Characterization of proteoglycans synthesized by rabbit articular chondrocytes in response to transforming growth factor-β (TGF-β)

Biochbnica et Biophysica Acta, 1093 (1991) 196-206 © 1991 Elsevier Science Publishers B.V. 0167-4889/91/$03.50 ADONIS 0i5748899100201R

196

BBAMCR 12960

Characterization of proteoglycans synthesized by rabbit articular chondrocytes in response to transforming growth factor-/3 (TGF-/3) Fran~oise Redini, Michelle Daireaux, Alain Mauviel, Philippe Galera, G6rard Loyau and Jean-Pierre Pujol Laboratoire de Biochimie &~ Tissu Conjonctif, CHU C~te de Nacre, Caen, France

(Received 30 October 1990)

Key words: Proteoglycans synthesis; Transforming growth factor; (Rabbit articular chondrocytes)

The effect of transforming growth factor-ll (TGF-fl, 1 n g / m l ) on proteoglycan synthesis by rabbit articular chondrocytes in culture was studied in the presence of fetal bovine serum. Exposure of confluent cells for 24 h to the factor resulted in a marked increase of asS-labeled sulfate incorporation in the newly synthesized proteoglycans (PG), as estimated by glycosaminoglycan (GAG) radioactivity ( + 58%). The onset was observed 6 h after addition of the factor but was significant after 12 h. TGF-fl also enhanced the uptake of laSS]sulfate by cbondrocytes, but had no effect on the release of PG by these cells. The effect of TGF-fi on the distribution of PG between the medium and the ceil layer was shown to be dependent on the serum concentration in the medium: the relative proportion of cell.layer associated GAG of TGF-~-treated cells decreased with increasing concentration of fetal bovine serum. The proportion of aggregated PC;, the hydrodynamic size of PG monomers and GAG chains were not modified by TGF-~, but the relative distribution of disaccharides 6- and 4-sulfate in GAG chains was altered by the factor:, the proportion of chondroitln 6.sulfate (C6S) was decreased while that of chondroitin 4-sulfate (C4S) was augmented in presence of TGF-~, leading to a decrease of the ratio C 6 S / C 4 S ( - 1 1 to -22%, P <0.01). The present study indicates that TGF.,8 promotes the synthesis of a modified extracellular matrix in cultured articular chondrocytes. This mechanism could be relevant to some aspects of cartilage repair in osteoarticular diseases.

Introduction Articular cartilage is a highly specialized connective tissue which consists of relatively few cells distributed throughout an abundant extracellular matrix. For an optimal macromolecular organization, special cartilaginous components have been developed during the course of evolution. These include collagen type !I, IX and X and the characteristic hyaluronate-proteoglycan complexes which are entrapped in a network of collagen fibers (reviewed Refs. in 1, 2). PG are important

Abbreviations: CPC, cetylpyridinium chloride; CS, chondroitin sulfate; DMEM, Dulbecco's modification of Eagle's medium; FBS, fetal bovine serum; GAG, glycosaminoglycan; KS, keratan sulfate; PBS, phosphate-buffered saline; PG, proteoglycan; PMSF, phenylmethyl sulfonyl fluoride; TGF-j0, transforming growth factor-/3. Correspondence: F. Redini, Laboratoire de Biochimie du Tissu Conjonctif, CHU C.6te de Nacre, Niv. 3, 14033 Caen Cedex, France.

components of the extracellular matrix of articular cartilage. Their ability to bind water molecules [3,4] plays a major role in the swelling pressure of articular cartilage. Cartilage PG monomers are large macromolecules (molecular mass (1-4) 10 ~ Da) in which chondroitin 4- and 6-sulfates and keratan sulfate (KS) are linked to an extended core protein of estimated molecular mass 200 kDa [5-7]. This latter represents 5-15% of the total molecule, the remainder consisting of GAG chains and oligosaccharides. In articular cartilage, chondroitin sulfate is the predominant GAG, with an average chain size of 20 kDa. More than 85% of the cartilage PG are found as large macromolecular aggregates formed by interaction of monomers with hyaluronic acid (HA) [8]. However, two forms of small interstitial PG, containing dermatan sulfate and keratan sulfate chains linked to a core protein of about 40 kDa, have recently been extracted from a variety of connective cell cultures including articular chondrocytes [9-11].

