234
Biochimica et Biophysica Acta, 582 (1979) 234--245 © Elsevier/North-Holland Biomedical Press
BBA 28761
E X T R A C E L L U L A R MATRIX METABOLISM BY CHONDROCYTES 5. THE P R O T E O G L Y C A N S AND GLYCOSAMINOGLYCANS SYNTHESIZED BY C H O N D R O C Y T E S IN HIGH DENSITY C U L T U R E S
C.J. HANDLEY and D.A. LOWTHER
Department of Biochemistry, Monash University, Clayton, Victoria 3168 (Australia) (Received June 9th, 1978)
Key words: Proteoglycan; Enzymic degradation; Hyaluronate; Extracellular matrix; Glycosaminoglycan; (Chondrocyte)
Summary Proteoglycans were extracted from the extracellular matrix of cultures of embryonic chick chondrocytes grown at high density and were purified by CsC1 density gradient centrifugation. The chemical, physical and hyaluronate binding properties of the proteoglycans were similar to those observed in proteoglycans from other hyaline cartilages. Proteoglycans in the media were also purified and on analysis showed three populations of proteoglycans to be present. One population had the physical characteristics of a typical proteoglycan subunit and b o u n d hyaluronate, the other two populations were unable to complex with hyaluronate b u t one had the physical characteristics of the proteoglycan subunit and the other was of smaller molecular weight. The small molecular weight appears to be a p r o d u c t of the enzymatic degradation of the larger molecular weight species. Introduction
Cartilage is a tissue made up of cells which secrete t w o main macromolecules, collagen and proteoglycan, which are subsequently organized into the extracellular matrix. In recent years there has been a growing interest in whether the extracellular concentrations of these macromolecules can interact with chondrocytes to regulate their own synthesis [1--5]. Permeability problems present difficulties in using cartilage tissue for these studies and have lead to the use of chondrocyte suspension cultures or monolayer cultures. In a system developed in this laboratory [6,7] chrondrocytes from embryonic chick epiphyseal cartilage are grown in high density cultures where the extracellular matrix of collagen and proteoglycan surrounding the cell is permeable to macromolecules added to the culture media [8]. This culture method, being a
235 closed system, is also ideal for the study of the synthesis and degradation of the macromolecules of the extracellular matrix of cartilage. In this study we report the chemical and physical characteristics of the proteoglycans and glycosaminoglycans that are synthesized by chondrocytes grown at high density. We also show that the chondrocytes in this culture system are capable of degrading under physiological conditions the endogenous proteoglycans present in the cultures grown at high density. Methods
Isolation and culture of chondrocytes. Chondrocytes were isolated from the epiphyses of the long bones from 13-day-old embryonic chicks and grown in high density cultures as previously described [4]. The growth medium was changed every 24 h. Extraction and purification of proteoglycans from chondrocyte cultures. The cell layers from 30 flasks of 20~lay-old chondrocyte cultures were extracted with 50 ml 4 M guanidine hydrochloride in 50 mM acetate buffer, pH 5.6, containing the following protease inhibitors; 25 mM EDTA, 10 mM N-ethylmaleimide, 0.1 mM phenylmethanesulphonyl fluoride and I mM benzamidine hydrochloride [9]. The extract was clarified by centrifugation at 20 000 X g for 30 min at 4°C. The supernatant was dialysed against 9 vols. of 50 mM Tris-HC1 buffer, pH 7.2 containing the inhibitors at 4°C for 24 h. The density of the dialysis residue was adjusted to 1.69 g/ml with solid CsC1 before being centrifuged at 90 000 X g at 20°C for 38 h in a MSE Superspeed 75 centrifuge using 10 X 10 ml head (cat. No. 59429) [10]. On completion of the centrifugation, the tubes were fractionated into three equal parts. The b o t t o m fraction from each tube were pooled and made to 4 M with 7.5 M guanidine hydrochloride, 50 mM Tris-HC1 buffer, pH 7.2, containing the inhibitors. The density of this fraction was adjusted to 1.50 g/ml with solid CsC1 before being centrifuged as described above. On completion of the run the tubes were fractionated into three equal parts [10]. The b o t t o m fractions from each tube were pooled and the proteoglycan preparation exhaustively dialysed against distilled water at 4 ° C.
Chemical and physical analysis of the proteoglycans from chondrocyte cultures. Two samples (approx. 1 mg) of the proteoglycan were hydrolysed with 6 M HC1 in sealed tubes under vacuum for 18 h at l l 0 ° C before being dried d o w n in vacuo and analysed for amino acid and amino sugars using a Jeol (Tokyo, Japan) amino acid analyser. Standard amino acid and amino sugar mixtures were hydrolysed at the same time and were included into each run enabling a correction to be made for losses due to decomposition of the amino acids in the course of the hydrolysis. Further samples of the proteoglycans were analysed for hexuronic acid [ 11 ], hexosamine [12] and protein [13]. The molecular weight of the c h o n d r o c y t e culture proteoglycans were determined using the meniscus depletion m e t h o d [14]. The determinations were carried o u t in a Beckman Model E ultracentrifuge at 20°C at 4609 rev./min in a J rotor with 12-mm cells. Density increments of the proteoglycans were calculated from their composition assuming a partial specific volume of 0.74 and
236 0.52 ml/g for the protein and polysaccharide components components, respectively [15]. Interaction of the proteoglycans with hyaluronate. Six chondrocyte cultures were grown for 20 days in the presence of Na23SSO4. The media from the cultures were changed every 24 h and replaced with fresh media containing 10 ~zCi of Na23SSO4 (carrier free). The spent media were collected, the prOtease inhibitors added at the appropriate concentration, and stored at--20°C. At the end of 20 days the cultures were extracted with 15 ml 4 M guanidine hydrochloride in 50 mM acetate buffer, pH 5.6, containing the protease inhibitors. The proteoglycans in the extract were purified by density gradient centrifugation in gradients of CsC1 as described above. The spent media were pooled and concentrated by ultrafiltration before being made to 0.4 M with 4 M guanidine hydrochloride in 50 mM acetate buffer, pH 5.6, containing the protease inhibitors. The proteoglycans in the media were then purified in CsC1 gradients as described above. The proteoglycans isolated from the cell layers and from the media were exhaustively dialysed against several changes of 10 mM Na2SO4. Samples of the t w o proteoglycan preparations were dialysed against 1 M guanidine hydrochloride (pH 5.6) before being applied to a column of Sepharose 2B (84 X 1.6 cm) which was eluted at a rate of 7.5 ml/h with 1 M guanidine hydrochloride in 50 mM acetate buffer, pH 5.6. Fractions (4 g) were collected and analysed for radioactivity. Further samples of the proteoglycans were mixed with hyaluronate (ex cock's comb) under associative conditions in the proportion of proteoglycan to hyaluronate of 100 : 1 (w/w) before being chromatographed on a Sepharose 2B column (84 × 1.6 cm) which was eluted with 0.5 M sodium acetate, pH 5.8, at 8.6 ml/h. 4 g fractions were collected and assayed for radioactivity. The hyaluronate used in this experiment was completely excluded from the Sepharose 2B column.
