266

Btochimtca et Btoplo'stca Aeta. 1051 I 1990l 266 275 I~lsevicr

BBAMCR 12642

Production of collagens, collagenase and collagenase inhibitor during the dedifferentiation of articular chondrocytes by serial subcultures V6ronique Lefebvre, Chantal Peeters-Joris and Gilbert Vaes Laboratoire de Chimie Physiologique (Connective Tissue Group), Uni~ersit~ de Louvain and lnternattonal Institute of Cellular and Molecular Pathology, Brussels (Belgium)

(Received 26 June 1989)

Key words: Chondrocyte dedifferentiation; Collagen synthesis: Collagenase; lnterleukin l; Tissue inhibitor of metalloproteinase: (Rabbit cell)

Rabbit articular chondrocytes were cultured in monolayer and the progressive loss of their differentiated phenotype was monitored from passage to passage. The cell densities achieved in confluent cultures decreased abruptly between the primoculture and the second or third subculture, and more slowly thereafter, reflecting parallel morphological changes. The synthesis of collagen (but not that of other proteins) decreased sharply, and a smaller proportion of collagen was incorporated into the matrix. Cells in primocuiture synthesized mainly the cartilage-specific collagens, types II and XI, which were mostly deposited in the matrix, but no type I nor |II collagen. With increasing passages, the synthesis of type II collagen decreased progressively while that of types I and III collagens increased, the latter being almost completely released in the culture medium. Simultaneously, the production of type XI collagen was apparently switched to that of type V. Fully differentiated confluent chondrocytes in primoculture produced the collagenase inhibitor TIMP (tissue inhibitor of metalioproteinases) but no detectable procoilagenase; their production of procollagenase was, however, induced by interleukin 1. The production of TIMP increased from passage to passage. A spontaneous production of procollagenase was only occasionally observed in confluent cultures of dedifferentiated chondrocytes. However, interleukin 1 induced an always higher production of procollagenase from dedifferentiated chondrocytes than from cells in primocuiture.

Introduction Although normally responsible for synthesizing the matrix of articular cartilage, chondrocytes can become activated to produce enzymes which degrade the collagens and proteoglycans of this tissue (reviewed in Ref. 1). This activation is thought to play a critical role in the joint destruction that occurs in arthritis. Therefore, unravelling the conditions leading to or accompanying such activation is of importance. In osteoarthritic cartilage, an increased production of collagenase has been reported [2] as well as the appearance of type I collagen, a sign of chondrocyte

Abbreviations: APMA, 4-aminophenylmercuric acetate; DMEM, Dulbecco's modified Eagle's medium; FCS, foetal calf serum; IL-1, interleukin 1; TIMP, tissue inhibitor of metalloproteinases. Correspondence: G. Vaes, Laboratoire de Chimie Physiologique, UCL 75.39, Avenue Hippocrate 75, B-1200 Bruxelles, Belgium.

phenotype modulation (often called dedifferentiation) towards a fibroblastic phenotype [3,4]. Contrary to non-activated chondrocytes [5], fibroblasts are a particularly good source of collagenase and related tissue metalloproteinases, such as proteoglycanase (stromelysin) [6]. This suggests that the production of collagenase by chondrocytes could be linked to their dedifferentiation. To investigate this hypothesis, we cultured rabbit articular chondrocytes in monolayer for several passages, allowing the progressive loss of their differentiated phenotype [7]. This was monitored by fluorographic visualization of the collagen type chains produced by the cells after their electrophoretic separation, and by quantitative evaluation of the collagen and non-collagenous proteins produced. In parallel, both the spontaneous and IL-l-induced [8] chondrocyte productions of procollagenase were evaluated. Because of its potentially important regulatory role on the activity of collagenase [9], the production of the collagenase inhibitor T I M P was also recorded. New assay procedures

0167-4889/90/$03.50 ,~5 1990 Elsevier Science Publishers B.V. (Biomedical Division)