197 In inflammatory joint diseases, such as rheumatoid arthritis, depletion of PG is an early feature that leads to alteration of the biophysical propc~ies of that tissue [12]. The cartilage breakdown is also currently present in degenerative osteoarticular pathologies, albeit the process is not inflammatory and more progressive in that case [13,14]. Cytokines such as interleukin-1 and tumor necrosis factor-a have been implicated in these matrix alterations since they trigger the chondrocytes to release degrading metalloproteinases [15,16]. However, some evidences indicate that in joint diseases, specially in degenerative pathology, a repair process may take place as a result of activated chondrocyte metabolism [17]. It is therefore tempting to speculate that systemic or local growth f:~ctors could play a role in the sequence governing the deposition of newly synthesized PG into the cartilage extracellular matrix. Outstanding among these factors is transforming growth factor type /3 (TGF-Il), a disulfide-linked dimeric peptide of 25 kDa which was originally found in neoplasic cells and then in non-neoplasic tissues [18]. Recent studies have shown that TGF-t exerts a number of regulatory activities on growth and differentiation of cells (reviewed in Ref. 19). TGF-t is present in bone, from which it has been extracted and de w scribed as CIF-A ('cartilage-inducing factor A') and induces chondrogenic differentiation of embryonic rat muscle cells [20]. It has also been reported that chondrocytes possess TGF-I mRNA [21]. Furthermore, TGF-/3 has been detected by immunohistochemistry in great amount in both chondrocytes in the articular cartilage and osteocytes in the bone [22]. During the last few years, T G F - t has been found to stimulate the synthesis of extracellular matrix macromolecules in many cell lines of several origins [23]. Recently, we demonstrated that TGF-Il could enhance the biosynthesis of collagen and proteoglycans in cultured rabbit articular chondrocytes [24]. It was of interest to extend this preliminary quantitative study and characterize the PG synthesized by the chondrocytes in response to TGF-18 in order to determine whether this factor could affect the phenotype of cartilage PG. Materials and Methods

Materials Dulbecco's modification of Eagle's essential medium (DMEM) and fetal bovine serum (FBS) were obtained from Serva (Heidelberg, F.R.G.). Collagenase and trypsin used for tissue dissociation were from Sigma (St. Louis, MO, U.S.A.). Guanidinium chloride, cetylpyridinium chloride, EDTA, 6-aminohexanoic acid, benzaminidium chloride, thin layer chromatography plates (HPTLC aluminium sheets) were purchased from Merck (Darmstadt, F.R.G.). Phenylmethylsulfonyl fluoride (PMSF), N-ethylmaleimide,

proteinase type XXV from Streptomyces griseus, chondroitinase ABC from Proteus vulgaris, keratanase from Pseudomonas species, chondroitin sulfate type A from bovine trachea, chondroitin sulfate type C from shark cartilage and hyaluronic acid from human umbilical cord were also purchased from Sigma. 4,5 unsaturated standards (A Di-0S, A Di-4S and A Di-6S) were obtained from Seikagaku Kyogo Co. (Tokyo, Japan). D[1-3H]Glucosamine (11 Ci/mmol; 0.40 TBq/mmol) was purchased from CEA (Saclay, France). [aSS]Sulfate (25 Ci/mg; 0.9 TBq/mg) came from Amersham (U.K.). Sepharose CL 2B, CL 6B and DEAE-Sephacel were from Pharmacia Fine Chemicals (France). Transforming growth factor-ill purified from human platelets was supplied by R and D systems (Mineapolis, MN, U.S.A.) and dissolved in 4 mM HCI containing 1 mg/ml bovine serum albumin.

Chondrocyte culture Articular cartilage slices were taken from the shoulders and the knees of 3-week-old rabbits. Chondrocytes were obtained by enzymatic dissociation [25] and cultured in DMEM supplemented with glutamine (2 raM), penicillin (100 IU/ml), streptomycin (100 /zg/ml), fungizone (0.25/zg/ml) and 10% FB5 (heatinactivated for 30 min at 56 ° C). The cells were grown at 37°C in a 5% CO 2 environment with medium change at 2-3 day intervals~ Depending on the experiment, cells were plated in Petri dishes (9.6 cm 2) at 2-105 cells/well, in 25 cm 2 Falcon flasks at 5.105 cells/flask or in 75 cm 2 Falcon flasks at 1.5" 106 cells/flask. After reaching confluem~ (5-7 days), the primary cultures were used for experiments in order to avoid dedifferentiation of the chondrocytes. Such cultures were previously shown to produce both collagen type II [26,27] and proteoglycans specific of cartilage [28,29].

Time-course labeling For kinetic experiments, when cells became confluent the culture medium was replaced with control medium (10% FBS-containing DMEM) containing TGF-/3 (1 ng/ml) and [35S]sulfate (5/zCi/ml), and the incubation was stopped at 1, 2, 4, 6, 12, 24 and 48 h. Cell layers were washed three times with phosphatebuffered saline (PBS). Samples of both medium and cell layer were treated with pronase as described under 'Assay of total labeled glycosaminoglycans'.