Isolation of degradation products from the proteoglycans of chondrocyte cultures. 12 c h o n d r o c y t e cultures were grown for the first 3 days of culture in growth media containing 30/~Ci of Na23SSO4 (carrier free). The media and isotope were changed every 24 h. After 3 days the cultures were grown for 20 days with media not containing the isotope. Every 24 h the media were replaced with fresh media and an aliquot of the spent media was counted for radioactivity. After 20 days, three chondrocyte cultures were incubated with normal growth media for a period of 72 h, three cultures were incubated with media containing 3.3 pg retinol/ml medium and another three cultures with media containing the protease inhibitors. The growth media in the cultures were changed every 24 h with the respective fresh media. The spent media were collected, and the protease inhibitors added where necessary to the appropriate concentration. These media were stored at --20°C. After 72 h the spent media from each treatment were pooled and desalted on a Sephadex G-25C column (70 × 3.5 cm) equilibrated" and eluted with 0.2 M Tris-HC1 pH 7.2, containing the protease inhibitors. The void volume material was concentrated by ultrafiltration and then chromatographed on a Sepharose 4B CL column (80 × 1.8 cm) which was eluted with 0.2 M Tris-HC1, pH 7.2, containing the protease
237 inhibitors at 12.4 ml/h. Fractions (5 g) were collected and assayed for radioactivity. The cell layers from the remaining three cultures were extracted with 4 M guanidine hydrochloride in 50 mM acetate buffer, pH 5.6, containing the protease inhibitors. The extract was desalted on Sephadex G-25C before an aliquot was chromatographed on Sepharose 4B CL as described above. Further samples of the proteoglycan isolated from the matrix were incubated with 1.0 mM NaOH for 24 h at 4°C and with foetal serum for 24 h at 37°C. These incubations were chromatographed on Sepharose 4B CL.
Isolation and analysis o[ the glycosaminoglycans synthesized by chondrocytes in culture. Ten chondrocyte cultures were grown in the presence of 20 pCi of [U-14C]glucose (spec. act. 3 Ci/mol) for 20 days. The media were changed every 24 h and replaced by fresh media containing the isotope. After 20 days the cell layers were digested with papain and the glycosaminoglycans purified on DEAE-cellulose as previously described [4]. The glycosaminoglycan fraction was dialysed against distilled water before being dried by lyophilization. The dried glycosaminoglycan fraction was made up to 2 ml with 0.05 M Tris-HC1 buffer, pH 8.0, and 0.01 M NaF before the addition of 1.2 units of chondroitin lyase ABC (EC 4.2.2.4) [16]. The digest was incubated at 37°C for 24 h and the digest monitored by measuring the increase in the absorbance at 232 nm. The resulting disaccharides were then analysed by chromatography on Whatman 3 MM paper using a descending solvent, isobutyric acid/2 M ammonia (5 : 3, v/v) as described by Saito et al. [17]. Disaccharides derived from hyaluronate, chondroitin, chondroitin 4-sulphate and chondroitin 6sulphate were run as standards on the chromatographs. After 40 h the chromatographs were dried and observed under ultraviolet light. The chromatographs were divided into 2-cm wide strips which correspond to each radioactive sample. These strips were then cut into 1-cm sections that were placed into vials containing 5 ml of h o t distilled water and were left for 24 h at room temperature. Scintillator was added to the vials, which were then counted for radioactivity.
Determination of chain size o f the glycosaminoglycans synthesized by chondrocytes in culture. The molecular weight distribution of glycosaminoglycans synthesized by chondrocytes was determined using the m e t h o d described by H o p w o o d and Robinson [18]. 20 flasks containing 20-day-old chondrocyte cultures were each treated with 3 ml 20 mM NaB3H4 (spec. act. 1.41 • 106 d p m / m m o l ) in 0.5 M KOH for 14 days at 4°C. The supernatant was then acidified and applied to a Biogel P4 column (50 × 4.5 cm). The void volume was applied to a DEAE-cellulose column and the glycosaminoglycans eluted with 1 M NaC1 in 50 mM Tris-HC1, pH 7.4 [4]. The glycosaminoglycan fraction was dialysed against two changes of distilled water, freeze-dried, made up in 1 ml of 0.02 M NaC1 buffered with 0.02 M imidazole-HC1 at pH 6.0 and applied to a column of Sephadex G-200F (90 X 2.5 cm) equilibrated and eluted with the same buffer. The column was eluted at a flow rate of 20 ml/h and 5-g fractions were collected. Each fraction was counted for radioactivity and hexuronic acid [11]. Radioactive counting. Radioactivity was assayed using a Philips Liquid Scintillation Analyser using a scintillation mixture described by Fox [19].
238
Results
Characterization o f the proteoglycan subunits synthesized by chondrocytes grown in high density cultures Table I shows the chemical composition of the proteoglycan subtmit isolated by dissociative CsC1 gradients from the matrix synthesized by 20-day-old cultures of chondrocytes. Approx. 95% of the hexuronic acid-containing material extracted from the cultures was recovered in the b o t t o m one third of the dissociative gradient. Sedimentation equilibrium studies revealed a molecular weight distribution of the proteoglycans of between 1.07 • 106 and 1.59 • 106. The amino acid profile of the proteoglycans is given in Table II. In order to obtain information about the distribution on Sepharose 2B, as well as the hyaluronate binding properties, of the proteoglycans that were secreted into the media of the cultures, it was necessary to isotopically label the proteoglycan since the foetal calf serum in this work contains a small but significant a m o u n t of proteoglycan. In this experiment cultures were grown in growth media containing [3SS]sulphate for the entire period that the cells were cultured. It can therefore be assumed that the a m o u n t of radioactivity is directly proportional to the a m o u n t of proteoglycan present. Fig. l a shows the distribution of the proteoglycan subunit isolated from the matrix of 20-day-old chondrocytes. It is evident that there is a single peak (Kay = 0.23) which was included into the gel. The radioactivity was found to co-chromatograph with hexuronic acid. On addition of hyaluronate to the proteoglycans under associative conditions, the majority of the radioactivity associated with the proteoglycans was now eluted in the void volume. This demonstrates that a major proportion (approx. 61% total radioactivity) of the proteoglycans were capable of complexing the hyaluronate. The distribution of the proteoglycans isolated from the growth media of the cultures was much more disperse than the molecules obtained from the matrix (Fig. lb). Two distinct peaks of radioactivity were observed (Kav = 0.23 and 0.70). On addition of hyaluronate to these proteoglycans it was clear that only a small portion of the proteoglycans from the media was capable of complexing with hyaluronate (approx. 20% of the total radioactivity).