267 were used, established [10] to improve the recovery of TIMP and the validity of the enzymatic assay of procollagenase in crude culture media which contain both procollagenase and TIMP. Our study provides an overview of both quantitative and qualitative changes occurring in collagen production during the chondrocyte dedifferentiation. It shows that these changes are accompanied by enhanced production of TIMP and by increased IL-l-induced production of procollagenase. Materials and Methods

Materials Recombinant human IL-la (specific activity 108 U/mg) was a gift from Dr. P.T. Lomedico (HoffmannLaRoche, Nutley, N J, U.S.A.). Mouse bone-conditioned culture medium containing latent tissue procollagenase was kindly donated by J.M. Delaiss6 from our laboratory (see Ref. 10). DAPI (4',6-diamidino-2-phenylindole) was from Serva Feinbiochemica (Heidelberg, F.R.G.); L-[-2,3-3H]proline and 14C-methylated proteins, from Amersham Belgium (Brussels); 4-aminopropionitrile fumarate, from Janssen Chimica (Beerse, Belgium); calf thymus DNA, N-ethylmaleimide and purified bacterial collagenase (type VII), from Sigma Chemical, MO, U.S.A.); pepsin, from Boehringer, Mannheim (F.R.G.); z-ascorbic acid and ammonium hydroxide, from Merck (Darmstadt, F.R.G.); X-ray films, from Fuji Photo Film Company (Japan). Other special chemicals, culture plates and culture media were from suppliers previously mentioned [10].

Articular chondrocyte cultures Cartilage pieces from rabbit humeri, femurs, patellae and tibiae were freed from adhering other tissues by successive digestions at 37 °C with hyaluronidase (0.05% (w/v) for 10 min) and trypsin (0.25% (w/v) for 30 min) in phosphate-buffered saline. The pieces were then digested overnight with crude bacterial collagenase (0.15% (w/v) in DMEM supplemented with 10%, v/v, FCS). The dissociated chondrocytes were washed and cultured following procedures described for synovial fibroblasts [11], except that the trypsin solution used to disperse the cells at each passage was supplemented with 0.05% (w/v) bacterial collagenase. The chondrocytes were plated at (0.8-1.0)- 105 cells per cm2 culture dish and cultured in DMEM supplemented with 10% FCS; the medium was renewed once or twice a week. Subcultures were done at 7-day intervals and at a split ratio of 1 : 2 in order to allow the progressive dedifferentiation of the chondrocytes [7]. Cell densities were determined at the end of each experiment by assaying the DNA content of the cultures with the DAPI fluorimetric method [12]. Calf thymus DNA (50 pg/ml) was used as a standard and it was assumed that 1 0 6 cells contain 6 #g of DNA.

