Biochimica et Biophysica Acta, 1093 (1991) 153-161 © 1991 Elsevier Science Publishers B.V. 0167-4889/91/$03.50 ADONIS 016748899100195T

153

BBAMCR 12965

Effects of glutathione depletion on the synthesis of proteoglycan and collagen in cultured chondrocytes Osami Habuchi, Toshiyuki Miyachi, Satoru Kaigawa, Satoko Nakashima, Chika Fujiwara and Miho Hisada Department of Chemistry, Aichi Kyoiku University, Kariya (Japan)

Key words: Proteoglycan; Type 1I collagen; Type X collagen; Glutathione; Buthionine sulfoximine (Chick chondrocyte)

We studied the effect of the depletion of glutathione on the synthesis of proteoglycan and collagen in cultured chick chondrocytes. When the cultured chondrocytes were incubated with I mM buthionine sulfoximine (BSO), a specific inhibitor of y-glutamyl-cysteine synthetase, the intracellular glutathione level markedly dropped within 12 h with no loss of cell viabiliUt. Incorporation of 3Sso42- into proteoglycan was lowered in the presence of BSO. When the 3ss-labeled proteoglycans were separated into two fractions by glycerol density gradient centrifugation, the inhibitory effect of BSO on the synthesis of proteoglycan was greater in the fast-sedimenting proteoglycan fraction, which consisted mainly of cartilage specific large proteoglycan (PG-H), than in the slowly sedimenting proteoglycan fraction. The inhibition by BSO of the synthesis of core protein-free glycosaminoglycan chains primed by p.nitrophenyl-/3-n-xyloside was smaller than the inhibition of the synthesis of proteoglycan. Analysis of glycosaminoglycans labeled with PH]glucosamine indicated that the treatment of chondrocytes with BSO resulted in a small increase in the" proportion of synthesis of hyaluronic acid to the synthesis of total glycosaminoglycan. The incorporation of PH]proline into collagen was also inhibited by BSO. Sodium dodecyi sulfate polyacrylamide gel electrophoresis of the 3H-labeled collagen showed that, in the presence of BSO, processing of Type I1 collagen appeared to slow down and the proportion of Type X collagen synthesis was reduced.

Introduction

Epiphyseal cartilage in developing chick embryo is made up of chondrocytes with different morphologies and different capacities for proteoglycan and collagen synthesis 1"1-3]. The rates of synthesis of proteoglycan and Type lI collagen are elevated at the zone of elongated and flattened cells (zone 2), while the synthesis of Type X collagen is initiated exclusively by the zone of hypertrophying chondroojtes (zone 3) [4,5]. The synthesis of proteoglycan and collagen in cultured chondrocytes has been investigated in relation to the differentiation of chondrocyte, and was found to be affected by various conditions including treatment with hormones and growth factors [6-10], subcultures [11],

Abbreviations: PG-H and PG-L b, cartilage specific large proteoglycan and minor cartilage proteoglycan containing dermatan sulfate, respectively; BSO, L-buthionine-(S,R)-sulfoximine; SDS, sodium dodecyl sulfate. Correspondence: O. Habuchi, Department of Chemistry, Aichi Kyoiku University, Kariya 448, Japan.

depletion of serum [12], addition of cations [13], ascorbic acid [14,15], or vanadate [16]. In addition to these conditions, exposure of chondroeytes to low oxygen tension was known to stimulate proteoglycan synthesis [17]. Oxygen toxicity is known to correlate closely to the level of intraceilular glutathione which is a major sulfhydryl compound in various cells [18]. BSO was reported as a specific inhibitor of ~,glutamyleysteine synthetase [19] and was used for decreasing intracellular glutathione level in tissues and cultured cells [20,21]. In this paper, we report the biosynthesis of proteoglycan and collagen in the presence of BSO. We show that depletion of glutathione with BSO reduces the rate of the biosynthesis of both proteoglycan and collagen and affects the processing of collagen. Materials and Methods Materials The following commercial materials were used: H~5SO4 (carrier free) from Japan Radioisotope Assc,ciation, Tokyo; D-[6-3H]glucosamine (27 Ci/mmol), L-