TABLEI CHEMICAL COMPOSITION OF PROTEOGLYCANS OLD CHONDROCYTECULTURES Percent dry weight Protein Galact osamine Gluc osamine Hexuronic acid Galactosamine/protein Glucosamine/protein Hexuronic acid/protein
7.6 24,1 1.6 23.9 3.43 0.21 3.14
ISOLATED
FROM
THE MATRIX OF 20-DAY-
239
T A B L E II AMINO
ACID
COMPOSITION
OF
P R O T E O G L Y C A N I S O L A T E D FROM T H E MATRIX
OF
20-DAY-
OLD CHONDROCYTE CULTURES Residues per I000
Lys His Arg Cys Asp Thr Set Glu Pro Gly Ala Val Met Ile Leu Tyr Phe
20 26 31 13 61 77 126 158 69 123 82 59 9 41 53 20 33
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no. ( 4 g )
Fig. 1. S e p h a r o s e 2B e l u t i o n p r o f i l e o f p r o t e o g l y c a n s u b u n i t i s o l a t e d f r o m t h e m a t r i x (a) a n d m e d i a (b) f r o m c h o n d r o c y t e s g r o w n in high d e n s i t y c u l t u r e s f o r 2 0 d a y s . P r o t e o g l y c a n c h r o m a t o g r a p h e d u n d e r dissociative c o n d i t i o n s ( ) and u n d e r associative conditions after interaction with hya]uronate
( ......
).
240
Evidence for the degradation of endogenous proteoglycans by chondrocytes grown in high density cultures. The loss of radioactive material into the media of chondrocyte cultures that were grown in the presence of [3SS]sulfate for the first 3 days in culture is shown in Fig. 2. Fig. 2 also shows the radioactively labelled proteoglycan that is released into the media from the extracellular matrix of the cultures expressed as a percentage of the original isotopically labelled proteoglycan. It is clear that with respect to time there is a small but significant release of radioactive material into the media. When chromatographed on Sepharose 4B CL the radioactivity was found to be associated with hexuronate-containing material that was eluted from the column in the void volume and also with material that was included into the column (Kay = 0.24) (Fig. 3a). These two peaks corresponded to the two proteoglycan peaks previously observed on Sepharose 2B with Kay values of 0.23 and 0.70. The proteoglycans extracted from the cell layers with 4 M guanidine hydrochloride and chromatographed on Sepharose 4B CL were excluded from the column (Fig. 3a). It was considered probable that the material included in the Sepharose 4B CL column was a product derived from the degradation of proteoglycans. In order to determine whether this was the case, chondrocyte cultures, which had been prelabelled with [3sS]sulphate for the first 3 days in culture and then grown in media n o t containing the isotope for 20 days were treated with media containing retinol and the protease inhibitors as described in Methods. The radioactive material appearing in the media was chromatographed on Sepharose 4B CL (Fig. 3b). It was found in chondrocyte cultures that were treated with media containing retinol, there was an increase in the release of [3SS]sulphate into the media (185% of control), and that the majority of this labelled material was included into Sepharose 4B CL when compared to the profile obtained from untreated cul-
8--
6
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oi E 4 _ u >_~
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Age of cultures (days)
Fig. 2. L o s s o f [ 3 5 S ] s u l p h a t e - c o n t a i n i n g m a t e r i a l i n t o t h e m e d i a (o e) o f c h o n d r o c y t e cultures that grown in the presence of [35S]sulphate f o r t h e first 3 d a y s in c u l t u r e . T h e r e l e a s e o f [ 3 5 S ] s u l p h a t e - c o n t a i n i n g m a t e r i a l f r o m t h e c u l t u r e s i n t o t h e m e d i a is also s h o w n (o o) a n d is e x p r e s s e d as a p e r c e n t a g e o f t h e o r i g i n a l n o n - d i a l y s a b l e [ 3 S S ] s u l p h a t e - c o n t a l n i n g m a t e r i a l o b s e r v e d in t h e c u l t u r e s had been
a f t e r t h e first 3 d a y s i n c u l t u r e .
241
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Q
1600
a
800
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10
15 20 25 Fraction no. (5g)
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~
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30
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Fig. 3. S e p h a r o s e 4B C L p r o f i l e s o f p r o t e o g l y c a n s i s o l a t e d f r o m t h e m e d i a a n d m a t r i x o f c h o n d r o c y t e c u l t u r e s w h i c h h a d b e e n g r o w n in t h e p r e s e n c e o f [ 3 5 S ] s u l p h a t e f o r t h e first 3 d a y s o f c u l t u r e . (a) [3 S S ] . s u l p h a t e - l a b e l l e d p r o t e o g l y c a n f r o m t h e m a t r i x (e o) a n d m e d i a (4 ~.) o f u n t r e a t e d 2 0 - - 2 3 d a y - o l d c h o n d r o c y t e c u l t u r e s a n d t h e s a m e p r o t e o g l y c a n s s u b j e c t e d t o f l - e l i m i n a t i o n (m a ) . (b) [ 3 5 S ] s u l p h a t e - l a b e l l e d p r o t e o g l y c a n a p p e a r i n g in t h e m e d i a o f u n t r e a t e d 2 0 - - - 2 3 - d a y - o l d c h o n d r o c y t e c u l t u r e s (A ~) a n d in t h e s a m e age c u l t u r e s t r e a t e d w i t h r e t i n o l (o o) a n d p r o t e a s e i n h i b i t o r s
(u
a).
tures. When the cells were treated with media containing the protease inhibitors there was a marked decrease in the release of [3SS]sulphate into the media (43% of control) and the majority of this labelled material was exluded from the gel relative to the control. The included and excluded peak when treated with alkali were both found to co-elute from Sepharose 4B CL in the same position as ~-eliminated proteoglycan. In order to determine whether the foetal calf serum contained proteolytic activity, a sample of the serum used in this work was incubated with proteoglycan isolated from the c h o n d r o c y t e layers
242 T A B L E III COMPOSITION
OF G L Y C O S A M I N O G L Y C A N S
SYNTHESIZED
BY 20-DAY-OLD CHONDROCYTE
CULTURES Percent radioactivity incorporated into glycosaminoglycans
59 17 12 3 8
Chondroitin 6-sulphate Chondroitin 4-sulphate Chondroitin H y a l u r o n i c acid Keratan s u l p h a t e
before it was analysed on Sepharose 4B CL. No change in the elution profile of the proteoglycan was observed.