Synthesis of collagen and non-collagenous proteins The proteins synthesized by confluent chondrocytes at successive passages were labelled for 24 h with L-[2,3H3]proline (15 /~Ci/ml) in fresh DMEM supplemented with 10% FCS, 4-aminopropionitrile fumarate (100 /xg/ml) and ascorbic acid (100 /~g/ml). The culture medium was then removed and centrifuged to sediment the floating cells. The matrix and cell layer was dissolved in 0.25 M NH4OH and combined with the floating cells. The production of collagen and non-collagenous proteins was quantified according to Peterkofsky et al. [13]. After extensive dialysis against water, the samples were incubated for 90 min at 37°C in a 50 mM Tris-HC1 buffer (pH 7.5) containing 150 mM NaCI, 5 mM CaC12, 0.2 mg of NaN3/ml, 0.05% (v/v) Triton X-100 and 3 mM N-ethyl-maleimide, and with or without 20 /zg of purified bacterial collagenase/ml. Undigested proteins were precipitated by 10% (w/v) cold trichloroacetic acid in the presence of 1 mg of bovine serum albumin/ml. After centrifugation (35000 g-min) the pellets were dissolved in 0.2 M NaOH, and the amount of radioactivity was determined in the pellets and in the supernatants. Collagenase sensitive and unsensitive proteins were considered as collagen and non-collagenous proteins respectively. The types of collagen were determined from samples treated with pepsin (0.2 mg/ml in 0.5 M acetic acid for 3 h at 15°C, followed by extensive dialysis against water). To facilitate the identification of the collagens or of their subtypes, aliquots of the samples were further digested either by purified bacterial collagenase (as described above), or by tissue collagenase (5 U / m l for 4 h at 25°C, in the same buffer as that used for the bacterial collagenase digestion). Types V [14] and XI collagens [15] are indeed resistant to the action of tissue collagenase while types I, II and III [16], as well as type I trimer [17], are cleaved into characteristic 3/4-1/4 fragments. Procollagenase-containing mouse bone-conditioned culture medium was used as a source of tissue collagenase, after the activation of procollagenase by trypsin [18]. Collagenase-digested and non-digested aliquots were concentrated 25-fold by dialysis and lyophilization, resuspended in electrophoresis buffer with or without dithiothreitol, and denatured at 100 °C for 2 min. Sodium dodecyl sulfate-polyacrylamide gel electrophoreses [19] were performed on 5-8% (w/v) polyacrylamide linear gradient gels. Fluorographs [20] were exposed for 2 or 8 days at - 8 0 ° C . Type I guinea-pig skin collagen, partially digested by animal collagenase, and 14C-methylated proteins were used respectively as collagen and protein standards. Individual collagen bands resolved from the samples were quantitated by scanning densitometry of the fluorographs.

268

Production of collagenase and collagenase inhibitor (TIMP) Collagenase and TIMP were evaluated as described [10] in conditioned culture media obtained (unless otherwise indicated) at cell confluence, in DMEM with or without 10% FCS, 10% NU serum or IL-1, as specified in the figure legends. Briefly, free TIMP was assayed by measuring the inhibition of the trypsinactivated collagenase present in a crude skin fibroblastconditioned medium. This assay underestimates the total amount of TIMP present because it does not consider the proportion of T I M P that binds to other metalloproteinases present in the fibroblast medium. Therefore, all parallel assays were done using the same fibroblast medium. TIMP bound to metalloproteinases was measured after destruction of the enzymes and dissociation of the complexes at pH 2 and 100°C for 30 min. Prior to the activation of procollagenase by either trypsin or APMA, the free TIMP present in the conditioned media was neutralized whenever needed (see the figure legends) by the binding of other tissue metalloproteinases (i.e., the proteoglycanase and gelatinase present in the culture medium of rabbit bone marrowderived macrophages [21]). Collagenase was then assayed at 2 5 ° C using soluble [3H]acetylated guinea-pig skin collagen as a substrate. The procollagenase values presented have been corrected for the stimulation ( × 1.5) exerted on collagenase activity by the macrophage-conditioned medium [10]. 1 U of collagenase degrades 1 ~g of collagen per min at 25 ° C and 1 U of TIMP inhibits 2 U of collagenase by 50%. Cultures of chondrocytes on collagen/proteoglycan-coated plates The capability of chondrocytes, at various passages, to degrade type I or type II collagen and cartilage proteoglycans was assayed by culturing the cells on the surface of [14C]collagen/[3H]proteoglycan films [11]. Type II collagen was prepared from calf articular cartilage [16] and 14C-acetylated [22]. Results

Morphological aspects and cell densities of confluent cultures Observed at low magnification (not shown), chondrocytes in primoculture appear as small polygonal cells surrounded by an abundant matrix. In some areas, the cells pile up in small multilayer 'cartilaginous nodules'. This morphology changes progressively with increasing passages. The abundance of extracellular matrix decreases and confluent cells appear larger and flattened, although still polygonal in shape, and they no longer pile up. In agreement with this evolution, the cell densities achieved in confluent cultures decrease from passage to passage. An abrupt diminution, approx. 50%

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Fig. l. Evolution of the cell densities achieved by confluent chondrocytes during their dedifferentiation by serial monolayer subcultures. Six different chondrocyte cell lines (referred to as lines l to VI in Figs. 2, 3, 5 and 6) were cultured on plastic for 6 to 10 passages to allow' their progressive dedifferentiation. The cellular densities achieved after 9 to 10 days of culture at various passages were evaluated by measuring the DNA content of the cultures; the results, expressed in 10 5 cells/cm 2, are the means of evaluations done on 1 to 6 of the different cell lines, as indicated by the corresponding figures on the graph; vertical bars correspond to + 1 S.D.

loss, is observed between the primoculture and either the second or third culture passage, depending upon the experiment. This is followed by a slower decrease thereafter (Fig. 1).