154 [2,3-3H]proline (51 Ci/mmol) and L-[4,5-3H]leucine (70 Ci/mmol) from Amersham Japan, Tokyo; Dulbecco's modified Eagle's medium and fetal bovine serum were obtained from GIBCO-Oriental, Tokyo; trypsin (from bovine pancreas, Type II), glutathione, glutathione reductase, L-buthionine-(S,R)-sulfoximine, streptomycin sulfate and penicillin G, from Sigma Chemical Co. St. Louis, MO; NADPH from Boeringer-yamanouchi, Tokyo; Pansorbin (Staphylococcus aureus cells)from Hoechst Japan, Tokyo; chondroitinase ABC, chondroitinase ACII, keratanase, heparinase and heparitinase from Seikagaku Kogyo Co., Tokyo; Pronase from Kaken Seiyaku, Tokyo; DEAESephacel from Pharmacia Japan; Tokyo; collagenase (Worthington, CLS II) from Funakoshi, Tokyo; and Enlightning from Du Pont/NEN, Boston, MA. Anti PG-H rabbit antibody and affinity purified anti PG-L h rabbit antibody were generously donated from Dr. T. Shinomura, Institute for Molecular Science of Medicine, Aichi Medical University.

Chondrocyte culture Chondrocytes were prepared from a mixture of zone 1, zone 2 and zone 3 of the distal end of the tibiotarsus of 12-day chick embryos and cultured as described [22-24]. Chondrocytes were plated in 6-cm culture dishes at a density of 2 8 . 104/5 ml of medium. The medium in which the cells were plated consisted of Dulbecco's modified Eagle's medium adjusted to pH 7.0 containing 2 g/! of v-glucose, penicillin (100 units/ml), streptomycin (50 /zg/ml) and 10% fetal bovine serum and cells were grown at 380C in 7% CO2/93% air. The medium was changed on days 2, 4, 7 and 9 with fresh medium at a pH of 7.4. On day 9, preincubation with 1 mM BSO was commenced. After preincubation, the medium was replaced with fresh medium containing 1 mM BSO and a radioactive precursor was added to the medium. BSO was omitted from the control culture during both preincubation and labeling periods. When the cultures were labeled with [aH]proline for determining collagen synthesis, the labeling medium was supplemented with 100 mg of flaminopropionitril, and 100 mg of L-ascorbate/l, and the concentration of fetal bovine serum was reduced to 0.5%. The number of cells were counted on a hematocytometer. Cell viability was determined as the percentage of cells excluding Trypan blue [25].

Determination of total glutathione Preparation of the samples from cultured chondrocytes and determination of glutathione was basically as described previously [26]. The dishes were chilled on ice and the medium was removed. The cell layer was

rinsed with ice-cold Tris-saline twice, and then removed from the dishes in 1 ml of 10 mM HCI with the aid of a rubber policeman. Collected cells were lysed by freezing in liquid nitrogen and then thawing; this freezing-thawing process was repeated three times. After centrifuging at 10000 x g for 5 rain, 50/zl of 10% (w/v) 5-sulfosalicylic acid was added to 100 ~I of the supernatant solution, and the protein was removed by centrifugation for 5 rain. The 25-t.d aliquots of the final supernatant solution was used for the enzymatic recycling assay for total glutathione [27].

