Matrisian, L.M. (1990)TGF-PI inhibition of transinistromelysin gene expression is mediated through a Fos binding sequence. Cell, 61 t267-278. King, S.M., and Witman, G.B. (1987) Structure of the CY and p heavy chains of the outer arm dynein from Chlumydomonus flagella. Masses of chains and sites of ultraviolet-induced vanadate-dependent cleavage. J. Biol. Chem., 262:17596-17604. Kulyk, W.M., Rodgers, B.J., Greer, K., Kosher, R.A. (1989) Promotion of embryonic chick limb cartilage differentiation by transforming growth factor-p. Dev. Biol., 135t424-430. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227t680-685. Malemud, C.J. (1985) Pathophysiological responses of articular cartilage in the arthritides. In: Proceedings of the XVI International Congress of Rheumatology. P.M. Brooks and J.R. York, eds., Elsevier Science Publishers, Amsterdam, pp. 79-83. Malemud, C.J., and Papay, R.S. (1984) The in vitro cell culture age and cell density of articular chondrocytes alter sulfated-proteoglycan biosynthesis. J. Cell. Physiol., 121.558-568.
Malemud, C.J., and Sokoloff, L. (1974) Some biological characteristics of a pituitary growth factor (CGF) for cultured lapine articular chondrocytes. J . Cell. Physiol., 84t171-179. Mason, A.J., Hayflick, J.S., Ling, N., Esch, F., Ueno, N., Ying, S.-Y., Guillemen, R., Niall, H., and Seeburg, P.H. (1985) Complementary DNA sequences of ovarian follicular fluid inhibin show precursor structure and homology with transforming growth factor-p. Nature, 318t659-663. Melching, L.I., and Roughley, P.J. (1989) The synthesis of dermatan sulfate Droteoelvcan bv fetal and adult human articular cartilage. BiocheG. J., 2%?:501-508. Morals, T.I., and Roberts, A.B. (1988) Transforming growth factor p regulates the metabolism of proteoglycans in bovine cartilage organ cultures. J. Biol. Chem., 263:12828-12831. Oegema, T.R., Jr., Hascall, V.C., and Dziewiatkowski, D.D. (1975) Isolation and characterization of proteoglycans from the Swarm rat chondrosarcoma. J . Biol . Chem., 250t6 151-61 59. Pulkkinen, L., Kainulainen, K., Krusius, T., Makinen, P., Schollin, J., Gustavsson, K.-H., and Peltonen, L. (1990) Deficient expression of the gene coding for decorin in a lethal form of Marfan syndrome. J. Biol. Chem., 265t17780-17785. Purchio, A.F., Cooper, J.A., Brunner, A.M., Lioubin, M.N., Gentry, L.E., Kovacina, K.S., Roth, R.A., and Marquardt, H. (1988) Purification of mannose 6-phosphate in two aspargine-linked sugar chains of recombinant transforming growth factor-pl precursor. J. Biol. Chem., 263:14211-14215. Ranchalis, J.E., Gentry L., Ogawa, Y., Seyedin, S.M., McPherson, J., Purchio, A., and Twardzik, D.R. (1987) Bone-derived and recombinant transforming growth factor p’s are potent inhibitors of tumor cell growth. Biochem. Biophys. Res. Commun., 148t783-789. Rapraeger, A. (1989) Transforming growth factor (Type p) promotes the addition of chondroitin sulfate chains to the cell surface proteoglycan (Syndecan) of mouse mammary epithelia. J. Cell Biol., 109t2509-2518. Redini, F., Galera, P., Mauviel, A,, Loyau, G., and Pujol, J.