Relative production of collagen and non-collagenous proteins The chondrocyte synthesis of collagen and non-collagenous proteins was characterized during the progressive dedifferentiation of several cell lines by serial monolayer subcultures. The results of a representative experiment, obtained with cell line I (see Figs. 1, 3, 5 and 6), are illustrated in Fig. 2 for the relative production of collagen and non-collagenous proteins, and in Fig. 3, for the collagen types produced. Collagen is actively synthesized in primoculture and first subculture where it is mainly incorporated in the matrix and cell layer. A sharp reduction of collagen synthesis occurs in subsequent passages, along with a decreased incorporation into the matrix and cell layers and a slightly increased release into the media (Fig. 2A and B). Inversely, the production of non-collagenous proteins increases steadily from the primoculture to the 10th passage. Most of these proteins are located in the matrix and cell layers but with increasing passages the amounts released into the media increase more, relative to those incorporated in the matrix and cell layers (Fig. 2A and B). Consequently, the ratio of newly synthesized collagen to newly synthesized total proteins decreases sharply during the first two passages and more slowly thereafter (Fig. 2C). The ratio of newly synthesized collagen incorporated into the matrix and cell layers relative to the total of newly synthesized collagen decreases progressively with increasing cell passages (Fig. 2D).

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Fig. 2. Production of collagen and non-collagenous proteins by dedifferentiating chondrocytes in monolayer culture. The chondrocyte cell line I (see Figs. 1, 3, 5 and 6) was characterized for its synthesis of collagen (m) and non-collagenous proteins (©) during its progressive dedifferentiation by serial monolayer subcultures. A and B present the accumulation of these newly synthesized proteins in either the matrix and cell layer (A) or the culture medium (B) as a function of the cell passage number. Results are expressed in cpm incorporated into protein per cell during the labelling period with [3H]proline (see Materials and Methods). Each point is the mean of three determinations; vertical bars correspond to _+1 S.D. (shown only if its magnitude exceeds the size of the symbol used for the experimental points). C shows the progressive reduction in the percent ratio of newly synthesized collagen to newly synthesized total proteins with increasing passage number. D shows the parallel reduction in the percentage of newly synthesized total collagen deposited in the matrix and cell layer, The former ratio only (C) was calculated on cpm values corrected according to Peterkofsky et al. [13] in order to include in the collagen determinations the collagenase-resistant propeptides of procollagens otherwise included in the non-collagenous proteins and to take account of the higher proline and hydroxyproline content of collagen relative to non-collagenous proteins. (COLL, collagen; NCP, non-collagenous proteins).

