Secretion of proteoglycans by chondrocytes

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 192, No. 1, January, pp. 148-15’7, 1979

Secretion Influence

of Proteoglycans

of Colchicine,

S. LOHMANDER,’ Department

of Histology,

Cytochalasin

K. MADSEN,

Karoliwka

by Chondrocytes B, and /I-D-Xyloside AND

A. HINEK2

In,stitu,tpt, S-704 01 Stockh,olm 60, Sweden

Received June 20, 1978; revised September

13, 1978

Chondrocytes obtained from epiphyseal cartilage of fetal guinea pigs or ear cartilage of young rabbits were cultured in monolayer. The influence of colchicine, cytochalasin B, and p-nitrophenyl-P-D-xylopyranoside on secretion of proteoglycans was investigated. Radioactive sulfate was used as a precursor. As observed previously in other systems, /%Dxylosides initiated the synthesis of free chondroitin sulfate chains, competing with the endogenous proteoglycan core protein acceptor. The molecular weights of the chondroitin sulfate chains synthesized both on the xyloside and on the core-protein acceptor in maximally stimulated cells were similar and significantly lower than in proteoglycans synthesized in the absence of xyloside. The size of the chondroitin sulfate chains synthesized on the xyloside was inversel) related to the concentration of this compound. This finding suggests that the chain length is dependent on the ratio between available acceptor and chain-lengthening enzymes or precursors. Cytochalasin B, a microfilament-modifying agent, inhibited proteoglycan synthesis, without any effect on secretion. Cells treated with cytochalasin B could be stimulated with P-D-xylosidc to synthesize free chondroitin sulfate chains to the same relative degree as cells with intact microfilaments. Colchicine, an antimicrotubular agent, partially inhibited synthesis and secretion of proteoglycan. However, cells treated with colchicine Lo synthesize and swrete free chondroitin sulfate could be stimulated with P-D-xykmide chains to about the same relative degree as cells with intact microtubules. The data suggest that microtubules may have a facilitatary rather than an obligatory role in the secretion of proteoglycans and that at least part of the effect of colchicine is located at or after the site of glycosaminoglycan synthesis.

Because colchicine inhibits insulin secretion, Lacy and co-workers (1) proposed, in 1968, that microtubules were involved in cell secretion. Colchicine and other antimicrotubular agents have similar effects on secretion of matrix components by fibroblasts, osteoblasts, and chondrocytes. Consequently, it has been suggested that microtubules play an active role in the secretion of collagen, elastin, and proteoglycan in these cell types (2-8). Morphological (9) and chemical (10) data

have suggested that the effects of microtubule-depolymerizing agents on matrix deposition and proteoglycan secretion in chondrocyte cultures are the result of functional disturbances in the microtubular system that cause secondary alterations in the Golgi complex. These changes in the Golgi complex, together with a retarded translocation of secretory vacuoles, could inhibit the secretion of proteoglycan. However, colchicine-treated chondrocytes retain a large part of their biosynthetic and secretory activities. This observation suggests that alternative pathways, independent of functional microtubules, exist for the secretion of matrix molecules and/or that the Golgi complex retains a major part of its function despite structural alterations (10). Microfilaments may play a role in intracel-

’ Present address: Laboratory of Biochemistry, National Institute of Dental Research, Building 30, Room 106, National Institutes of Health, Bethesda, Maryland 20014. To whom all correspondence shuuld be addressed. 2 On leave from the Department of Anatomy, Medical Academy, Warsaw, Poland. 0003-9861/‘79/010148-10$02.00/O Copyright G 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.

148

SECRETION

OF PROTEOGLYCANS

Mar transport phenomena (1, 11,12) as well as in cell-surface events such as pinocytosis or exposure of cell-surface hormone receptors (13-15). Furthermore, microfilaments may act as a cytoskeleton and thereby maintain cell shape and intracellular organization (16, 1’7). Cytochalasin B, a microfilament modifier, inhibits cellular uptake of glucose and glucosamine (18, 19). Possibly, this ability influences the synthesis of glycosaminoglycans in fibroblasts (19, 20). p-D-Xylosides can act as exogenous acceptors for the first galactosyltransferase in the synthesis of chondroitin sulfate chains in chondrocytes by competing with endogenous proteoglycan core protein acceptor (21, 22). These compounds permit study of glycosaminoglycan synthesis independent of proteoglycan core-protein synthesis. This study was undertaken to determine further details of the possible role of microtubules and microfilaments in the secretion of proteoglycans in chondrocytes. P-DXylosides were used to stimulate glycosaminoglycan synthesis in an attempt to localize intracellular sites where inhibitory effects of colchicine and cytochalasin B on proteoglycan secretion occur and to determine the capacity of secretory pathways independent of microtubules. Some of these results have been presented in preliminary form (23, 24). EXPERIMENTAL