-P. (1988) Transforming growth factor p stimultes collagen and glycosaminoglycan biosynthesis in cultured rabbit articular chondrocytes. FEBS Lett., 234t172-176. Reiss, M., and Dibble, C.L. (1988) Reinitiation of DNA synthesis in quiescent mouse keratinocytes; regulation by polypeptide hormones, cholera toxin, dexamethasone and retinoic acid. In Vitro Cell. Dev. Biol., 24.537-544. Roberts, A.B., Anzano, M.A., Lamb, L.C., Smith, J.M., and Sporn, M.B. (1981)New class of transforming growth factors potentiated by epidermal growth factors: Isolation from non-neoplastic tissues. Proc. Natl. Acad. Sci. U.S.A., 78t5339-5343. Roberts, A.B., Anzano, M.A., Wakefield, L.M., Roches, N.S., Stern, D.F., and Sporn, M.B. (1985) Type p transforming growth factor: A bifunctional regulator of cellular growth. Proc. Natl. Acad. Sci. U.S.A., 82.119-123. Rosen, D.M., Stempien, S.A., Thompson, A.V., and Seyedin, S.M. (1988) Transforming growth factor-p modulates the expression of osteoblast and chondroblast phenotypes in vitro. J. Cell. Physiol., 134t337-346. Sawhney, R.S., Hering, T.M., and Sandell, L.J. (1991) Biosynthesis of small proteoglycan I1 (Decorin) by chondrocytes and evidence for a procore protein. J . Biol. Chem. (in press). Seyedin, S.M., Rosen, D.M., and Segarini, P.R. (1988) Modulation of chondroblast phenotype by transforming growth factor-p. Pathol. Immunopathol. Res., 7t38-42. Seyedin, S.M., Segarini, P.R., Rosen, D.M., Thompson, A.Y., Bentz, H., and Graycar, J . (1987) Cartilage-inducing-factor+ is a unique protein structurally and functionally related to transforming growth factor-p. J. Biol. Chem., 262t1946-1949. Seyedin, S.M., Thompson, A.Y., Bentz, H., Rosen, D.M., McPherson, J.M., Contin, A,, Siegel, N.R., Galluppi, G.R., and Piez, K.A. (1986) Cartilage-inducing factor-A. Apparent identify to transforming growth factor-p. J . Biol. Chem., 2615693-5695. Shuckett, R., and Malemud, C.J. (1988) Qualitative changes in human osteoarthritic hip cartilage proteoglycan synthesis during longterm explant culture. Mech. Ageing Dev., 46:3345. Sokoloff, L., Malemud, C.J., and Green, W.T. Jr. (1970) Sulfate incorporation by articular chondrocytes in monolayer culture. Arthritis Rheum., 13t118-124. Srivastava, V.M.L., Malemud, C.J., and Sokoloff, L. (1974) Chondroid expression by lapine articular chondrocytes in spinner culture following monolayer growth. Connect. Tissue Res., 2t127-136. tenDijke, P., Hansen, P., Iwata, K., Pieler, C., and Foulkes, J.G. (1988) I
TGF-P STIMULATION OF CHONDROCYTE PROTEOGLYCANS
Identification of another member of the transforming growth factor type p gene family. Proc. Natl. Acad. Sci. U.S.A., 85t47154719. Tucker, R.F., Shipley, G.D., Moses, H.L. and Holley, R.W. (1984) Growth inhibitor from BSC-1 cells closely related to platelet type p transforming growth factor. Science, 226:705-707. Tucker, R.F., Volkenant, M.E., Branum, E.L. and Moses, H.L. (1988) Comparison of intra- and extracellular growth factors from nontransformed and chemically transformed mouse embryo cells. Cancer Res., 43:1581-1586. Vertel, B.M. and Hitti, Y. (1987) Biosynthetic precursors of cartilage chondroitin sulfate proteoglycan. Collagen Rel. Res., 7:57-75.