Collagen types produced The matrix and cell layers as well as the culture media of the chondrocyte cell line presented above (Fig. 2) were monitored from the primoculture to the seventh subculture to determine the types of collagen synthesized (Fig. 3) and their relative proportions (Table I). In primoculture, the most abundant neosynthesized collagen found in the medium (Fig. 3A) and, in considerably larger amounts, in the matrix and cell layer (Fig. 3B), has a migration similar to that of the al(I) chain standard. No a2(I) chains are detected. This indicates the absence of type I collagen with a chain composition of [al(I)]2.a2(I), but the presence of s o m e [al(I)] 3 cannot be excluded. Thus, the major collagen synthesized appears to be type II, a typical product of differentiated chondrocytes, consisting of three al(II) chains which are transformed by a crude preparation of tissue collagenase (Fig. 3C and D) into fragments of the size of the 3 / 4 ~1 and 3 / 4 a2 standards or smaller (discussed below). This material is followed by two bands, visible almost exclusively in the matrix layer, which migrate between the 3 / 4 fl(I) and the al(I) standards. These t w o b a n d s p e r s i s t a f t e r d i g e s t i o n w i t h c r u d e t i s s u e coll a g e n a s e as d o e s a m i n o r c o m p o n e n t o f t h e b a n d m i g r a t i n g a t t h e a l ( I ) level. T h e s e t i s s u e c o l l a g e n a s e - r e sistant b a n d s a p p e a r to be c o l l a g e n o u s by their c o m plete removal by bacterial collagenase (not shown). T h e y c o n t a i n m a i n l y t h e a l ( u p p e r ) , c~2 ( c e n t r e ) a n d a 3

chains [lower band, migrating together with the al(II) chains] of type XI collagen. This is another product of differentiated chondrocytes existing predominantly under a heterotrimer [al(XI).a2(XI).a3(XI)] form [23]. TABLE I

Relative proportions of collagen types synthesized by chondrocytes during their progressive dedifferentiation in monolayer culture The table was established by compiling the data obtained from densitometry of fluorographs presented in Fig. 3. Type III collagen was considered to correspond to band A of the culture medium, since the contribution of types I and II collagens to this band, visualized after dithiothreitol reduction (not shown), was found to be minimal. All other collagen types were evaluated from both the medium and the cell and matrix compartment: type I collagen [(al)2.a2 ] was estimated equal to 3-fold the values of bands F; type XI, to 3-fold the values of bands D; type V, to 1.5-fold the difference between bands C and D; type II (and type 1 trimer), to the difference between bands E and the values estimated for the participation of types I, V and XI collagens to these bands. Cell passage number 0 1 2 3 5 7

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The presence of some newly synthesized cd(V) and c~2(V) chains, comigrating respectively with the M(XI) and the ~3(XI) chains is possible, because the ratio of M(XI) to ~2(XI) or c~3(XI) to ~2(XI) chains is greater than 1:1. The absence of reducible "},-components in the gels indicates that type III collagen is not synthesized by the cells in primoculture. Identical collagen bands were observed in gels run under reducing conditions (not shown).

With increasing cell passages, the pattern of collagen types synthesized changes progressively. Chondrocytes in the first passage still produce types II and Xl (possibly also type V) collagens but no type I. They have initiated synthesis of type Ili collagen, visible almost exclusively in the media as y-components transformed by dithiothreitol reduction (not shown) into al components comigrating with the M(I) chains. With further passages, the synthesis of type III collagen increases

271 dedifferentiated in the first passage and we occasionally saw chondrocyte cultures that remained essentially differentiated in the second passage.

progressively as well as that of type I collagen, demonstrated by the presence of ct2(I) chains in both matrix layers and media. Conversely, the synthesis of type II collagen decreases progressively. Types I and III collagens are digested by a crude mouse bone collagenase preparation but, in contrast to type II collagen, they produce only typical 3 / 4 ct fragments. Interestingly, the 3 / 4 etl fragments of (pepsinized) type II collagen are further degraded into smaller pieces by either the collagenase or, more likely, by contaminating proteinases such as proteoglycanase (stromelysin) or gelatinase, known to be present in the crude collagenase preparation [24]. The disappearance of these smaller pieces after the fifth passage provides evidence for the virtual switching off of type II collagen synthesis after this passage. A progressive reduction in type XI collagen synthesis occurs in parallel, shown by the disappearance of the a2(XI) chains after the fifth passage as well. The simultaneous disappearance of al and ct3 chains of type XI collagen is likely but presumably masked by a progressive increase in the synthesis of type V collagen. Several unidentified peptides, resistant to bacterial collagenase (not shown) as well as to pepsin, are produced by chondrocytes after the second passage in parallel with their dedifferentiation (Fig. 3, bands G, H and I). It should be noted that, from one experiment to another, the dedifferentiation of chondrocytes may be initiated at slightly different passages. We frequently observed chondrocyte populations which were half-