Characterization of the glycosaminoglycans synthesized by chondrocytes grown in high density cultures The chondrocytes were grown in culture over a period of 20 days with [U14C]glucose, and the glycosaminoglycans present in the matrix isolated and digested with chondroitin lyase ABC. The resulting disaccharides were analysed by paper chromatography. As the cells had been grown with isotopic glucose since the time of inoculation, the amount of radioactivity in each disaccharide was used as a direct measure of the amount of the corresponding glycosaminoglycan present in the matrix. Table III shows the distribution of glycosaminoglycans isolated from the matrix of 20-day-old cultures. About 8% of the total radioactivity was observed to remain at the origin of the chromatograph and was presumed to be keratan sulphate, which is not digested with chondroitin
] i E
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~
100C
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--4O o
80C
60C
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-2o
40
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20C
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rr
20
30
40
50 Fraction
60 no.
70
8O
(Sg)
Fig. 4. S e p h a d e x G - 2 0 0 e l u t i o n profile c h o n d r o c y t e cultures. R a d i o a c t i v i t y ( e weight (o-o) are s h o w n .
of
[3H]-end-labelled glycosaminoglycans from 20-day-old e ) , h e x u r o n i c acid ( m i ) and c a l c u l a t e d m o l e c u l a r
243 lyase ABC. Further evidence that the isotopic label is directly associated with keratan sulphate is that the ratio of glucosamine to galactosamine in the isolated proteoglycans from the matrix was observed to be 1 : 15 (Table I). The hexuronic acid and radioactivity profiles on Sephadex G-200 of the [3H]-end-labelled glycosaminoglycans isolated from the matrix of the cultures are shown in Fig. 4. From the specific activity of the borohydride, the radioactivity and the hexuronate appearing in each fraction, the molecular weight of the glycosaminoglycan in that fraction was calculated and is also shown in Fig. 4. The weight average molecular weight was 3.08 • 104 and the numberaverage molecular weight 2.60 • 104. Discussion
It was found that the physical and chemical properties of the proteoglycans isolated from the matrix of 20-day-old chondrocyte cultures were similar to those reported by other workers for proteoglycans studied in other cartilages [20--22]. Furthermore, the chemical composition and amino acid profile of the proteoglycans isolated from the matrix closely resembles those reported by Hascall et al. [23] and Deluca et al. [24] for proteoglycans synthesized by cultures of limb bud chondrocytes from 4-day-old embryonic chicks. The amino acid profile of the proteoglycans would suggest that the protein core of the proteoglycans contains both a hyaluronate binding region and a glycosaminoglycan-rich region [25]. The presence of the hyaluronate binding region was verified in the hyaluronate binding experiments where approx. 61% of the proteoglycan, isolated by gradient centrifugation under dissociative conditions, aggregated with hyaluronate. This raises the question as to the origin of the proteoglycan observed in the media. There is a population of proteoglycans that have the physical characteristics of typical proteoglycan subunits in that they are capable of forming aggregates with hyaluronate and are eluted early from Sepharose 2B. They must represent native proteoglycans that have diffused from the matrix of the cultures either as subunits or possibly as aggregates with hyaluronate. It must be pointed o u t that the physical dimensions of the cultures, a very high surface area to thickness ratio, would favour such a phenomenon. Weibkin and Muir [26] have recently shown that chondrocytes from adult pig laryngeal cartilage grown in suspension culture secrete into the media proteoglycans which can form complexes with hyaluronate. Another group of proteoglycans observed to be present in the media of the c h o n d r o c y t e cultures co-eluted with the large molecular weight proteoglycans b u t were unable to bind to hyaluronate. These proteoglycans could represent a group of proteoglycans that prior to secretion into the extracellular compartment are modified enzymatically so that they do n o t possess a hyaluronate binding region and will have the tendency to diffuse o u t of the matrix into the media. This has been suggested by Hardingham et al. [25] to explain the nature of non-aggregating proteoglycans extracted from cartilage by isotonic saline. Alternatively this group of non-aggregating proteoglycans could represent products from the first stage in the degradation of the proteoglycan complex where the hyaluronate binding region is cleaved from the proteoglycan subunit
244 prior to further degradation of the macromolecule. This has been suggested by Sandy et al. [27] to explain the appearance of high molecular weight non-aggregating proteoglycans in the media of organ cultures of rabbit articular cartilage. A population of small non-aggregating proteoglycans which is included into both Sepharose 2B and Sepharose 4B CL was also observed in the media of the chondrocyte cultures. These proteoglycans probably reflect the products of normal degradation of proteoglycan by proteolytic enzymes present in chondrocytes [28]. The addition of retinol to cultures of cartilage has been reported to increase the release of lysosomal enzymes from chondrocytes [29]. Addition of retinol to these chondrocyte cultures causes an increase in the appearance of these small molecular weight proteoglycans in the media. Addition of the protease inhibitor cocktail to the media of the chondrocyte cultures decreased the appearance of these small molecular weight proteoglycans in the media. The protease inhibitor cocktail does have an appreciable cytotoxic effect on the chondrocytes in that protein and proteoglycan synthesis is inhibited (Handley, C.J. and Lowther, D.A., unpublished data). It is therefore possible that the inhibitor cocktail is not only inhibiting protease activity, but also the synthesis and release of the enzymes from the cells. The possibility that the proteolytic activity was originating from the foetal calf serum was discounted, since no degradative activity was found in the serum towards proteoglycan. These observations coupled with the fact that the media of the cultures was maintained between pH 6.8 and 7.2 would suggest that a neutral proteoglycanase is present in the cultures and is active against the endogenous proteoglycan. Similar observations have been made for rat costal cartilage in organ culture by Wasteson et al. [30]. The possibility of free sulphate being incorporated into a low molecular weight proteoglycan that does not bind hyaluronate seems remote since very little free isotopic sulphate was observed to be present in the cultures after 20 days of culture. The spectrum of glycosaminoglycans synthesized by chondrocyte cultures after 20 days is similar to that reported for 13
Biochimica et Biophysica Acta, 582 (1979) 234--245 © Elsevier/North-Holland Biomedical Press
BBA 28761