Uptake experiments with [ ~SS]sulfate These were performed in serum-containing medium at different time periods (0.25, 0.5, 1, 2, 4, 6 and 24 h) following addition of 5/zCi/ml [aSS]sulfate. After removing the medium and rinsing the cell layer three times with PBS, 1 ml of 0.2% Triton X-100 prewarmed at 70 ° C was added. The suspension plus a rinse with 1

198 ml Triton X-t00 solution was sonieated (30 s) and treated with 4 vol. of ethanol to precipitate the macromolecules. After centrifugation (1 h, 12000×g) and washing of the pellet with ethanol/0.15 M NaCI (80:20, v/v), the radioactivity of the supernatant was estimated by scintillation couating (free intracellular pool of [3SS]sulfate), whereas the pellet was treated as described under 'Assay of total labeled glycosaminoglycans' ([35S]sulfate bound to GAG).

Pulse-chase labeling Spent medium was replaced with fresh medium containing 20/~Ci/ml of [35S]suifate with or without TGF(1 ng/ml) and 'pulse' incorporation was allowed for 2 h. The radiolabeled medium was then removed and the cell layer washed gently with pre-warmed control medium. Medium containing or not TGF-/3 was added and cultures were incubated for various 'chase' periods (0.5, i, 3, 6, 12 and 24 h). The amount of radiolabeled GAG in both medium and cell layer was determined as described under 'Assay of total labeled glycosaminogly¢~fl.S'.

Effect of FBS on the TGF-IJ-induced stimulation of GAG synthesis The medium of confluent cultures was replaced by DMEM containing 0, 2, 5 and 10% FBS, supplemented with 5 ~ C i / m l [sSS]sulfate in presel:ee or absence of TGF-~8 (1 ng/ml). After 24 h, the cells were washed three times with PBS and samples of both medium and cell layer were treated with pronase as described below.

Assay of total labeled glycosaminoglycans Total amount of labeled GAG was estimated by precipitation of media and cell extracts with cetylpyridinium chloride (CPC) [30]. Briefly, at the end of incubation, the media were removed, the cell layers washed three times with PBS at 37"C and the washes were added to the media. The cell layers and 1 ml of medium samples were separately digested with pronase (I mg/ml) for 24 h at 55 °C. GAG were precipitated by addition of CPC (1%, w/v) in presence of carriers (HA, C4S and C6S, 100/~g/'ml) allowing the mixture to stand for 1 h at 37 ° C. After centrifugation (15 rain, 10000 × g), the pellet was dissolved in 0.8 ml of 2 M MgC! 2 and precipitated again with 5 vol. of ethanol for 16 h at 4"C. The final pellet was dissolved in 0.5 ml of 75 mM NaCI and the radioactivity of an aliquot was assayed by liquid scintillation counting in a Betamatic counter (Kontron, France), using ACS scintillating mixture (Amersham, U.K.).

Gel filtration of PG aggregates In order to study the effect of TGF-/3 on the proporfon of PG eluting as aggregates, 25 cm 2 flasks (in

triplicate) of primary confluent articular chondroeyte cultures were labeled for 24 h with 2.5 /zCi/ml [3H]glucosamine and 5/~Ci/ml [35S]sulfate in presence or in absence of TGF-/3 (1 ng/ml). Then the medium was removed and dialyzed against 0.5 M sodium acetate pH 6.8 containing protease inhibitors (EDTA: 50 raM, PMSF: 1 raM, N-ethylmaleimide: 10 raM, 6aminohexanoic acid: 100 raM, benzamidinium chloride: 10 raM) for 24 h at 4 ° C. Cell-layer associated PG were extracted with 0.05 M sodium acetate (pH 5~8), containing 4 M guanidinium chloride and proteinase inhibitors for 16 h at 4 ° C [31]. The cell extracts were then dialyzed as described above and the samples were subjected to size-exclusion chromatography in associative conditions on a Sepharose CL 2B column (1 × 56 era, flow rate 6 m l / h , 0.4 ml fractions) equi!ibrated and eluted with the same buffer. The radioactivity was determined in all fractions.

Ion-exchange chromatography of labeled PG 75 cm e flasks of confluent cultures v.,ere labeled for 24 h with [3H]glucosamine (2.5 /LCi/ml) and [3SS]sulfate (5 /~Ci/ml), with or without TGF-/3 (1 ng/ml). Then the medium was dialyzed against 0.1 M Tris-HCi, 7 M Urea buffer (pH 7.5), containing proteinase inhibitors for 24 h at 40C. The cells were washed twice with 1 mi of PBS and the cell-layer associated PG were extracted and dialyzed as described above. The dialyzed samples from the culture medium and cell extracts were then applied to a DEAE-Sephacel column (T × 10 can) equiiiLr~ted with 0.1 M Tris-HCl, 7 M Urea buffer (pH 7.5). After loading, the column was washed with 15 ml of this buffer, followed by elution with a linear gradient of 0-1 M NaCI in 0.1 M Tris-HCl, 7 M Urea buffer (pH 7.5). A total volume of 100 ml of eluting buffer was passed through the column at a flow rate of 10 ml/h. Fractions (0.4 ml) were collected and radioactivity determined on all samples.