Production of active collagenase and collagenase inhibitor Free active collagenase was not detectable in the medium from non-stimulated chondrocytes (not shown). The fact that no active collagenase (nor proteoglycanase) was produced by these cultures, was ascertained by culturing chondrocytes from different passages on 14C-labelled type II or type I collagen/ [3H]proteoglycan-coated plates. Regardless of passage number, the cells did not degrade either substrate (Fig. 4A), although they all did after stimulation with IL-1 (shown for first-passage cells in Fig. 4B). In a unique case (not shown), we observed degradation of type II collagen by non-stimulated chondrocytes in the 9th passage, but not by chondrocytes in the 2nd passage, when the cells were cultured in the same experiment on type II collagen-coated plates. The absence of active collagenase in the medium can be related to the presence of the inhibitor TIMP produced by the chondrocytes [10]. This production was always higher in cultures with serum, either FCS or NU serum, than in its absence (compare cell lines I and III to the others in Fig. 5). Regardless of the culture media, the production of TIMP increased steadily from passage to passage (Fig. 5I-VI). The inhibitor was almost completely in the free form as its amount did not increase

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Fig. 5. Production of procollagenase and TIMP by chondrocytes during their dedifferentiation in monolayer culture. The 6 chondrocyte cell lines (Fig. 1) were considered independently for their procollagenase and collagenase inhibitor production (I VI). In 1, conditioned media were produced from the 2nd to the 9th day of culture in DMEM with 10% FCS; before assays, they were freezed and thawed several times in order to denature their a2-macroglobulin content. In II to VI, conditioned media were produced at or near confluence for 2 days in DMEM without (full lines) or with (dotted lines) 10% NU serum (a serum devoid of active collagenase inhibitors). Free TIMP was measured in all media without other treatment (o) or after optimal prometalloproteinase activation ( o ) with APMA alone (IV to VI) or APMA and trypsin (I). In II to VI, procollagenase (~) was measured after neutralization of the free inhibitor present by binding to active tissue metalloproteinases added to the media prior to activation of the proenzyme with trypsin. Results are expressed as a function of cell passage number, in I, in U / m l , as the cell numbers were increasing during conditioned media production, and in II to VI, in U / m l per 106 cells, as the latter cell lines were studied at or near confluence.

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Fig. 6. Production of procollagenase by chondrocytes stimulated with IL-1 during their dedifferentiation in monolayer culture. In parallel with the experiments reported for non-stimulated cells in Fig. 5, the chondrocyte cell lines I1, II1, IV and VI, cultured at or near confluence in DMEM, were stimulated for 2 days with 100 U of lL-1/ml. Procollagenase was assayed in the conditioned media as done for the corresponding non-stimulated cells. Results are expressed as a function of cell passage number in U / m l per 106 cells.

273 by heating the media at acid pH (not shown). The absence of bound TIMP suggests again that only minimal or no active collagenase ( a n d / o r other tissue metalloproteinases) was formed in the cultures.

Spontaneous production of procollagenase and response to IL-1 Treatment of media with APMA a n d / o r trypsin (which activates the proenzymes of collagenase and other tissue metalloproteinases under conditions where it does not inactivate TIMP [10]) did not elicit measurable collagenase activity in either differentiated primocultures or dedifferentiated cultures from later passages. The inactivation of free TIMP by complex formation with an exogenous metalloproteinase, before activation of procollagenase by trypsin, did not elicit collagenase activity in media from differentiated primocultures analyzed at or near confluence (Fig. 5II-VI). With increasing passage numbers and cell dedifferentiation, collagenase became detectable, after this treatment, in only two (lines II and III) out of the five cell lines tested at or near confluence. However, preliminary evidence (not shown) suggests that low levels of collagenase can be produced transiently by differentiated cells, and in relatively larger quantities by dedifferentiated cells, before approaching confluence. When collagenase was detected (as for cell lines II and III of Fig. 5), its activity remained lower than that of the free TIMP present in the same medium. In the four other cell lines (Fig. 5I, IV, V and VI), treatment of the media with APMA, followed or not by trypsin, decreased the amount of free TIMP measurable in late passage cultures but not in primocultures. Production of procollagenase could be induced by IL-1 in all chondrocyte cultures; it was optimal using an IL-1 concentration of 100 U / m l regardless of the cell passage number (not shown). However, the procollagenase response of differentiated chondrocytes in primoculture was always lower than that of dedifferentiated cells in later passage cultures (Fig. 6). The chondrocyte production of TIMP was unaffected by IL-1 [25] and usually lower (in U / m l ) than that of procollagenase. Discussion