E X T R A C E L L U L A R MATRIX METABOLISM BY CHONDROCYTES 5. THE P R O T E O G L Y C A N S AND GLYCOSAMINOGLYCANS SYNTHESIZED BY C H O N D R O C Y T E S IN HIGH DENSITY C U L T U R E S
C.J. HANDLEY and D.A. LOWTHER
Department of Biochemistry, Monash University, Clayton, Victoria 3168 (Australia) (Received June 9th, 1978)
Key words: Proteoglycan; Enzymic degradation; Hyaluronate; Extracellular matrix; Glycosaminoglycan; (Chondrocyte)
Summary Proteoglycans were extracted from the extracellular matrix of cultures of embryonic chick chondrocytes grown at high density and were purified by CsC1 density gradient centrifugation. The chemical, physical and hyaluronate binding properties of the proteoglycans were similar to those observed in proteoglycans from other hyaline cartilages. Proteoglycans in the media were also purified and on analysis showed three populations of proteoglycans to be present. One population had the physical characteristics of a typical proteoglycan subunit and b o u n d hyaluronate, the other two populations were unable to complex with hyaluronate b u t one had the physical characteristics of the proteoglycan subunit and the other was of smaller molecular weight. The small molecular weight appears to be a p r o d u c t of the enzymatic degradation of the larger molecular weight species. Introduction
Cartilage is a tissue made up of cells which secrete t w o main macromolecules, collagen and proteoglycan, which are subsequently organized into the extracellular matrix. In recent years there has been a growing interest in whether the extracellular concentrations of these macromolecules can interact with chondrocytes to regulate their own synthesis [1--5]. Permeability problems present difficulties in using cartilage tissue for these studies and have lead to the use of chondrocyte suspension cultures or monolayer cultures. In a system developed in this laboratory [6,7] chrondrocytes from embryonic chick epiphyseal cartilage are grown in high density cultures where the extracellular matrix of collagen and proteoglycan surrounding the cell is permeable to macromolecules added to the culture media [8]. This culture method, being a
235 closed system, is also ideal for the study of the synthesis and degradation of the macromolecules of the extracellular matrix of cartilage. In this study we report the chemical and physical characteristics of the proteoglycans and glycosaminoglycans that are synthesized by chondrocytes grown at high density. We also show that the chondrocytes in this culture system are capable of degrading under physiological conditions the endogenous proteoglycans present in the cultures grown at high density. Methods
Isolation and culture of chondrocytes. Chondrocytes were isolated from the epiphyses of the long bones from 13-day-old embryonic chicks and grown in high density cultures as previously described [4]. The growth medium was changed every 24 h. Extraction and purification of proteoglycans from chondrocyte cultures. The cell layers from 30 flasks of 20~lay-old chondrocyte cultures were extracted with 50 ml 4 M guanidine hydrochloride in 50 mM acetate buffer, pH 5.6, containing the following protease inhibitors; 25 mM EDTA, 10 mM N-ethylmaleimide, 0.1 mM phenylmethanesulphonyl fluoride and I mM benzamidine hydrochloride [9]. The extract was clarified by centrifugation at 20 000 X g for 30 min at 4°C. The supernatant was dialysed against 9 vols. of 50 mM Tris-HC1 buffer, pH 7.2 containing the inhibitors at 4°C for 24 h. The density of the dialysis residue was adjusted to 1.69 g/ml with solid CsC1 before being centrifuged at 90 000 X g at 20°C for 38 h in a MSE Superspeed 75 centrifuge using 10 X 10 ml head (cat. No. 59429) [10]. On completion of the centrifugation, the tubes were fractionated into three equal parts. The b o t t o m fraction from each tube were pooled and made to 4 M with 7.5 M guanidine hydrochloride, 50 mM Tris-HC1 buffer, pH 7.2, containing the inhibitors. The density of this fraction was adjusted to 1.50 g/ml with solid CsC1 before being centrifuged as described above. On completion of the run the tubes were fractionated into three equal parts [10]. The b o t t o m fractions from each tube were pooled and the proteoglycan preparation exhaustively dialysed against distilled water at 4 ° C.
Chemical and physical analysis of the proteoglycans from chondrocyte cultures. Two samples (approx. 1 mg) of the proteoglycan were hydrolysed with 6 M HC1 in sealed tubes under vacuum for 18 h at l l 0 ° C before being dried d o w n in vacuo and analysed for amino acid and amino sugars using a Jeol (Tokyo, Japan) amino acid analyser. Standard amino acid and amino sugar mixtures were hydrolysed at the same time and were included into each run enabling a correction to be made for losses due to decomposition of the amino acids in the course of the hydrolysis. Further samples of the proteoglycans were analysed for hexuronic acid [ 11 ], hexosamine [12] and protein [13]. The molecular weight of the c h o n d r o c y t e culture proteoglycans were determined using the meniscus depletion m e t h o d [14]. The determinations were carried o u t in a Beckman Model E ultracentrifuge at 20°C at 4609 rev./min in a J rotor with 12-mm cells. Density increments of the proteoglycans were calculated from their composition assuming a partial specific volume of 0.74 and
236 0.52 ml/g for the protein and polysaccharide components components, respectively [15]. Interaction of the proteoglycans with hyaluronate. Six chondrocyte cultures were grown for 20 days in the presence of Na23SSO4. The media from the cultures were changed every 24 h and replaced with fresh media containing 10 ~zCi of Na23SSO4 (carrier free). The spent media were collected, the prOtease inhibitors added at the appropriate concentration, and stored at--20°C. At the end of 20 days the cultures were extracted with 15 ml 4 M guanidine hydrochloride in 50 mM acetate buffer, pH 5.6, containing the protease inhibitors. The proteoglycans in the extract were purified by density gradient centrifugation in gradients of CsC1 as described above. The spent media were pooled and concentrated by ultrafiltration before being made to 0.4 M with 4 M guanidine hydrochloride in 50 mM acetate buffer, pH 5.6, containing the protease inhibitors. The proteoglycans in the media were then purified in CsC1 gradients as described above. The proteoglycans isolated from the cell layers and from the media were exhaustively dialysed against several changes of 10 mM Na2SO4. Samples of the t w o proteoglycan preparations were dialysed against 1 M guanidine hydrochloride (pH 5.6) before being applied to a column of Sepharose 2B (84 X 1.6 cm) which was eluted at a rate of 7.5 ml/h with 1 M guanidine hydrochloride in 50 mM acetate buffer, pH 5.6. Fractions (4 g) were collected and analysed for radioactivity. Further samples of the proteoglycans were mixed with hyaluronate (ex cock's comb) under associative conditions in the proportion of proteoglycan to hyaluronate of 100 : 1 (w/w) before being chromatographed on a Sepharose 2B column (84 × 1.6 cm) which was eluted with 0.5 M sodium acetate, pH 5.8, at 8.6 ml/h. 4 g fractions were collected and assayed for radioactivity. The hyaluronate used in this experiment was completely excluded from the Sepharose 2B column.