Extraction and separation of 3SS-labeled proteoglycan After preincubation with 1 mM BSO, proteoglycan was labeled with 3sSO42- in the presence of BSO. The concentration of radioactive sulfate and the period of preincubation and labeling were indicated in the individual experiments. After labeling, proteoglycan from the culture medium and the cell layer was extracted with 4 M guanidine chloride containing proteinase inhibitors as previously described [24]. The final proteoglycan fractions were dissolved in 0.5 ml of 4 M guanidine chloride solution with the proteinase inhibitors and 50/zl aliquots were used for determining the total proteoglycan synthesis. To the 4 M guanidine chloride solution of the 35Slabeled proteoglycan obtained from three culture dishes, 3 vols of ethanol containing 1.3% potassium acetate was added. The precipitates were suspended in water and precipitation with the ethanol solution was repeated three times. The final pellets were dissolved in 0.02 M Tris-HCl (pH 7.2)/7 M u r e a / 0 . 2 % Triton X-100 and applied to a column of DEAE-Sephacel (bed volume, 1 ml). After washing the column with 5 ml of the 7 M urea solution, the adsorbed materials were eluted with 30 ml of a solution in which the concentration of NaCl in the 7 M urea solution increased linearly from 0 to 0.75 M. 3sS-labeled materials eluted in a single peak around 0.4 M of NaCI. The recovery of radioactivity from this column was above 80%. The peak fractions were pooled and proteoglycan was precipitated with three volume of ethanol containing 1.3% potassium acetate. The pellets were dissolved in 0.5 ml of the 4 M guanidine chloride solution. Glycerol density gradient centrifugation in the presence of 4 M guanidine chloride was carried out as described by Kimata et al. [28]. After centrifugation was performed at 24 000 rev/rpm, 20°C for 44 h using an Hitachi RPS 27-3 rotor, 0.6-ml fractions were collected. The radioactivity of 20/L! of each fraction was measured. Proteoglycan fractions indicated by the bars in Fig. 3, were pooled separately, precipitated with 3 vols of ethanol containing 1.3% potassium acetate and dissolved in 50 mM Hepes buffer (pH 8.0)/2% SDS/10 mM EDTA.

155

Preparation of glycosaminoglycans labeled with [3H]glucosamine After preincubation with the medium containing 1 mM BSO for 12 h, chondrocyte cultures were labeled with [3H]glucosamine (10 /~Ci/ml) for 12 h in the presence of 1 mM BSO. After labeling, the culture medium was collected, and the cell layer was washed with 1 ml Tris-saline. The medium and washes were combined. To the combined medium fraction was added 5 M NaOH at a final concentration of 0.5 M. The rinsed cell layer was covered with 1 ml of 0.5 M NaOH. The reaction with alkali was carried out at 4°C for 24 h. After neutralization, the alkali-treated samples were digested with pronase (1.0 mg/ml) in 0.05 M Tris-HCI (pH 8.0) for 2 h at 37°C. To the pronase digests were added 1/10 vol of 50% trichloroacetic acid and the mixtures were chilled on ice for 30 min and the resulting precipitates were removed by centrifugation for 10 rain at 10000×g. The supernatant fluids were dialyzed against water and lyophilized.

Immunoprecipitation of 35S-labeled proteoglycans Immunoprecipitation was carried out using rabbit antiserum raised against PG-H and PG-L b [29]. As a carrier of protein A, S. aureus cells were used as described previously [30]. 20 /~1 of the proteoglycan solution was added to 400/~1 dilution buffer (50 mM Hepes (pH 7.4)/1% Nonidet P-40/1% sodium deoxycholate/0.15 M sodium chloride/10 mM EDTA/0.1 M 6-aminocaproic acid/10 mM N-ethyl maleimide/5 mM benzamidine hydrochloride) and 10 ~! antibody solution was added. After 16 h at 4°C, 150/xl 10% S. aureus cell suspension was added and shaken for 3 h at 4°C. The immunocomplexes bound to S. aureus were precipitated, washed with the dilution buffer once, extracted with 0.5 M NaOH and the radioactivity was counted.

Enzymatic analysis of the ssS- and "~H-labeled glycosaminoglycans After being precipitated with 75% ethanol to remove SDS, the 3sS-labeled proteoglycans were digested with pronase (1 mg/ml) in 0.05 M Tris-HCI (pH 8.0) at 37°C for 2 h. The reaction was stopped by heating for 5 min. To the pronase digests 3 vols. of ethanol containing 1.3% potassium acetate was added, and resulting precipitates were collected by centrifugation. 35Slabeled glycosaminoglycan fractions thus obtained were separated into four parts and each part was digested with any one of the following enzyme combinations for 4 h at 37°C: (1) chondroitinase ACII (0.8 unit/ml) [31]; (2) chondroitinase ACII (0.8 unit/ml) and chondroitinase ABC (0.8 unit/ml) [32]; (3) chondroitinase ACII (0.8 unit/ml), chondroitinase ABC (0.8 unit/ml), heparinase (0.08 unit/ml) [33] and heparitinase (0.08 unit/ml) [33]; and (4) chondroitinase ACII (0.8