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Vivien, D., Galera, P., Lebrun, E., Loyau, G., and Pujol, J.-P. (1990) Differential effects of transforming growth factor-p and epidermal growth factor on the cell cycle of cultured rabbit articular chondrocytes. J. Cell. Physiol., 143t534-545. Wasteson, A. (1971) A method for the determination of the molecular weight and molecular-weight distribution of chondroitin sulphate. J. Chromatogr., 59:87-97. Yanaeishita., M.., and Hascall. V.C. (1979) Biosvnthesis of Droteoelvcans by rat granulosa cells cultured in vitro. J. Biol. Chem., 254:12355-12364. I
I "
Enhanced Sulfated-Proteoglycan Core Protein Synthesis by Incubation of Rabbit Chondrocytes With Recombinant Transforming Growth Factor$, CHARLES J. MALEMUD*, WENDY KILLEEN, THOMAS M. HERINC, AND ANTHONY F. PURCHIO Departments of Medicine (C.).M., W.K., J.M.H.) and Andtomy (C.I.M., T.M.H.), Case Western Reserve University, Cleveland, Ohio 44 106; Oncogen Corporation, Seattle, Washington 98 195 (A.F.P.) Rabbit articular chondrocytes were incubated with recombinant transforminggrowthiactor-p, (rhTGF-P,) and its effect on newly synthesized proteoglycan measured. rhTGF-P, stimulated proteoglycan synthesis at a concentration as low as 5 ngiml without further increases in radiosulfate incorporation up to 50 ngiml. The quantitative increase in radiosulfate incorporation in rh-TGF-P, -treated chondrocytes was greater in the cell-associated culture compartment than in the medium compartment. rhTGF-P, promoted an increased proteoglycan retention in the cell-associated compartment as evidenced by an increase in the t% of retention from 8 h to 11 h. Specific enhanced synthesis of [35S]-methioninelabeled core proteins was seen in rh-TGF-fi-treated chondrocytes. rh-TGF-P, increased the synthesis of the 2 core proteins derived from hydrodynamically large proteoglycans. They possessed apparent molecular weights of > 480 kD and 390 kD after 3-5% acrylamide gel electrophoresis. A compartmental analysis revealed that the cell-associated culture compartment contained only the larger of the 2 core proteins derived from large proteoglycans. Two other core proteins with apparent molecular weights 52 kD and 46 kD were also stimulated by rhTGF-P,. These results indicated that TGF-P probably plays a significant role in stimulating proteoglycan core protein synthesis in articular chondrocytes and therefore may be an important growth factor in the restoration of cartilage extracellular matrix after injury.
Recent studies have shown that TGF-P, belongs to a family of closely related growth-modulatory molecules including TGF-P2 (Seyedin et al., 1987; Cheifetz et al., 1987; Ikeda et al., 19871, TGF-P, (tenDijke et al., 1988; Derynck et al., 1988; Jakowlew et al., 1988a1, TGF-P, (Jakowlew et al., 1988b), Mullerian inhibitory substance (Cate et al., 1986), and the inhibins (Mason et al., 1985). TGF-P, is a molecule capable of evoking multiple pleiotypic responses when incubated with cells derived from embryonic mesenchyme. Numbered among these responses are inhibition of cell proliferation (Tucker et al., 1984; Roberts et al., 1985; Ranchalis et al., 1987; Coffey et al., 1988; Reiss and Dibble, 19881, stimulation of cell growth (Tucker et al., 1988; Roberts et al., 19811, and upregulation of expression of several extracellular matrix macromolecules, namely fibronectin and collagen (Ignotz and Massague, 1986; Centrella et al., 1987). The effects of TGF-P, on proteoglycan synthesis in organ culture and in cell cultures appears partially dependent on whether or not cells are in a developmental stage or are fully differentiated. For example, in articular chondrocytes isolated from fetal rat bones, purified TGF-P suppressed proteoglycan and Type I1 collagen synthesis, which could be modulated to normal 0 1991 WILEY-LISS. INC.
levels by the addition of fibronectin and agents that interact with the cytoskeleton (Rosen et al., 1988). The marked change in cell shape induced by TGF-P, suggested that, at least in part, the effects of TGF-P, were mediated through its interaction with the cytoskeleton whose structure significantly alters the surrounding pericellular matrix. Furthermore, the effects of TGF-P, on Type I1 collagen synthesis appears to involve alteration in Type I1 collagen gene promoter andior enhancer region activity (Horton et al., 1989). In studies using whole chick limb bud micromass cultures (Gay and Kosher, 19841, different results were obtained (Kulyk e t al., 1989). In this study, TGF-P, (1-10 ngiml) elicited a n increase in Alcian blue staining, radionuclide incorporation into glycosaminoglycans (GAG), and mRNA transcription as measured by hybridization of mRNA to cDNAs encompassing the Type I1 collagen gene and chick proteoglycan core protein gene. By contrast with the variable results obtained with embryonal chondrocytes, in mature chondrocyte culReceived February 5, 1991; accepted June 3, 1991.