Gel filtration of PG monomers One aliquot of the pooled and concentrated PGcontaining fractions from the ion-exchange chromatography was dialyzed against 0.5 M sodium acetate, 4 M guanidinium chloride buffer (pH 6.8), containing protease inhibitors. Samples were then subjected to sizeexclusion chromatography under dissociative conditions on a Sepharose CL 2B column (1 × 56 cm, flow rate 6 ml/h, 0.4 ml fractions) equilibrated and eluted with the same buffer. The radioactivity was determined in all fractions.

Characterization of GAG chains An .other aliquot of PG from ion-exchange chromatography was dialyzed against sodium acetate buffer and samples were precipitated with 5 vol. of ethanol

199 for 16 h at 4 °C in presence of carriers. The pellet obtained after centrifugation (15 min, 10000 × g) was dried and subjected to alkaline borohydride treatment [32]. Free GAG chains were en~nymatically digested as follows: aliquots obtained after borohydride treatment were dialyzed against distilled water and lyophilized. The samples were then dissolved in 50/zl of 0.05 M Tris-HCl/0.05 M NaCI/0.06 M sodium acetate/0.001 M sodium fluoride (pH 8.0). The samples were incubated with chondroitinase ABC or keratanase (0.05 U) for 4 h at 37 ° C; then 0.025 unit of respective enzymes was further added and the ~eaction pursued for another 20 h at 37 ° C [33]. Starting material and respective digested samples were chromatographed on a Sepharose CL 6B column (1 x 50 ¢m, flow rate 6 ml/h~ 0.4 ml fractions) equilibrated and eluted with 0.5 M sodium acetate (pH 7) containing protease inhibitors.

GAG production by articular chondrocytes in a dosedependent manner within the range 0.1 to 10 ng/ml [24]. Since 1 n g / m l of the factor led to a significant increase of the PG synthesis, this concentration was chosen for the present study. In 10% FBS-containing DMEM, TGF-/~ increased the total amount of newly synthesized GAG over a 48 h-period (Fig. 1A). The effect was present at 12 h after the addition of the factor. Then, the total incorporation in TGF-/i-treated cells was regularly enhanced, reaching twice the control value after 48 h. The distribution data show that TGF-/3 augmented the GAG content in both culture medium (Fig. 1B) and cell-layer associated (Fig. 1C) compartments, but the effects ef the factor on the cell-layer GAG fraction was significant only after 48 h of exposure. The stimulation of GAG synthesis by TGF-/~ cannot be due to the enhancement of cell proliferation since the growth factor had no effect on either the cell

Disaccharide composition of the GAG chabzs

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Aliquots of samples digested with chondroitinase ABC were lyophilized and dissolved in H~O. Samples were chromatographed on HPTLC cellulose sheets (20 x 20 cm) in presence of A Di-4S, d Di-6S and A Di-0S as references [34]. ChondroRinase-resistant radioactivity accounted for about 5-8% of the material chromatogtaphed on HPTLC cellulose sheets. The spots were cut up and eluted in counting vials with 0.4 ml of 0.01 M HCI for 12 h at 60 °C and radioactivity determined after addition of 4 ml of scintillation fluid.

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Statistical analysis Results were generally expressed as mean ± S.E. of triplicates. The Student's t-test was used to compare the r~eans from different groups of experiments.

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In our preceeding report [24], experiments were performed under serum-free conditions during the incubation period with TGF-/3 in order to reduce interference with growth factors or hormones from the serum and to prevent any binding of TGF-~ to serum proteins [35]. However, the synthesis of PG was considerably less than in the presence of serum and led to PG with molecular properties different than those of the cartilage components, as already reported by Malemud and Papay [36]. Therefore, we performed the present study in serum-containing medium.

Effeo" of TGF-I3 on the time-course synthesis of GAG Kinetic experiments were carried out in order to know whether TGF-/3 could influence the incorporation of [3SS]sulfate as a precursor of GAG biosynthesis. We previously reported that TGF-/3 enhanced the total

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Fig. 1. Effect of TGF-~ on the time-course synthesis of glycosaminoglycans. Confluent cultures of articular chondroeytes were incubated in presence (e) or in absence (0) of TGF-~ (1 n g / m b in medium containing [35S]sulfate (5 t~Ci/ml). GAG radioactivity was estimated as [35S]sulfate-labeled material precipitated by CPC as described in Material and Methods, in the culture medium (B) and in the cell-layer associated (C) material. A =: total amount (B + C). The bars represent S.E. values (n = 3). ** P < 0.01; *** P < 0.001.