unit/ml), chondroitinase ABC (0.8 unit/ml) and keratanase (36 units/ml) [34]. 3H-labeled glycosaminoglycan fractions extracted from chondrocyte cultures were digested with the combination (1) and (2). The buffer used for these enzymatic reactions was 50 mM Tris-acetate (pH 7.5), containing 0.1 mg/ml bovine serum albumin except that 1 mM C a C I 2 w a s supplemented in the combination (3) reaction mixture. After digestion, the samples were spotted on strips (2.5 cm × 57 cm) of Whatman No. 3 chromatography paper. Unsaturated disaccharides formed from repeating units of chondroitin 6-sulfate, chondroitin 4-sulfate, chondroitin and hyaluronic acid were separated by developing the paper with butan-l-ol/acetic acid/1 M NH~ (2: 3 : 1, v/v) for 20 h. The dried strip was cut into 1.25 cm segments which were counted in a scintillation fluid containing 5 g of diphenyl oxazole and 0.5 g dimethyl 1,4-bis(2-(5-phenyloxazolyl))benzene per liter of toluene. The proportion of dermatan sulfate, heparan sulfate and keratan sulfate was calculated from the difference in the radioactivity remaining at the paper origin between the combinations (1) and (2), between the combinations (2) and (3), and between combinations (2) and (4), respectively.

Extraction and SDS polyacrylamide gel electrophoresis of [3H]proline-labeled collagen After the cultures were preincubated with 1 mM BSO for 12 h, they were incubated with [3H]proline (5 /zCi/ml) for 12 h in the presence of 1 mM BSO. Culture medium collagen pool and cell matrix collagen pool were extracted and precipitated with 30% ammonium sulfate by the methods of Schmid and Conrad [35]. Precipitates with ammonium sulfate were dissolved in 3 ml of 0.15 M potassium phosphate buffer (pH 7.6) and dialyzed against 150 mM NH4HCO 3 containing 2 mM sodium EDTA. Samples of 50/zl of the dialyzed solution were used for determining radioactivity. From the residual cell layer, intracellular protein was extracted with 1% SDS. The incorporation of the radioactivity into the intraeellular protein fraction, however, was not included in the collagen synthesis, since the intracellular protein fraction showed no protein bands after limited pepsin digestion in SDS polyacrylamide gel electrophoresis (data not shov, n). SDS-polyacrylamide gel electrophoresis was carried out with 4% (w/v) stacking gel and 6% (w/v) separating gel [36] before or after limited pepsin digestion [35]. Samples were electrophoresed after disulfide-bond reduction. After gel electrophoresis, the gel was fixed with methanol/acetic acid/water (25:7:68, v/v), immersed in a diluted solution of Enlightning (Enlightning/water/ethanol, 5:3:2, v/v), and exposed to XOmat AR film (Eastman Kodak) for 2 weeks at -80°C. The fluorogaram was scanned with a densitometer

156 (Bio.Rad Model 165011) and the relative intensity of the bands in each lane was calculated.

Determination of total protein synthesis After preincubation with 1 mM BSO for 12 h, [3H]leucine (10 /tCi/ml) was added to the culture medium. After labeling for 12 h in the presence of 1 mM BSO, the culture medium was removed and the cell layer was washed with Tris-saline. The washed cell layer was scraped from the dishes in a total of 2 ml of Tris-saline and homogenized with a glass homogenizer. To the culture medium and the homogenate of the cell layer, 50% trichloroacetic acid containing 5 mM leucine was added to a final concentration of 5%. The mixtures were centrifuged at 1000 x g for 5 rain. The precipitates were washed with 5% trichloroacetic acid containing 1 mM leucine three times, and the final precipitates were dissolved in 0.5 ml of 0.2 M NaOH. The radioactivity of 0.1 ml aliquots of the solution was determined.