Isolation of degradation products from the proteoglycans of chondrocyte cultures. 12 c h o n d r o c y t e cultures were grown for the first 3 days of culture in growth media containing 30/~Ci of Na23SSO4 (carrier free). The media and isotope were changed every 24 h. After 3 days the cultures were grown for 20 days with media not containing the isotope. Every 24 h the media were replaced with fresh media and an aliquot of the spent media was counted for radioactivity. After 20 days, three chondrocyte cultures were incubated with normal growth media for a period of 72 h, three cultures were incubated with media containing 3.3 pg retinol/ml medium and another three cultures with media containing the protease inhibitors. The growth media in the cultures were changed every 24 h with the respective fresh media. The spent media were collected, and the protease inhibitors added where necessary to the appropriate concentration. These media were stored at --20°C. After 72 h the spent media from each treatment were pooled and desalted on a Sephadex G-25C column (70 × 3.5 cm) equilibrated" and eluted with 0.2 M Tris-HC1 pH 7.2, containing the protease inhibitors. The void volume material was concentrated by ultrafiltration and then chromatographed on a Sepharose 4B CL column (80 × 1.8 cm) which was eluted with 0.2 M Tris-HC1, pH 7.2, containing the protease
237 inhibitors at 12.4 ml/h. Fractions (5 g) were collected and assayed for radioactivity. The cell layers from the remaining three cultures were extracted with 4 M guanidine hydrochloride in 50 mM acetate buffer, pH 5.6, containing the protease inhibitors. The extract was desalted on Sephadex G-25C before an aliquot was chromatographed on Sepharose 4B CL as described above. Further samples of the proteoglycan isolated from the matrix were incubated with 1.0 mM NaOH for 24 h at 4°C and with foetal serum for 24 h at 37°C. These incubations were chromatographed on Sepharose 4B CL.
Isolation and analysis o[ the glycosaminoglycans synthesized by chondrocytes in culture. Ten chondrocyte cultures were grown in the presence of 20 pCi of [U-14C]glucose (spec. act. 3 Ci/mol) for 20 days. The media were changed every 24 h and replaced by fresh media containing the isotope. After 20 days the cell layers were digested with papain and the glycosaminoglycans purified on DEAE-cellulose as previously described [4]. The glycosaminoglycan fraction was dialysed against distilled water before being dried by lyophilization. The dried glycosaminoglycan fraction was made up to 2 ml with 0.05 M Tris-HC1 buffer, pH 8.0, and 0.01 M NaF before the addition of 1.2 units of chondroitin lyase ABC (EC 4.2.2.4) [16]. The digest was incubated at 37°C for 24 h and the digest monitored by measuring the increase in the absorbance at 232 nm. The resulting disaccharides were then analysed by chromatography on Whatman 3 MM paper using a descending solvent, isobutyric acid/2 M ammonia (5 : 3, v/v) as described by Saito et al. [17]. Disaccharides derived from hyaluronate, chondroitin, chondroitin 4-sulphate and chondroitin 6sulphate were run as standards on the chromatographs. After 40 h the chromatographs were dried and observed under ultraviolet light. The chromatographs were divided into 2-cm wide strips which correspond to each radioactive sample. These strips were then cut into 1-cm sections that were placed into vials containing 5 ml of h o t distilled water and were left for 24 h at room temperature. Scintillator was added to the vials, which were then counted for radioactivity.
Determination of chain size o f the glycosaminoglycans synthesized by chondrocytes in culture. The molecular weight distribution of glycosaminoglycans synthesized by chondrocytes was determined using the m e t h o d described by H o p w o o d and Robinson [18]. 20 flasks containing 20-day-old chondrocyte cultures were each treated with 3 ml 20 mM NaB3H4 (spec. act. 1.41 • 106 d p m / m m o l ) in 0.5 M KOH for 14 days at 4°C. The supernatant was then acidified and applied to a Biogel P4 column (50 × 4.5 cm). The void volume was applied to a DEAE-cellulose column and the glycosaminoglycans eluted with 1 M NaC1 in 50 mM Tris-HC1, pH 7.4 [4]. The glycosaminoglycan fraction was dialysed against two changes of distilled water, freeze-dried, made up in 1 ml of 0.02 M NaC1 buffered with 0.02 M imidazole-HC1 at pH 6.0 and applied to a column of Sephadex G-200F (90 X 2.5 cm) equilibrated and eluted with the same buffer. The column was eluted at a flow rate of 20 ml/h and 5-g fractions were collected. Each fraction was counted for radioactivity and hexuronic acid [11]. Radioactive counting. Radioactivity was assayed using a Philips Liquid Scintillation Analyser using a scintillation mixture described by Fox [19].
238
Results
Characterization o f the proteoglycan subunits synthesized by chondrocytes grown in high density cultures Table I shows the chemical composition of the proteoglycan subtmit isolated by dissociative CsC1 gradients from the matrix synthesized by 20-day-old cultures of chondrocytes. Approx. 95% of the hexuronic acid-containing material extracted from the cultures was recovered in the b o t t o m one third of the dissociative gradient. Sedimentation equilibrium studies revealed a molecular weight distribution of the proteoglycans of between 1.07 • 106 and 1.59 • 106. The amino acid profile of the proteoglycans is given in Table II. In order to obtain information about the distribution on Sepharose 2B, as well as the hyaluronate binding properties, of the proteoglycans that were secreted into the media of the cultures, it was necessary to isotopically label the proteoglycan since the foetal calf serum in this work contains a small but significant a m o u n t of proteoglycan. In this experiment cultures were grown in growth media containing [3SS]sulphate for the entire period that the cells were cultured. It can therefore be assumed that the a m o u n t of radioactivity is directly proportional to the a m o u n t of proteoglycan present. Fig. l a shows the distribution of the proteoglycan subunit isolated from the matrix of 20-day-old chondrocytes. It is evident that there is a single peak (Kay = 0.23) which was included into the gel. The radioactivity was found to co-chromatograph with hexuronic acid. On addition of hyaluronate to the proteoglycans under associative conditions, the majority of the radioactivity associated with the proteoglycans was now eluted in the void volume. This demonstrates that a major proportion (approx. 61% total radioactivity) of the proteoglycans were capable of complexing the hyaluronate. The distribution of the proteoglycans isolated from the growth media of the cultures was much more disperse than the molecules obtained from the matrix (Fig. lb). Two distinct peaks of radioactivity were observed (Kav = 0.23 and 0.70). On addition of hyaluronate to these proteoglycans it was clear that only a small portion of the proteoglycans from the media was capable of complexing with hyaluronate (approx. 20% of the total radioactivity).