Serial subcultures allowed us to establish a parallel between the loss of the differentiated phenotype of rabbit articular chondrocytes, as expressed by their production of collagens, and the modulation of their production of collagenase inhibitor and procollagenase. The differentiated phenotype expressed by chondrocytes in primoculture (Table I) was characterized by abundant synthesis of types II and XI collagens [26]. Apparently some type V collagen was also produced, in agreement with other reports [27,28]. Pepsin-resistant

fragments from type IX collagen [29] were not identifiable in our fluorographs, unless sometimes after long exposure (not shown). This absence was apparently not due to the technical procedure used (unpublished data). It is therefore likely that our chondrocytes did not synthesize significant amounts of type IX collagen, at least during the labelling period with [3H]proline. Differentiated chondrocytes simultaneously produced other, non-collagenous proteins among which the collagenase inhibitor TIMP, but no or very low levels of procollagenase. Thus, their metabolic behaviour is oriented towards the synthesis and preservation of the cartilage matrix collagen. As expected [26,30,31], the subsequent monolayer cultures of chondrocytes caused their progressive dedifferentiation. This was manifested by changes in the collagen types synthesized, as described previously by others [4,7,26,27,30,31]. Furthermore, it involved a reduction in the ratio of newly synthesized collagen to newly synthesized non-collagenous proteins. This reduction is explained by the progressive disappearance of the abundant synthesis of types II and XI collagens and its replacement by only low productions of types I, III and V collagens. Moreover, the ratio of newly synthesized collagen incorporated into the matrix to that released into the culture medium decreased in parallel. This decrease manifested principally the shifts from types II and XI collagens, mainly deposited in the matrix and cell layers, to types I and V collagens, more soluble in the culture medium, and to type III collagen, almost exclusively present in the medium. Nevertheless, the proportions,of type II plus type I collagens and of type XI plus type V collagens remained constant at all passages, the former representing about 60% of the total collagen produced and the latter about 30-40%. The proportion of type III collagen never exceeded 10% of the total collagen synthesized. Changes in production of collagenase inhibitor and procollagenase and shifts in collagen synthesis were evaluated in the same chondrocyte cultures while they progressively dedifferentiated throughout subcultures. Articular chondrocytes have been previously shown to produce TIMP [10,32,33] but they produce only very low or undetectable levels of collagenase, unless activated by cytokines (most notably, IL-1) or other agents [1,5,8,25,33,34]. However, in most of these studies, the phenotype of the chondrocytes was not controlled, although the shift from a differentiated to a modulated phenotype was sometimes suspected [34]. Also, problems arising in enzymatic assays of collagenase or TIMP [10], due to the possible simultaneous presence of (pro)collagenase and inhibitor in the culture medium, were generally not discussed. Using modified assay procedures established to improve the recovery of both TIMP and procollagenase in medium where they co-exist [10], we have shown here