200 protein content or the proliferative rate after a 24 h-treatment under the present conditions, i.e., confluency and presence of 10% FBS (results not shown). In such case, we never observed change in cell number over 24 h of incubation. This is different from our previous study, perfonned in absence of serum, where TGF-/3 generally produced a slight increase of cell number after 24 h of incubation.

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Experiments were performed in order to study the effect of TGF-/~ on the kinetics of [35S]sulfate uptake by the chondrocytes. Ethanol-soluble and ethanol-insoluble cellular extracts were examined as respective free-intracellular pool of the radiolabeled precursor (Fig. 2A) and macromoleeule-bound fraction (Fig. 2B). The intraceilular level of free [35S]sulfate reached a plateau at 3 h and the value remained constant up to 24 h, indicating that the pool was rapidly stabilized. TGF-//significantly increased the uptake as early as 6 h after the beginning of the experiment and the level of radioactivity remained 50% higher than in control cultures. The incorporation of [35S]sulfate into GAG was greatly stimulated in presence of TGF-~ (Fig. 2B) from 12 to 24 h, with a 50% augmentation at the end of the experiment.

To determine whether TGF-/3 could affect the processing of newly synthesized PC] in these cultures, pulse-chase experiments were carried out with radiolabeled sulfate. No significant difference could be detected in the release of GAG between control and treated-cultures whenever TGF-/~ was absent (Fig. 3A) or present during the pulse period (Fig. 3B). It was concluded that TGF-/3 did not affect the rate of intracellular processing and subsequent release of the newly synthesized PG of chondrocytes.

Effect of FBS on the TGF-~-induced stimulation of GAG synthesis As fetal bovine serum contains several growth factors which could interfere with the effect of TGF-/~ on GAG production, we performed experiments with increasing concentrations of FBS in the culture medium and estimated the total amount of [35S]sulfate-labeled GAGs produced over 24 h in such conditions. Fig. 4 illustrates the effect of 0, 2, 5 and 10% FBS on the TGF-/]-induced increase of total (Fig. 4A), medium (Fig. 4B) and cell-layer associated GAG (Fig. 4B) production. Fig. 4A shows that TGF-/3 exerted its enhancing effect on total GAG production whatever the concentration of FBS present in the culture medium: + 51,

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:~ect of TGF-/Y on [35S]sulfate uptake. The effect was

estimated at different time periods following addition of 5 #Ci/ml [ 3SS]sulfate. The medium from control cultures (0) or TGF-/3-treated cells (e) was removed and the cell layer was washed three times with PBS. Then the intracellular-free [3sS]fraction (A) and the fraction of ~5S incorporated into macromolecnles (B) were prepared as described in Material and Methods. The bars represent S.E. values (n = 3). ** P < 0.01; *** P < 0.001.

+38, +49 and +32% for 0, 2, 5 and 10% FBS respectively. However, there was no more difference in the amount of cell-layer associated GAG between controls and TGF-//-treated cells in 10% FBS-containing medium (Fig. 4C). This experiment has been repeated four times and gave the same pattern of results. The distribution of GAG among the medium and the cell layer was significantly changed; in TGF-//-treated cultures, the proportion of medium GAG increased from 78% in the absence of FBS to 83% in presence of 10% FBS (P < 0.01), whereas in control cultures the proportion of medium GAG was only slightly modified: it represents about 75% of the total GAG both in serum-free and 10% FBS-containing medium.

201 151

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Effect of TGF-fl on the charge density of newly synthesized PG Fig, 6 shows elution profiles on DEAE-Sephacel column of medium and cell-layer associated PG from control (A and C) and TGF-fl-treated cultures (B and D). Glycoproteins were eluted with the isocratic buffer in the first peak whereas sulfated PG were eluted at 0.5 M NaCI. Medium samples from cultures exposed to TGF-/3 eluted with a pattern similar to that of controls, indicating that the factor did not affect the overall charge density of the PG (Fig. 6A and B). TGF-fl did not modify either the charge density of cell-layer asso-

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Fig. 3. Effect of TGF-fl on the secretion of [35S]sulfate-labeled glycosaminoglycans. Confluent cultures of chondrocytes in 9.6 cm 2 dishes were pulsed in the absence (A) or presence (B) of TGF-fl in medium supplemented with [35S]sulfate (20/zCi/ml) for 2 13. In both cases the cells were then ci~ased in cold control medium (o) or in TGF-/3-comaining medium (e). The radioactivity remaining in the cell layer was determined as CPC-precipitable material. Data represent the mean_+ S.E. (n = 3).