TABLEI CHEMICAL COMPOSITION OF PROTEOGLYCANS OLD CHONDROCYTECULTURES Percent dry weight Protein Galact osamine Gluc osamine Hexuronic acid Galactosamine/protein Glucosamine/protein Hexuronic acid/protein
7.6 24,1 1.6 23.9 3.43 0.21 3.14
ISOLATED
FROM
THE MATRIX OF 20-DAY-
239
T A B L E II AMINO
ACID
COMPOSITION
OF
P R O T E O G L Y C A N I S O L A T E D FROM T H E MATRIX
OF
20-DAY-
OLD CHONDROCYTE CULTURES Residues per I000
Lys His Arg Cys Asp Thr Set Glu Pro Gly Ala Val Met Ile Leu Tyr Phe
20 26 31 13 61 77 126 158 69 123 82 59 9 41 53 20 33
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Fig. 1. S e p h a r o s e 2B e l u t i o n p r o f i l e o f p r o t e o g l y c a n s u b u n i t i s o l a t e d f r o m t h e m a t r i x (a) a n d m e d i a (b) f r o m c h o n d r o c y t e s g r o w n in high d e n s i t y c u l t u r e s f o r 2 0 d a y s . P r o t e o g l y c a n c h r o m a t o g r a p h e d u n d e r dissociative c o n d i t i o n s ( ) and u n d e r associative conditions after interaction with hya]uronate
( ......
).
240
Evidence for the degradation of endogenous proteoglycans by chondrocytes grown in high density cultures. The loss of radioactive material into the media of chondrocyte cultures that were grown in the presence of [3SS]sulfate for the first 3 days in culture is shown in Fig. 2. Fig. 2 also shows the radioactively labelled proteoglycan that is released into the media from the extracellular matrix of the cultures expressed as a percentage of the original isotopically labelled proteoglycan. It is clear that with respect to time there is a small but significant release of radioactive material into the media. When chromatographed on Sepharose 4B CL the radioactivity was found to be associated with hexuronate-containing material that was eluted from the column in the void volume and also with material that was included into the column (Kay = 0.24) (Fig. 3a). These two peaks corresponded to the two proteoglycan peaks previously observed on Sepharose 2B with Kay values of 0.23 and 0.70. The proteoglycans extracted from the cell layers with 4 M guanidine hydrochloride and chromatographed on Sepharose 4B CL were excluded from the column (Fig. 3a). It was considered probable that the material included in the Sepharose 4B CL column was a product derived from the degradation of proteoglycans. In order to determine whether this was the case, chondrocyte cultures, which had been prelabelled with [3sS]sulphate for the first 3 days in culture and then grown in media n o t containing the isotope for 20 days were treated with media containing retinol and the protease inhibitors as described in Methods. The radioactive material appearing in the media was chromatographed on Sepharose 4B CL (Fig. 3b). It was found in chondrocyte cultures that were treated with media containing retinol, there was an increase in the release of [3SS]sulphate into the media (185% of control), and that the majority of this labelled material was included into Sepharose 4B CL when compared to the profile obtained from untreated cul-
8--
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Age of cultures (days)
Fig. 2. L o s s o f [ 3 5 S ] s u l p h a t e - c o n t a i n i n g m a t e r i a l i n t o t h e m e d i a (o e) o f c h o n d r o c y t e cultures that grown in the presence of [35S]sulphate f o r t h e first 3 d a y s in c u l t u r e . T h e r e l e a s e o f [ 3 5 S ] s u l p h a t e - c o n t a i n i n g m a t e r i a l f r o m t h e c u l t u r e s i n t o t h e m e d i a is also s h o w n (o o) a n d is e x p r e s s e d as a p e r c e n t a g e o f t h e o r i g i n a l n o n - d i a l y s a b l e [ 3 S S ] s u l p h a t e - c o n t a l n i n g m a t e r i a l o b s e r v e d in t h e c u l t u r e s had been
a f t e r t h e first 3 d a y s i n c u l t u r e .
241
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3200
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Q
1600
a
800
5
4000 1
10
15 20 25 Fraction no. (5g)
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35
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Fig. 3. S e p h a r o s e 4B C L p r o f i l e s o f p r o t e o g l y c a n s i s o l a t e d f r o m t h e m e d i a a n d m a t r i x o f c h o n d r o c y t e c u l t u r e s w h i c h h a d b e e n g r o w n in t h e p r e s e n c e o f [ 3 5 S ] s u l p h a t e f o r t h e first 3 d a y s o f c u l t u r e . (a) [3 S S ] . s u l p h a t e - l a b e l l e d p r o t e o g l y c a n f r o m t h e m a t r i x (e o) a n d m e d i a (4 ~.) o f u n t r e a t e d 2 0 - - 2 3 d a y - o l d c h o n d r o c y t e c u l t u r e s a n d t h e s a m e p r o t e o g l y c a n s s u b j e c t e d t o f l - e l i m i n a t i o n (m a ) . (b) [ 3 5 S ] s u l p h a t e - l a b e l l e d p r o t e o g l y c a n a p p e a r i n g in t h e m e d i a o f u n t r e a t e d 2 0 - - - 2 3 - d a y - o l d c h o n d r o c y t e c u l t u r e s (A ~) a n d in t h e s a m e age c u l t u r e s t r e a t e d w i t h r e t i n o l (o o) a n d p r o t e a s e i n h i b i t o r s
(u
a).
tures. When the cells were treated with media containing the protease inhibitors there was a marked decrease in the release of [3SS]sulphate into the media (43% of control) and the majority of this labelled material was exluded from the gel relative to the control. The included and excluded peak when treated with alkali were both found to co-elute from Sepharose 4B CL in the same position as ~-eliminated proteoglycan. In order to determine whether the foetal calf serum contained proteolytic activity, a sample of the serum used in this work was incubated with proteoglycan isolated from the c h o n d r o c y t e layers
242 T A B L E III COMPOSITION
OF G L Y C O S A M I N O G L Y C A N S
SYNTHESIZED
BY 20-DAY-OLD CHONDROCYTE
CULTURES Percent radioactivity incorporated into glycosaminoglycans
59 17 12 3 8
Chondroitin 6-sulphate Chondroitin 4-sulphate Chondroitin H y a l u r o n i c acid Keratan s u l p h a t e
before it was analysed on Sepharose 4B CL. No change in the elution profile of the proteoglycan was observed.