*To whom reprint requests should be addressed.
TGF-p STIMULATION OF CHONDROCYTE PROTEOGLY CANS
tures and cartilage explants, TGF-P, has been reported to stimulate proteoglycan synthesis (Redini et al., 1988; Morales and Roberts, 19881, while a t the same time decreasing proteoglycan turnover and degradation (Chandrasekhar and Harvey, 1988). In this regard, TGF-P, may inhibit stromelysin synthesis (Kerr et al., 1990) by causing a reduction in c-fos interaction with the AP-1 site of the stromelysin gene, which regulates stromelysin gene transcription (Kerr et al., 1988). An increase in proteoglycan synthesis after exposure of chondrocytes to TGF-PI as measured by 35S04 or [3H1-glucosamine incorporation could reflect increased GAG chain length or stimulated GAG chain initiation caused by indirect effects of TGF-P, on galactosyltransferase, xylosyltransferase, or other GAG chain initiation recognition signals. In this context, Rapreager (1989) showed that TGF-P, causes the addition of increased GAG to the cell surface proteoglycan (i.e., syndecan) of mouse mammary epithelial cells. In the present study, we have used recombinant TGF-P, (rhTGF) to assess whether or not increased proteoglycan synthesis by chondrocytes results from stimulating production of more core proteins for eventual GAG chain addition or by a stimulation of GAG chain initiation or elongation.
153
from 1 h to 24 h. In most experiments, proteoglycan synthesis was measured after 24 h since we had previously shown that all the isoforms in the proteoglycan repertoire synthesized by rabbit chondrocytes are found in the cell cultures a t that time (Srivastava e t al., 1974; Malemud and Papay, 1984). Newly synthesized proteoglycans deposited in the pericellular matrix (cell-associated fraction, CAF) and secreted into the medium (M) were measured separately in 3 experiments. In 3 additional experiments, radiosulfate incorporation was measured on the combined CAF and M compartments. Analysis of newly synthesized proteoglycans The CAF and M compartment were extracted with 4M GuHCl/O.lM sodium acetate buffer, pH 5.8, for 24 h at 4°C. The extraction buffer contained a mixture of protease inhibitors (Oegema et al., 1975).The extracted material was dialyzed exhaustively against distilled deionized water (Q = 17 ohms) and the retained radioactivity counted. In most cases, each group contained 6 culture wells. After obtaining quantitative data on radiosulfate incorporation, the CAF or M fractions were pooled for each group and subjected to density gradient equilibrium centrifugation in CsCl under dissociative conditions (Malemud and Papay, 1984).The starting density was 1.5 gCsClim1. The most dense fraction (p = 1.6 gCsCl/ml) termed D1 was analyzed further.