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Effect of TGF.fl on the amount of aggregated PG In the culture medium, the [3H]glucosamine- and [35S]sulfate-labeled material eluted as one peak, near the void volume of the column, representing the aggregated PG (Fig. 5A). This peak eluted at the same position of large cartilage proteoglycans. TGF-/3 treatmerit did not influence the aggregation of these PG since no smaller, more-retarded, peak was seen after incubation with the factor (Fig. 5B). Cell-layer associated PG eluted mainly as aggregates at the same position as those of the medium (Fig. 5C). As can be seen on Fig. 5D, TGF-/3 did not alter the proportion of PG eluting as aggregates. We further isolated and purified PG by ion-exchange chromatography on a DEAE-Sephacel column before carrying out studies on the hydrodynamic size of PG monomers and GAG chains.

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FBS ('1,) Fig. 4. Effect of FBS on the TGF-,B-iuduced stimulation of glycosaminoglycan synthesis. Confluent cultures of articular cho~drocytes were incubated for 24 h in culture medium containing various concentrations of FBS, in the presence ( B ) or absence ( l I ) of TGF-/3 (1 ng/ml). GAG radioactivity was estimated as [35Slsulfatelabeled CPC-precipitated material in the medium (B) and the cell layer (C). (A) total amount (B + C). The bars represent S.E.M. values ( n = 3 ) . ** P<0.01; *** P <0.001.

202 A

matography were pooled, concentrated, dialyzed against 0.5 M sodium acetate/4 M guanidinium chloride buffer (pH 6.8) and subjected to gel-filtration on a Sepharose CL 2B column equilibrated with the same buffer. Elution profiles of PG monomers from the culture medium of chondrocytes treated (Fig. 7B) or not (Fig. 7A) with TGF-/] showed that the PG were eluted as a predominating peak at Kav= 0.2 followed by some

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ciated PG as shown in Fig. 6C and D. It could be only noted that TGF-/3 produced a change in the relative distribution of PG: the amount of medium PG was increased whereas that of cell-layer associated PG remained the same, confirming the data from Fig. 4B and C

Effect of TGF-IJ on the relative hydrodynamic size of PG monomers To test for an effect of TGF-/3 on monomer size, PG-containing fractions from the ion-exchange chro-

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40 60 80 Fraction number Fig. 6. Effect of TGF-/3 on the charge density of proteoglycans in the medium (A, 13) and in the cell layer (C, D). Double labeled glycoconjugates isolated from chondrocyte cultures incubated without (A and C) or with (I~ and D) TGF-/3 (1 ng/ml) were applied to DEAE Sephacel column (1 x 10 cm) in 0.1 M Tris-HCI, 7 M urea (pH 7.5). A 100 ml linear gradient of 0-1 M NaC! was superimposed starting at fraction 15. 400 /zl aliquots were taken from the fractions for scintillation counting, o, [3H]glucosamine; e, [35S]sulfate.

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Effect of TGF-/3 on the relative hydrodynamic size of GAG chains PG macromolecules from medium and cell layer were subjected to alkaline borohydride treatment and subsequent digestion by chondroitinase ABC and keratanase. The samples were then analyzed by Sepharose CL 6B column chromatography. GAG chains from culture media were eluted as one peak at Kay = 0.47 (Fig. 8A). The elution profiles of the digestion products are presented on the same fig-

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i 0.4 0.6 O.B 1 rov Fig. 7. Effect of TGF-//on the relative hydrodynamicsizeof proteoglycanmonomersin the medium(A, B) and in the cell layer(C, D). PG isolated on DEAE-sephacelwere subjected to size exclusion chromatography in dissociativeconditions on a Sepharos¢CL 2B columnelutedwith 0,5 M sodiumacetatebuffer (pH 6.8),containing 4 M guanidinihmhydrochloride.A, C; control cultures;B, D, TGF0

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polydispersity. TGF-fl increased the total amount oi PGs in that fraction but did not affect the hydrodynamic size of these monomers since the elution profile was not modified under TGF-/~-treatment (Fi~. 7B). Cell-layer associated PG were smaller in size than secreted PG as reflected by their elution at Kay = 0.5 on a Sepharose CL 2B column (Fig. 7C). Here again, the hydrodynamic size of PG monomers was not affected by TGF-/~ treatment as shown on Fig. 7D.

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0.4

0.6 0.8 Kov Fig. 8. Effect of TGF-/3 on the relative hydrodynamicsize of glycosaminoglycan chains in the medium (A, B) and in the cell layer (C, D). The radiolabeied GAG chains were obtained by alkaline borohydride treatment of PG isolated on DEAE-Sephacel (Fig. 6). One aliquot was digested by keratanase (o), another by the chondroitinase ABC (n).The starting material (o) and the digestion products were subjected to chromatography on a Sepharose CL 6B column (1 x50 cm). A, C, control cultures; B, D, TGF-//-treated culture.