Characterization of the glycosaminoglycans synthesized by chondrocytes grown in high density cultures The chondrocytes were grown in culture over a period of 20 days with [U14C]glucose, and the glycosaminoglycans present in the matrix isolated and digested with chondroitin lyase ABC. The resulting disaccharides were analysed by paper chromatography. As the cells had been grown with isotopic glucose since the time of inoculation, the amount of radioactivity in each disaccharide was used as a direct measure of the amount of the corresponding glycosaminoglycan present in the matrix. Table III shows the distribution of glycosaminoglycans isolated from the matrix of 20-day-old cultures. About 8% of the total radioactivity was observed to remain at the origin of the chromatograph and was presumed to be keratan sulphate, which is not digested with chondroitin
] i E
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vt
~
100C
- 5 o
--4O o
80C
60C
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-2o
40
b
o
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20C
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rr
20
30
40
50 Fraction
60 no.
70
8O
(Sg)
Fig. 4. S e p h a d e x G - 2 0 0 e l u t i o n profile c h o n d r o c y t e cultures. R a d i o a c t i v i t y ( e weight (o-o) are s h o w n .
of
[3H]-end-labelled glycosaminoglycans from 20-day-old e ) , h e x u r o n i c acid ( m i ) and c a l c u l a t e d m o l e c u l a r
243 lyase ABC. Further evidence that the isotopic label is directly associated with keratan sulphate is that the ratio of glucosamine to galactosamine in the isolated proteoglycans from the matrix was observed to be 1 : 15 (Table I). The hexuronic acid and radioactivity profiles on Sephadex G-200 of the [3H]-end-labelled glycosaminoglycans isolated from the matrix of the cultures are shown in Fig. 4. From the specific activity of the borohydride, the radioactivity and the hexuronate appearing in each fraction, the molecular weight of the glycosaminoglycan in that fraction was calculated and is also shown in Fig. 4. The weight average molecular weight was 3.08 • 104 and the numberaverage molecular weight 2.60 • 104. Discussion
It was found that the physical and chemical properties of the proteoglycans isolated from the matrix of 20-day-old chondrocyte cultures were similar to those reported by other workers for proteoglycans studied in other cartilages [20--22]. Furthermore, the chemical composition and amino acid profile of the proteoglycans isolated from the matrix closely resembles those reported by Hascall et al. [23] and Deluca et al. [24] for proteoglycans synthesized by cultures of limb bud chondrocytes from 4-day-old embryonic chicks. The amino acid profile of the proteoglycans would suggest that the protein core of the proteoglycans contains both a hyaluronate binding region and a glycosaminoglycan-rich region [25]. The presence of the hyaluronate binding region was verified in the hyaluronate binding experiments where approx. 61% of the proteoglycan, isolated by gradient centrifugation under dissociative conditions, aggregated with hyaluronate. This raises the question as to the origin of the proteoglycan observed in the media. There is a population of proteoglycans that have the physical characteristics of typical proteoglycan subunits in that they are capable of forming aggregates with hyaluronate and are eluted early from Sepharose 2B. They must represent native proteoglycans that have diffused from the matrix of the cultures either as subunits or possibly as aggregates with hyaluronate. It must be pointed o u t that the physical dimensions of the cultures, a very high surface area to thickness ratio, would favour such a phenomenon. Weibkin and Muir [26] have recently shown that chondrocytes from adult pig laryngeal cartilage grown in suspension culture secrete into the media proteoglycans which can form complexes with hyaluronate. Another group of proteoglycans observed to be present in the media of the c h o n d r o c y t e cultures co-eluted with the large molecular weight proteoglycans b u t were unable to bind to hyaluronate. These proteoglycans could represent a group of proteoglycans that prior to secretion into the extracellular compartment are modified enzymatically so that they do n o t possess a hyaluronate binding region and will have the tendency to diffuse o u t of the matrix into the media. This has been suggested by Hardingham et al. [25] to explain the nature of non-aggregating proteoglycans extracted from cartilage by isotonic saline. Alternatively this group of non-aggregating proteoglycans could represent products from the first stage in the degradation of the proteoglycan complex where the hyaluronate binding region is cleaved from the proteoglycan subunit
244 prior to further degradation of the macromolecule. This has been suggested by Sandy et al. [27] to explain the appearance of high molecular weight non-aggregating proteoglycans in the media of organ cultures of rabbit articular cartilage. A population of small non-aggregating proteoglycans which is included into both Sepharose 2B and Sepharose 4B CL was also observed in the media of the chondrocyte cultures. These proteoglycans probably reflect the products of normal degradation of proteoglycan by proteolytic enzymes present in chondrocytes [28]. The addition of retinol to cultures of cartilage has been reported to increase the release of lysosomal enzymes from chondrocytes [29]. Addition of retinol to these chondrocyte cultures causes an increase in the appearance of these small molecular weight proteoglycans in the media. Addition of the protease inhibitor cocktail to the media of the chondrocyte cultures decreased the appearance of these small molecular weight proteoglycans in the media. The protease inhibitor cocktail does have an appreciable cytotoxic effect on the chondrocytes in that protein and proteoglycan synthesis is inhibited (Handley, C.J. and Lowther, D.A., unpublished data). It is therefore possible that the inhibitor cocktail is not only inhibiting protease activity, but also the synthesis and release of the enzymes from the cells. The possibility that the proteolytic activity was originating from the foetal calf serum was discounted, since no degradative activity was found in the serum towards proteoglycan. These observations coupled with the fact that the media of the cultures was maintained between pH 6.8 and 7.2 would suggest that a neutral proteoglycanase is present in the cultures and is active against the endogenous proteoglycan. Similar observations have been made for rat costal cartilage in organ culture by Wasteson et al. [30]. The possibility of free sulphate being incorporated into a low molecular weight proteoglycan that does not bind hyaluronate seems remote since very little free isotopic sulphate was observed to be present in the cultures after 20 days of culture. The spectrum of glycosaminoglycans synthesized by chondrocyte cultures after 20 days is similar to that reported for 13