274 that fully differentiated, non-proliferating chondrocytes in primoculture secrete some T I M P but no detectable procollagenase. Interestingly, preliminary evidence suggests that low levels of procollagenase are produced by proliferating chondrocytes before they approach confluence, but the significance of this observation remains to be established. The production of procollagenase by differentiated cells can, however, be induced by IL-1. In parallel with their dedifferentiation, the chondrocytes progressively increase their production of TIMP. Dedifferentiated chondrocytes also respond to IL-1 by greater production of procollagenase than differentiated cells, suggesting an increased sensitivity to the cytokine. A 'spontaneous' (i.e., not induced by exogenous IL-1) induction of procollagenase production was observed in some cell lines after the first or second subculture and this production increased with further dedifferentiation. This could result from an autocrine stimulation of the cells by IL-1, since this cytokine can be produced by chondrocytes [35]. Indirect evidence was obtained suggesting that, even in the absence of a detectable 'spontaneous' induction of procollagenase, the synthesis of other latent tissue metalloproteinases can occur in dedifferentiated chondrocyte cultures. Treatment of their medium with A P M A followed (or not) by trypsin, under conditions where these agents do not destroy T I M P but activate the zymogens of tissue metalloproteinases [10], caused a reduction in the amount of free TIMP. This effect, which was not observed with medium from differentiated primocultures, is likely due to the activation of some tissue metalloproteinases (such as proteoglycanase or gelatinase) that upon activation form irreversible complexes with free T I M P [10]. Due to the excess of free T I M P present, it was, however, not possible to assay these enzymes in the medium after the activation of their latent proenzymes, because the exogenous metalloproteinase preparation used to neutralize T I M P prior to the assay of procollagenase is itself active on proteoglycans and gelatin [21]. Taking evidences altogether, it appears that dedifferentiated chondrocytes are more likely to switch towards a 'catabolic' behaviour characterized by enhanced production of procollagenase in response to IL-1, possibly even by a spontaneous induction of zymogen production. This could be relevant to the pathological events occurring in osteoarthritic cartilage, where foci of type I collagen-producing dedifferentiated chondrocytes coexist [3,4] with collagenase production and matrix degradation [2,36]. To degrade the cartilage matrix constituents, these proenzymes need to be activated and the level of active enzymes must be higher than that of the surrounding TIMP. We did not find active collagenase in the procollagenase-containing culture fluids from non-stimulated chondrocytes, nor did we find evidence for significant amount of T I M P bound to collagenase or to other metalloproteinases. This indicates that the

zymogens were not significantly activated by the chondrocytes in culture and that they did not 'autoactivate' in the media (a fact confirmed in nonreported experiments). Moreover, in non-stimulated subcultures. the level of free collagenase inhibitor recovered was always higher than that of procollagenase when the latter was found. It is thus not surprising that, even after full activation of the proenzyme, no collagenase activity could be detected in the media unless we had neutralized the inhibitor before the activation of procollagenase. It is interesting that non-stimulated chondrocytes from various passages, cultured on [14C]collagencoated plates, did not degrade collagen, as would be expected if T I M P was in excess over the potential collagenase activity produced by the cells. Thus, the balance between production of procollagenase, its rate of activation into collagenase (a problem not investigated in the present study) and the production of TIMP, is obviously important for maintaining the collagen matrix surrounding cells. We observed here that nonstimulated dedifferentiated chondrocytes can sometimes increase their production of procollagenase, although they usually keep the balance in favour of preserving the surrounding matrix by maintaining the production of inhibitor at a higher level than that of procollagenase. It was only through IL-I stimulation that more proenzymes than T I M P were produced [25] and that active metalloproteinases appeared and directly degraded the [14C]collagen and [3H]proteoglycans coated on the culture plates.

Acknowledgements This work was supported by the Fund for Medical Scientific Research (Belgium) and by the Belgian StatePrime Minister's Office Science Policy Programming (interuniversity attraction poles, grant No. 7bis and concerted actions, grant No, 88/93-122). V.L. was a Research Fellow of the Institut pour l'Encouragement de la Recherche Scientifique dans l'Industrie et l'Agriculture ' I R S I A ' . We gratefully acknowledge the expert technical assistance of F. Chenut and B. Dieudonn6 and the skillful secretarial work of Y. Marchand. Our thanks also go to Dr. K. Willard-Gallo for checking the manuscript.

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