MATERIALS AND METHODS rhTGF rhTGF (rhTGF-P,) was purified from serum-free medium conditioned by clone 17 cells (a clone of ChiHydrodynamic size of D 1 proteoglycans nese hamster ovary cells) transfected with a cDNA The size of the proteoglycan i n the D1 fraction was clone encoding TGF-PI (Gentry e t al., 1987; Purchio by chromatography on Sepharose CL-2B et al., 1988) as previously described (Gentry et al., assessed (Malemud and Papay, 1984). 1988). The final preparation was at a concentration of 170 pgiml in a solution containing 30% acetonitrile/ GAG chain size 0.05% trifluoracetate and stored a t 4°C. Dilute stock The size of GAG chains was assessed by first removsolutions of rhTGF-PI were prepared in Dulbecco’s modified Eagle’s medium (DME) on the day of its use ing the newly synthesized GAG chains from the core and further dilutions made to arrive a t various concen- protein via alkaline borohydride reduction (Yanagishita and Hascall, 1979) followed by Sepharose CL-6B trations used in the study. chromatography (Malemud and Papay, 1984). C h o n d r o c y t e cultures Assessment of proteoglycan retention Primary cultures of young immature rabbit articular in the C A F chondrocytes were prepared by enzymatic dissociation Chondrocytes were radiolabeled with 35S0, for 24 h of pooled shoulder, hip, and knee cartilage, as previously described (Sokoloff et al., 1970). The cultures in DME containing fetal bovine serum (10%). The cells were initiated in Ham’s F12 medium supplemented were washed free of unincorporated radioactivity and with fetal bovine serum (10%; Hazelton Biologics Inc., rhTGF-P, (50 ngiml) added without serum. Controls did Hazelton, PA), antibiotics, and antimycotics (Gibco, not receive rhTGF-pl. At 1, 6, 12, 24, and 48 h, the Grand Island, NY). The cultures were maintained a t amount of incorporated radiosulfate retained in the 37°C in a n atmosphere of 10% C02/90% air) subpas- CAF was measured after exhaustive dialysis of 4 M saged once and replated at high-density (2 x lo5 GuHCl extracts of the CAF. cellsil.2 cm diameter culture well) in DME suppleEffect of r h T G F on proteoglycan core protein mented with fetal bovine serum (10%) and maintained We compared 35S04-labeled D1 fractions with D1 a t 37°C for 2-3 days, at which time rhTGF-pl was fractions radiolabeled with [35S]-methionine (sp. act. added for 24 h. 1333 Ciimmol; Amersham) in minimal essential meRadiosulfate incorporation dium without methionine. D1 fractions were digested The incorporation of Na,35S0, (sp. act. Carrier Free; with chondroitin lyase ABC and keratanase (0.125 43 Ciimg at loo%, isotopic enrichment; ICN) was used units ABCiO.l unit keratanase; Seikagaku America to assess the effect of rhTGF-P, on proteoglycan syn- Inc.) to remove GAG chains, subjected to 3-16% gradithesis (Srivastava et al., 1974). Cultures were incu- ent slab gel electrophoresis containing 0.1% SDS (SDSI bated with rhTGF-PI (1-50 ngiml) in the presence of PAGE) (Laemmli, 19701, and visualized by autoradiogfetal bovine serum (10% viv) for periods of time ranging raphy.
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Resolution of isoforms of the large proteoglycan Chondroitin lyase ABClkeratanase-digested D1 fractions were electrophoresed on 3-5% acrylamide gels as previously described (King and Witman, 1987). The a and p forms of the heavy chain of dynein (480 kD and 440 kD, respectively) were used to mark the migration rate of a known protein from the top of the running gel to the 200 kD marker protein.
RESULTS Effect of rhTGF-Pl on 35S04 incorporation Incubation of rabbit chondrocytes a t high-density with rhTGF-P, stimulated proteoglycan synthesis (Fig. 1).The effect was demonstrable at a concentration of 5 nglml and was apparently saturable since no further increase was apparent a t concentrations above 5 ng/ml. The stimulation of proteoglycan synthesis was characterized predominantly by increased deposition of newly synthesized proteoglycan into the CAF. At a rhTGF-p, concentration of 50 nglml, the increase over control values for CAF 35S04incorporation was 32.9%, 55.9%, and 210% in 3 separate experiments. Furthermore, in label-chase experiments, rhTGF-P, treatment resulted in a n increase in the retention of the newly synthesized proteoglycan in the CAF (Fig. 2). These differences were statistically significant a t 12 h and 24 h after addition of rhTGF-p, but not so at 48 h. In this regard, rhTGF-p, increased the tb of retention of newly synthesized proteoglycan from 8 h to 11h. Effect of rhTGF-p, on D1 hydrodynamic size and GAG chain length After 24 h incubation with rhTGF-p, (50 ng/ml) the K,, of newly synthesized proteoglycan in control cultures was 0.34 compared to 0.37 for rhTGF-&-treated cultures as measured by Sepharose CL-2B chromatography. In order to examine whether or not rhTGF-P, altered the size of newly synthesized proteoglycans, D1 fractions were examined in label-chase studies. The K,, of the D1 fraction recovered from the medium compartment of control cultures after 24 h of labeling with 35s04 during rhTGF-P, treatment was 0.34. After 6 h of chase in control medium or medium with rhTGF-p,, the K,, of the D1 fraction was 0.32 for control cultures and 0.34 for rhTGF-P,-treated cultures. No changes were seen up to 48 h. The results show that rhTGF-6, did not modify the glycosylated intracellular newly synthesized proteoglycan. Treatment with rhTGF-p, did not alter the GAG chain length of newly synthesized proteoglycan. The sodium borohydride reduced product of both control and rhTGF-p, cultures eluted on Sepharose CL-6B at a K,, of 0.54, corresponding to a M, of 16,500, according to the data of Wasteson (1971).