PROCEDURES

Isolation nrld culture of’chomdrocytes. Chondrocytes were isolated from epiphyseal cartilage of guinea pig fetuses at 40-50 days gestation or from ear cartilage of l-kg rabbits by digestion with collagenase and DNase as described previously (10). For both types of cartilage, a 30.min preliminary digestion was used to remove perichondral cells, which were then discarded. With fetal epiphyseal cartilage, the concentration of collagenase was decreased from 0.5 to 0.1%. Cells were grown in medium F-12 (25) supplemented with 10%’ fetal calf serum, 0.3% tryptose phosphate broth, and with 50 pg of L-ascorbic acid, 150 pg of streptomycin sulfate, and 150 U of benzylpenicillin per milliliter. Variations in pH were minimized by buffering the medium with 10 mM Hepes” and 10 mM Tes buffers (26). An atmosphere of 5% CO, in air was used. :’ Abbreviations used: Hepes, 4-&hydroxyethyl)-lpiperazineethane sulfonic acid; Tes, 2-{ [2-hydroxy-I, 1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid.

BY CHONDROCYTES

149

Cells were cultured in 35-mm petri dishes or ‘75.cm’ tissue culture bottles (Falcon Plastics, Los Angeles, California) at initial densities of about 1.5 X loj cells/ cm2. Cells were allowed to attach to the dishes for about 18 h. The medium was then changed and the cells were incubated with drugs and isotope. Preincubation with drugs continued for 90 min, the isotope was added, and incubation was continued for another 4 h in the presence of the same drug at the same concentration. A concentration of 2-10 PCi of Nai’ SO, or 5 PCi of L-[c-“Hlproline was used per milliliter. Isolation, qunntitution, and characterization o.f glycosaminoglycans. These procedures have been described in detail previously (IO). Briefly, cell layers and media were digested with papain (27) and the glycosaminoglycans were fractionated by column chromatography on ECTEOLA-cellulose (28, 10). Alternatively, the cell layer was extracted with 1 ml of 4 M guanidine’HC1, 0.05 M NaAc, pH 5.8, for 1 h at 4”C, after which the unextractable residue was digested with papain (27). After separation of aliquots of medium, extract, and papain digest on columns of Sephadex G-25 in 4 M guanidine.HCl (44), the incorporation of “sSO, into macromolecular material was determined. The 4 M guanidine. HCl extract contains proteoglycan from the matrix surrounding the cells, while the papain digest represents mainly intracellular proteoglycan (44). Glycosaminoglycans were characterized by paper chromatography (29) after digestion with chondroitinase ABC or AC. The size of papain-digested glycosaminoglycans was assessed by chromatography on Sephadex G-200 (Lot 462, Pharmacia Fine Chemicals, Uppsala. Sweden). Isolation axd charactetization o~“labeled proteoglycans. The procedures were essentially as described previously (10). In brief, proteoglycans were isolated from culture media and guanidine. HCl extracts (30) of cell layers by ultracentrifugation in CsCl gradients under associative conditions (31). Proteolysis was minimized by including the following protease inhibitors during extraction and ultracentrifugation: 0.01 M Na,’ EDTA, 0.1 M 6-aminohexanoic acid (Kabi, Stockholm, Sweden), and 0.005 M benzamidine.HCl (32). Three fractions were collected from the density gradients, Al (density above 1.70 g/ml), A2 (density 1.70-1.63 g/ml), and A3 (density below 1.63 g/ml). Proteoglycan fractions were chromatographed on columns of Sephadex G-200. Quantitation of labeled collagen. This was performed by ion-exchange chromatography of labeled hydroxyproline and proline (10). RESULTS

AND DISCUSSION

The details of the culture system utilized in this study have been described previously (9, 10). The chondrocytes attach to the bottom of the flask within 4-6 h. After 24 h

150

LOHMANDER,

MADSEN, AND HINEK TABLE I

INHIBITIONOFPROTEOGLYCAN

SYNTHESISANDSECRETIONBY

COLCHICINE~

Cell layer

Control Colchicine

1O-8M IO-’ M Io-6M lo-5 M

Medium

cm

SD

A%

w

7829 8272 5127 6206 4406

4921 1544 1565 855 1814

+6 -35 -21 -44

4638 4246 2124 2292 1740

SD 2184 919 701 450 719

A%

-8 -53* -51**

-62***

a Guinea pig chondrocytes were preincubated with colchicine at the given concentration for 90 min, 5 PCi of N~35S04/ml of medium was added, and the incubation was continued for 4 h with the same concentration of colchicine. Incorporation of radioactivity into glycosaminoglycans of papain-digested aliquots of medium and cell layer was then determined. The statistical significance of the difference of means between control and treated groups - _ was tested with Wilcoxon’s rank sum test. The different P values are indicated by asterisks. Each value represents the mean and SD for 10 culture plates. * P < 0.05. **p < 0.01. *** P < 0.001.