204 ure. They showed that GAG chains were mainly composed of chondroitin sulfate chains since the [35S]sulfate radioactivity associated with the initial peak was then eluted at Kay = 0.75 after chondroitinase ABC treatment, whereas no change was seen after keratanase treatment. The composition of the medium GAG chains was not altered by TGF-/3 treatment as shown in Fig. 8B. In cell extracts, GAG were eluted as a single peak at Kay = 0.57 (Fig. 8C) and were also composed of chondroitin sulfate chains. In the presence of TGF-/3, the elution profiles of the GAG chains and of the digestion products on Sepharose CL 6B were similar to those observed in control cultures (Fig. 8D). The slight difference in eluting position of GAG chains from medium and cell layer fractions may contribute to the smaller size of monomers observed in the cell-layer associated PG (Fig. 7C). However, the results indicate that TGF./3 did not modify the molecular weight and the overall composition of GAG chains either in the culture medium compartment or in the cell layer fraction.

Analysis of GAG chain composition To further study the chemical composition of GAG chains produced in presence of TGF-/3, we analyzed by thin-layer chromatography the radioactive disaccha-

TABLE !

Effect of TGF. [J on the Di-4S and Di,6S composition of glycosamino-

~6'ca~,chai,s

The radiolabeled GAG chains were treated by chondroitinase ABC and the released disaccharides were separated on thin-layer chromatography as described in Materials and Methods. Radioactivity of the spots was d~termined by liquid scintillation after elution. Results from four different experiments are presented as % of total ['~S]sulfate incorporation,

Control

TGF-/3

6S/4S

66,75 33,25 2

63.95 36.05 1.77(- 11%)

Expt, II Di-6S Di-4S 6S/4S

73.2 26,8 2,73

70.25 29.75 2.36( - 14%)

Expt. Ill Di,6S Di-4S

66,8 33,2

2,01

63.45 36.55 1.74(- 14%)

66.1 33.9 1,95

60.25 39.75 1.52(- 22%)

Expt, I Di-6S Di-4S

6,$/4,$

Expt. IV Di-6S Di-4S 6S/4S

rides obtained after chondroitinase treatment. As the relative proportion of KS and chondroitin O sulfate were not significantly modified under TGF-fl treatment, only the relative proportion of Di-6S and Di-4S in four independent experiments are shown in Table I. Despite of some variability between experiments, it is clear that TGF-13 produced a significant decrease of the ratio Di-6S/Di-4S in the newly synthesized proteoglycans, varying from 11% to 22% of the control value. This was due to a reduction in the amount of Di-6S and a concomitant augmentation of the Di-4S level (not shown). Discussion

5i~ce TGF-/3-1ike peptides have been shown to be abundant in cartilage as well as in bone [21,22], it was of interest to characterize the PG synthesized by articular chondrocytcs under TGF-/3 treatment to know whether this factor could modify the macromolecular organization of the cartilage matrix. Results presented here show that TGF-/31 may influence the expression of the chondroitin sulfate PG synthesized by rabbit articular chondrocytes in culture. We previously reported that TGF-/3 stimulated in a dose-dependent manner the production of GAG by chondrocytes cultured in serum-free conditions [24]. In the present study performed in the presence of serum to favor the synthesis of PG specific of articular cartilage, we found that this factor at 1 ng/ml increased the total GAG production by about 58%. It has been reported that some TGF-~ could bind to a2-macroglobulin (a2-M) present in the serum [35]. Nevertheless, the factor did show activity on GAG synthesis under our experimental conditions. However, it is interesting to note that in presence of 10% FBS we observed that the distribution of the newly synthesized PG was apparently modified by TGF-/3: in that case, the GAG content of the cell-layer associated fraction was not changed, compared to controls so that the ratio of medium/cell-layer associated GAG was increased by approx. 25%. These data contrast with our preceeding study showing that the distribution of PG was not affected by TGF-/3 in the absence of serum [24]. This phenomenon is probably related to the maturation process that affects the PG after release from the chondrocytes. Indeed, it has been shown that PG monomers synthesized by rabbit articular chondrocytes in primary culture are secreted in a form in which the HA binding domain is not maximally functional. Conversion of this form to one of higher binding affinity is achieved by maturation in the cell layer matrix of cultures or by addition of HA to the purified monomers [37]. It may be postulated that this cell layer maturation process of PG could reach its maximum level when