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Chase (hr.) Fig. 2. rhTGF-p, increases the retention of newly synthesized proteoglycan in the CAF compartment. First passage chondrocytes were radiolabeled with "SO4 for 24 h, in the presence of fetal bovine serum (10%viv) washed and incubated with or without rhTGF-p, (50 ngiml) for up to 48 h without serum. The amount of retained radioactivity in the CAF was measured after 4 M GuHCl extraction and dialysis. Mean i S.D. n = 6; "PI< 0.051.0, without rhTGF-PI; with rhTGF-P,. t%, time in which 50% of "SO4 is retained in CAF.
be discerned. The proteoglycan subpopulation a t the top of the running gel is the hydrodynamically large proteoglycan (PG,), whereas the two other proteoglycan subpopulations migrated a s a broad band; one SDWPAGE of newly synthesized proteoglycan subpopulation migrated slower and the other migrated Newly synthesized proteoglycan transported into the to a similar position as the 200 kD marker protein. M compartment, radiolabeled with 35S0, or [35Sl-me- After enzyme digestion, a faint radiosulfate signal was thionine was displayed on 3-16% gradient slab gels seen, corresponding to the core proteins of the large (Fig. 3). The separation of 35S04-labeledproteoglycans proteoglycans containing sulfated-oligosaccharide before and after digestion with chondroitin lyase ABCI stubs. keratanase is shown (Fig. 3A; lanes 1-4; lanes 5-8, The migration of partially deglycosylated core prorespectively). Three proteoglycan subpopulations can tein was visualized by autoradiographs of [35Sl-methio-
TGF-p STIMULATION OF CHONDROCYTE PROTEOGLYCANS
A
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Fig. 3. Three-16% gradient SDS/PAGE slab gel of newly synthesized proteoglycan. Chondrocytes were radiolabeled with either 3 s S 0 , or LJ5S1-methioninefor 24 h in the presence of 0-50 ng/ml rhTGF-p,. The CAF and M compartments were extracted with 4 M GuHCl and dense proteoglycan fraction D1 generated by equilibrium density centrifugation under dissociative conditions. The D1 fractions were digested or not with chondroitin lyase ABCikeratanase prior to SDSIPAGE. (A)
lanes 1-4, M compartment, 0,5, 10,50 ngiml rhTGF-PI,no digestion; lanes 5-8, after enzyme digestion; (B)same conditions as in (A)but D1 radiolabeled with P5SJ-methionine. Molecular weight markers are, myosin (200kD), phosphorylase B (92.5kD), albumin (66kD), ovalbumin (46kD),carbonic anhydrase (30kD), and soybean trypsin inhibitor (21.5kD).