Effects of glutathione depletion on biosynthesis of total proteoglycan, collagen and total protein Fig. 2 shows the incorporation of radioactivity into total proteoglycan (A), collagen (B) and total protein (C), when the late log cultures of chondrocytes were preincubated with 1 mM BSO for 12 h and labeled with 35SO2- (20 /zCi/ml), [3H]proline (10 /~Ci/ml) and [3H]leucine (10 ~tCi/ml) for 12 h in the presence of 1 mM BSO. Incorporation into the sum of the cell layer (for collagen, cell matrix) and the culture medium was inhibited by 36%, 30% and 51% for total proteoglycan, collagen and total protein, respectively. The incorporation into total proteoglycan of the culture medium was more affected by the addition of BSO than that of the cell layer, while the incorporation into total protein was more inhibited in the cell layer than in the culture medium. The incorporation into the cell matrix collagen and the incorporation into the culture medium collagen was inhibited by BSO to a similar extent.

Results

Analysis of SsS-labeledproteoglycans produced by chondrocytes in the presence or absence of BSO

Effects of BSO on intracellular levels of glutathione in cultured chondrocytes

Proteoglycan labeled with 35SO42- (70/zCi/ml) for 3 h in the presence of 1 mM BSO after 24 h preincubation with 1 mM BSO was subjected to glycerol density gradient centrifugation, and both the cell layer proteoglycan and the medium proteoglycan were separated into two fractions with different sedimentation rates (Fig. 3). The fast-sedimenting fractions obtained from the cell layer and the culture medium are denoted by C-PG-I and M-PG-I, respectively, and the slowly sedimenting fractions from the cell layer and the culture medium are denoted by C-PG-II and M-PG-II, respectively. The sedimentation rate of C-PG-I was the same as that of M-PG-I and was not altered in the presence of BSO. M-PG-II sedimented as a distinct peak but C-PG-II showed no peak. As shown in Table I, most of the C-PG-I and M-PG-I was precipitated with anti PG-H antibody. Table II shows that C-PG-I and MPG-I contained keratan sulfate in addition to chondroitin sulfate. These results indicate that C-PG-I and M-PG-I largely consisted of PG-H. M-PG-II reacted not only to anti PG-H antibody but also to anti PG-L b antibody and contained dermatan sulfate in addition to chondroitin sulfate, suggesting that M-PG-II contained PG-L b contaminated by PG-H. C-PG-II, on the other hand, contained dermatan sulfate and heparan sulfate in addition to chondroitin sulfate. It is not clear whether these glycosaminoglycans contained in C-PG-II attached to the same proteoglycan or not. The incorporation of 35S radioactivity into these proteoglycan fractions was calculated from the separation pattern shown in Fig. 3 and presented in Table I. The syntheses of all of these proteoglycans were inhibited by the addition of BSO; however, the degree of

Fig. 1 shows a time course of the level of intracellular glutathione in the presence or absence of 1 mM BSO from day 9 to day 10. In the presence of BSO, the contents of glutathione decreased linearly up to 8 h, and was maintained at a low level hereafter; after 24 h, the level of glutathione became 12% of the control. When chondrocytes were exposed to 1 mM BSO from day 9 to day 10, the cell number on day 10 was almost the same as that of the control culture and viability of chondrocytes were more than 90% on day 10.

2O

%

t 0

. 4

I 8

l 12 Time

I 16

I 20

I 24

(h)

Fig. I. Time course of reduction of intracellular glutathione level of cultured chondtocytes by BSO. On day 9 of the culture, the culture medium was replaced with fresh medium containing 0 mM BSO (o) or 1 mM BSO (e). At the indicated time, extracts were prepared from the cell layer and assayed for glutathione as described in Materials and Methods.

157 A

4

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06

06

C -PG-I

J.