205 a limiting concentration of serum factor(s) is attained. As an alternative, some structural or quantitative changes may affect the cell surface proteoglycans or adhesion proteins [38] leading to altered binding with the other matrix components. Work specially devoted to the study of the pericellular proteoglycan fraction is presently in progress to get insight into this question. In the present study, TGF-/3 was shown to increase sulfate uptake. We do not know what could be the mechanism involved in this ~,io~ss but it is worth noticing that it has been already reported that TGF-/3 was able to stimulate glycolysis and uptake of nutrients such as amino acids [39] and glucose [40] in several fibroblastic cells. It is not clear whether this elevation of intracellular sulfate pool may contribute to the stimulation of PG synthesis by TGF-/3 since a number of precursor steps possibly influence the biosynthesis of GAG portions of chondroitin sulfate PG, including the amount and specific activity of the sulfate donor PAPS (3'-phosphoadenylphosphosulfate) [41]. Our results are in agreement with those obtained on rabbit and chick growth-plate chondrocytes, showing that TGF-/~ potentiated the GAG synthesis [42,43]. Nevertheless, the culture conditions may apparently influence the effect of TGF-/~ on the production of GAG by chondrocytes since it has been reported that rabbit articular chondrocytes cultured in agarose respond to TGF-~ by a decrease of GAG synthesis [44]. However, in that case, the cultures were exposed to TGF-B for 7 days and the assay was then performed after .mother 7 days. It must be noted also that the results, obtained by the Aician Blue-dye method, only display small differences between treated and control cultures. The present data are also consistent with the reported stimulative effect of TGF-/3 on PG synthesis in bovine articular cartilage organ culture [45]. However, none of the preceeding studied addressed the question whether TGF-/3 influences the structure and composition of newly synthesized PG. We demonstrate that TGF-/3 did not modigg the molecular size of the PG monomers nor that of the chondroitin sulfate chains. The factor did not induce reduction of aggregating potentiality of these proteoglycans, as judged by gelfiltration on Sepharose CL-2B. By contrast, Bas~ols and Massagu6 [46] have reported that TGF-/3 induced an increase in the molecular size of the GAG chains of the chondroitin/dermatan sulfate small PG synthesized by NRKo49F cells. Moreover, a recent report indicates that TGF-/3 may induce differential regulation of the biosynthesis of individual members of the small proteoglycan family in a human osteosarcoma cell line [47]. The authors demonstrate that the synthesis of PG-I was stimulated by TGF-/3 whereas that of PG-II was not. Although TGF-/3 increased the production of the main specific chondroitin sulfate PG in our

chondrocyt~ cultures, we cannot rule out that some differential effect of the factor could be exerted on synthesis of the or cell-associated PG present in articular chondrocytes [9]° The most interesting result obtained here was the TGF-/3-induced modification of polysaccharidic chain composition: the relative proportion of chondroitin 6sulfate was decreased, whereas that of chondroitin 4-sulfate was augmented. These effects led to a diminution of the ratio Di-6S/Di-4S in GAG chains in the presence of TGF-fl. These data are consistent with a recent report indicating that TGF-/3 also decreased the ratio Di-6S/Di-4S from 2.1 to 1.1 in the cell-layer associated GAG of rabbit growth-plate chondrocytes [48]. Interestingly, this evolution contrasts with the situation which is generally observed in ageing human adult cartilage, where an increase in 6-sulfation relative to 4-sulfation has been reported [49]. Similarly, cultured chondrocytes from cartilage undergoing matrix disorganization were shown to synthesize PG which are more undersulfated and carry most of the sulfate at the carbon-6 position [50]. The mechanism underlying the TGF-/3-induced decrease of Di-65/Di-4S ratio is not clear. This effect could be interpreted as indicating that Di-4S were synthesized much more rapidly than Di-6S but the possible causes for these qualitative changes are not well understood. Biosynthesis of sulfated PG is known to be a complex process involving several post-translational steps and sequential enzymatic additions. It is not possible to relate precisely these TGF-/3-induced changes in the Di-6S/Di-4S ratio of newly synthesized PG to some biosynthetic step. It seems unlikely that the effect could be the result of a differential action on each of the specific sulfotransferases responsible for addition of sulfate either in position 4 or position 6 [51]. Whatever the mechanism underlying the changes produced by TGF-/3 on the sulfation of chondrocyte PG, we cannot yet appreciate the conformational modifications they could cause in the PG molecules and the resulting functional changes they may lead to. Even if the in vitro data presented here cannot be extrapolated to the in situ context, they may help understanding the role played by TGF-/3 on the chondrocyte metabolism. The stimulating effect produced by the growth factor on the matrix synthesis may be relevant to cartilage repair attempts observed in osteoarthritis. For example, TGF-/3 may be released in response to the early injury of cartilage and trigger the chondrocytes to produce new extracellular matrix. Moreover, we may speculate that the factor may also originate from the bone matrix, especially in the later stages of the disease when endochondral bone is degraded. However, further research is required to better appreciate the eventual role of TGF-/3 in this reparative process.

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