used, the LIIiLI ratio fluctuated somewhat (range, 0.80-0.92). Two additional core proteins generated from the PG Subuouulation 5 50 small proteoglycan subpopulations (Fig. 3B; lanes 1 4 , LT +8.8 +13.6 +5R.F, .. ~. arrowheads) are seen migrating somewhat slower than t42.0 +55.1 f87.3 the 46 kD marker. Both small proteoglycans core “PG-S,” t24.0 +41.2 +77.0 proteins were stimulated equally by rhTGF-p, “PG-So” f14.1 +31.9 +66.0 (Table 1). IThe results in Figure 3 8 (lanes 5-8) were analyzed by scanning laser densitometry To determine more accurately the apparent size of (Shuckett and Malemud, 1988). “PG-S,” and “PG-S,” refer to the 2 cure proteins shown by arrowheads in Figure 3. the large proteoglycan core proteins LI and LII, the D1 fraction was electrophoresed in 3-5% acrylamide. The a and p chains of dynein (480 kD and 440 kD, respectively) were used to mark protein migration between the beginning of the running gel and the 200 kD nine-labeled D1 fractions. These results are shown in marker protein. The results shown in Figure 4 display methionine-labeled core proteins genFigure 3B. There was a n substantial increase in the the 2 large [35S] radioactive signal in both core proteins (LI, LII) gener- erated after chondroitin lyase/keratanase digestion of ated from the large proteoglycan (PG,) in rhTGF- D1 fractions derived from chondrocytes in the absence p,-treated cultures (Fig. 3B). The laser densitometric and presence of (5 ng/ml) rhTGF-p,. The apparent size scanning (Shuckett and Malemud, 1988) results are of the core protein L, was in excess of 480 kD, since i t summarized in Table 1. Increased radioactivity in L, migrated slower than the dynein markers. The size of was seen almost exclusively at higher concentrations of L,, was about 390 kD. Laser densitometric scanning rhTGF-p,, (50 ngiml). However, lower concentrations showed that rhTGF-P1 did not alter the migration rate of rhTGF-p, (5 ng/ml) resulted in augmented LII syn- of either LI or LII. In the CAF, only L, was found (Fig. 5). Treatment thesis. Over the range of rhTGF-p, concentrations TABLE 1. rhTGF-pl Stimulates proteoglycan core protein synthesis’ [rhTGF-Pl] ng/ml A% 10
MALEMUD ET AL.
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Distance (mm)
Distance (mm)
Fig. 4. Three-5% acrylamide gel electrophoresis of [3sSl-methioninelabeled D1 derived from chondrocytes treated with rhTGF-P, (5 ng/ml). D1 was digested with chondroitinase ABCikeratanase. The w and p chains of dynein (generously supplied by Dr. Stephen King, Worcester Foundation) was used as protein markers between the top of the running gel and the 200 kD protein marker. The gels were exposed to BetaMax film and then scanned by laser densitometry (Shuckett and Malemud, 1988).(- 1, no additions, (+)-rhTGF-p, (5 ngiml). L, and L,, are the two core proteins generated from PG,.
with rhTGF-p, (50 ngiml) augmented the synthesis of this core protein.
DISCUSSION Stimulation of chondrocyte proteoglycan synthesis would be advantageous to cartilage after injury or other traumatic insult. In the early stages of osteoarthritis, abortive cellular proliferation and increased synthesis of proteoglycans and collagen has been reported (Malemud, 1985). However, these early events are not sustained. The possible failure of available growth factors to affect the outcome of the osteoarthritic process should be considered. It seems plausible that TGF-P is involved in cartilage responses to injury, since it has been extracted and purified from articular cartilage (Seyedin et al., 1986, 19881, and recent studies suggest a n autocrine feedback loop, whereby TGF-P upregulates the transcription of mRNA for TGF-P (Joyce et al., 1990; Malemud and Purchio, unpublished data). The mechanism for activation of the pre-ipro-form of TGF-P in cartilage remains to be elucidated but may involve proteolytic cleavage which has been shown to activate the pre-/pro-form of TGF-P (Gentry et al., 1988).A pH in the range 2.5-3.5, which can result in test-tube