C - P G - II

24

% 3 "

04

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04 16

2

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Fig. 2. Effects of BSO on the synthesis of total proteoglycan, collagen and total protein. Incorporation of 3SSO~- into proteoglycan (panel A), ['~H]proline into collagen (panel B) and ['~H]leucine into total protein (panel C) was determined in the presence or absence of 1 mM BSO as described in Materials and Methods. The open bars represent the incorporation into cell layer fractions (for collagen in panel B, cell matrix fractions) and the hatched bars represent the incorporation into the culture medium fractions. Each value is the mean of duplicate cultures.

the inhibition was significantly larger in the fast-sedimenting proteoglycans (C-PG-I and M - P G - I ) than in the slowly sedimenting preteoglycans ( C - P G - I I and MPG-II). Relative values of 35SO42- incorporation into various glycosaminoglycans derived f r o m the 3sS-labeled proteoglycans were d e t e r m i n e d (Table II). Although changes in the relative values caused by the addition of BSO were small, the proportion o f chondroitin 6-sulfate

TABLE I

Effects of BSO on the incorporation of 3SSOJ- into two distinct proteoglycan fractions and immunoreactivity of these proteoglycan fractions incorporation of 35S radioactivity into each proteoglycan fraction was determined by the sedimentation profiles shown in Fig. 3. The methods of immunoprecipitation are described under Materials and Methods. Incorporation values represent averages+ ranges of duplicate determinations. Fraction

C-PG-I C-PG-II /.t-PG-I M-PG-1[

BSO

+ + + +

35S incorporation a (cpm x 10-5/ 106 cells) 7.48 + 0.35 2.97+0.10 (0.40) 0.37 +_0.07 0.21 + 0.02 (0.57) 2.05 + 0.11 0.56 + 0.03 (0.27) 0.45+0.01 0.27+0.01 (0.60)

Ratio of precipitates formed with antibodies against PG-H

PG-Lb

0.89 0.86 0,46 0.34 0.89 0.87 0.17 0.11

< 0.02 < 0.02 0.03 < 0.02 < 0.02 < 0.02 0.15 0.15

a Each value in parenthesis represents a ratio of the incorporation in the presence of BSO to the incorporation in the absence of BSO in each proteoglycan fraction.

I 5

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I

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15

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B

M - PG - I I

M-PG-I

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0 I 5

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Bottom

I

I

I

15

20

25

number

Top

Fig. 3. Separation of 35S-labeled proteoglycans with glycerol density gradient centrifugation. Proteoglycans labeled with 35SO4- were extracted from the cell layer (panel A) and the culture medium (panel B) in the presence ( e - - - e ) or absence (0 - - - o ) of 1 mM BSO. The fractions indicated by bars were pooled and used for immunoprecipitation and analysis of glycosaminoglycans.

in C - P G - I I and M - P G - I ! had a tendency to decrease when BSO was added. T h e proportion of heparan sulfate in C - P G - I I and the proportion of dermatan sulfate in M - P G - I I were slightly increased. These small changes observed in C - P G - I I and M - P G - I I , however, may only reflect the decrease of the contamination of P G - H as shown in the immunoprecipitation experiments of T a b l e I. After digestion of C-PG-1I with chondroitinase ABC, heparinase, heparitinase and keratanase, more than 10% of the digests remained at the p a p e r origin, but the resistant material was not further examined.

Effect of BSO on the synthesis of proteoglycan in the presence of p-nitrophenyl-fl-D-xyloside T o examine w h e t h e r depletion of glutathione inhibited glycosaminoglycan chain elongation directly or affected glycosaminoglycan synthesis indirectly through

158 TABLE II

Analysis of the incorporation of 3SSOZ4- into rarious glycosaminoglycans contained in the proteoglycan fractions Glycosaminoglycans were prepared from the proteoglycan fractions presented in Table I. The methods of analysis of the incorporation into various glycosaminoglycans are described under Materials and Methods. Values represent averages of duplicate determinations. C6S, C4S, DS, HS and KS represent chondroitin 6-sulfate, chondroitin 4-sulfate, dermatan sulfate, heparan sulfate and keratan sulfate, respectively. Fraction

Percent of the incorporation of 3Sso2- into various glycosaminoglycans ~)

BSO

C-PG-I

+ + + +

C-PG-I! M-PG-! M-PG-II

C6S

C4S

DS

HS

73.9 72.0 57.9 51.4 73.1 71.6 47.1 42.2

20.6 22.6 19.1 21,9 21,2 23,5 31.8 33.4

< 2,0 < 2,0 5.2 6,5 < 2.0 2.2 14.9 16.7

< 2.0 < 2.0 4.8 6.9 < 2.0 < 2.0 < 2.0 < 2.0

KS

< < < <