activation of the precursor form of TGF-61 (Gelb e t al., 19901, seems less likely to occur in cell culture. This suggests that the only active form of TGF-PI came from rhTGF-P, and not a combined effect of TGF-p, activated in fetal bovine serum and rhTGF-6,. Gelb et al. (1990)could not find any detectable active TGF-P in the conditioned medium of growth plate chondrocytes maintained serum free for 24 h. In adult rabbit articular chondrocytes, purified TGF-P was previously shown to stimulate proteoglycan synthesis (Redini et al., 1988). The present studies revealed that the rhTGF-p, had a similar effect. The studies indicated that, in all likelihood, rhTGF-P, caused a n upregulation of core protein synthesis, which then became available for GAG chain initiation. It was noteworthy that rhTGF-P, stimulation of core protein synthesis was concentration-dependent. The L, form of core protein required a higher concentration of rhTGF-p, than did L,, to stimulate its synthesis. The apparent M, of 390 kD for L,, was in good agreement with the cell-free translation product from chick sternal cartilage mRNA immunoprecipitated with antisera to core protein (Vertel and Hitti, 1987) and the hydrogen fluoride-treated newly synthesized chick proteo-
TGF-6 STIMULATION OF CHONDROCYTE PROTEOGLYCANS
M,X
480,440 (dynein)
200 -
93 69 Fig. 5. Three -5% acrylamide gel electrophoresis of 135S1-methionine labeled D1 from the CAF of chondrocytes treated with rhTGF-p, (50 ngiml). D1 was digested with chondroitinase ABCikeratanase prior to electrophoresis. The L, form is the only core protein seen in the cell-associated (C) compartment. L,, is the expected migration position of this core protein.
157
drocytes and other cell types (Fisher et al., 1989; Melching and Roughley, 1989; Pulkkinen et al., 1990). We have, a t present, been unable to unequivocally characterize either of these molecules. Neither antidecorin antibody (Sawhney et al., 1991) nor antisera made against synthetic peptides derived from the amino acid sequences of DS-PG-I or DS-PG-I1 core protein (Fisher et al., 1989) immunoprecipitated the chondroitinase ABC-digested “small” rabbit proteoglycans. Significant differences may exist between the rabbit form of these “small” proteoglycans and “small” proteoglycans from other species. Although the molecular mechanism for the rhTGFP1 effect on rabbit chondrocyte proteoglycan remains unknown, a recent report sheds some light on how the cell cycle may play a role in this stimulation. TGF-6, increases the time chondrocytes spend in the interface between the S phase and mitosis (Vivien et al., 1990). We have previously shown that proteoglycans are synthesized by rabbit chondrocytes in both the G, and G, phase of the cell cycle (Malemud and Sokoloff, 1974). These results are noteworthy since the proliferative capacity of chondrocytes participating in repair of cartilage lesions may be directly related to their capacity to also synthesize extracellular matrix macromolecules. Thus, TGF-P, may act to stimulate proteoglycan synthesis only when early proliferative events consonant with the initial stages of attempts at cartilage repair after trauma cease or become less prominent.
ACKNOWLEDGMENTS This work was supported in part by grant AG-02205 from the National Institute on Aging (USPHS). LITERATURE CITED
glycan core protein (Campbell et al., 1990). Recent studies using rotary shadowing electron microscopy of chick core protein (Dennis et al., 1990) revealed a core protein size larger than predicted by a cDNA clone for the rat chondrosarcoma core protein (Doege et al., 1987). The larger form of core protein we have observed (> 480 kD) has not been previously described in chondrocytes, but large chondroitin sulfate-containing core proteins of this size or somewhat smaller (> 400 kD) have been reported in brain tissue (Gowda et al., 1989). It is not known at present whether or not the 480 kD core protein represents a n alternative spliced form of the large proteoglycan gene or a unique gene product. Other studies have reported that TGF-6, stimulates decorin synthesis in rat mesangial cells (Border et al., 1990). A recent report by Breuer et al. (1990) found no effect of TGF-P on decorin in human skin fibroblasts and a human osteosarcoma cell line. Using laser densitometric scanning, a distinct increase in the radioactive signal was seen in both of the 2 “small” proteoglycans. The larger of these 2 core proteins (PG-S,) migrated slower than PG-S, presumably due to more glycosaminoglycan stubs. It may be structurally similar to DS-PG-I (biglycan) synthesized by bovine chon-
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