in culture, the cells form a partly confluent layer of dividing cells, with most cells flattened and polygonal in shape. Electron microscopic studies (9, 10, 34) have shown that the cells have a prominent endoplasmic reticulum and Golgi complex and that they are surrounded by an intercellular matrix containing collagen, proteoglycan, and, in the case of cells from elastic cartilage, elastin. Fetal guinea pig epiphyseal chondrocytes and rabbit ear chondrocytes cultured under conditions similar to those used here synthesize and secrete proteoglycan with properties similar to those present in hyaline cartilage (10, 33; Madsen and Lohmander, unpublished work). In the present study, both cell types were used and, unless otherwise stated, gave similar results. Quantitative Effects of Drug Treatment on Synthesis and Secretion of Proteoglycan

The results of a dose-response experiment on the effect of colchicine on synthesis and secretion of proteoglycan are given in Table I. A statistically significant inhibition was reached at 10m7M colchicine. Increasing the colchicine level loo-fold did not significantly increase the level of inhibition, nor did changing the time of preincubation with

colchicine between 1 and 4 h alter cellular response (Table II). In subsequent experiments a colchicine concentration of 10m5M was used to allow comparison with earlier studies (9, 10). At lop5 M colchicine the intracellular levels of macromolecular label were significantly increased while those of both intercellular matrix and medium were significantly decreased (Table III). After a 40-min incubation with 10-j M colchicine, no microtubules could be observed in the chondrocytes (9). This suggests that, in the absence of microtubules, there is a partial inhibition of secretion of proteoglycans in chondrocytes. It has previously been shown that there are no structural changes in the chondroitin sulfate chains and only minor changes in the structure of the proteoglycans synthesized in chondrocytes treated with colchicine (10). Evidently, chondrocytes partially retain their ability to synthesize and secrete proteoglycan molecules in the absence of functional microtubules. Cytochalasin B inhibited total proteoglycan synthesis by about 30% when used at a concentration of 1 pg/ml (Table III). However, there was no significant increase in the intracellular level of radioactivity. This observation suggests that cytochalasin B does not affect the secretion of proteoglycan. By contrast, colchicine seems to retard

151

SECRETIONOFPROTEOGLYCANSBYCHONDROCYTES TABLE

II

EFFECT OF INCREASING PREINCUBATION TIME WITH COLCHICINE ON PROTEOGLYCAN SYNTHESIS AND SECRETION” Medium

Cell layer Control

Treated

Control

Treated

cpm

SD

cpm

SD

A%

cpm

SD

cpm

SD

A%

0 min 30 min

3068 2778

1945 435

1302 1426

407 477

-58** -4g***

1087 1370

342 271

545 390

150 95

-50*** -72***

60 min 90 min

2300 2583

612 505

1567 1473

435 483

-32* -43**

1375 1599

510 365

616 578

174 136

-55** -64***

4h 24 h

2954 5407

1359 1604

1138 1558

364 334

-61** -71***

1592 2502

954 685

475 542

70 130

-7o*** -78***

Preincubation

a Guinea pig chondrocytes were preincubated with or without lo-” M colchicine for O-24 h followed by a 30min labeling with Na,%O, in the presence or absence of lo-” M colchicine. The incorporation of radioactivity into glycosaminoglycans of papain-digested aliquots of medium and cell layer was then determined. Each value represents the mean and SD for five culture plates. See Table I, Footnote n and asterisk footnotes, for an explanation of the P values.

TABLE

III

EFFECTS OF COLCHICINE, CYTOCHALASIN B, AND p-D-XYLOSIDE ON GLYCOSAMINOGLYCAN SYNTHESIS AND SECRETION” Cell layer Extract

Residue

Medium

cpm

SD

A%

cpm

SD

A%

cpm

SD

(A) Control (B) Colchicine, 10m9M (C) Cytochalasin B, 1 pgiml

368 430 393

88 49 42

+ 17* +7

3234 2274 2078

544 475 178

-3o** -36***

1583 840 1125

197 66 84

(D) Colchicine + cytochalasin

538

35

+46**

1817

325

-44***

566

92

-64*“*

186

21

-50***

2300

19.5

-2g***

9659

720

+510***

275

53

-25**

2961

423

-9

4489

800

+184***

190

51

-49**

2052

375

-37***

6662

1184

+321***

B

(E) P-D-Xyloside, 5 x 10m4M (F) P-D-Xyloside + colchicine (G) /?-D-Xyloside + cytochalasin B F G F G

vs. vs. vs. vs.

E E B C

+48** +2 -36** -52***

+29** -11* -30** -1

A% -47*** -2g***

-54*** -31** +434*** +493***

‘I Rabbit chondrocytes were preincubated with or without drugs for 90 min, 10 &i of Na,““SO,/ml of medium was added, and the incubation was continued for 4 h. The incorporation of radioactivity into glycosaminoglycans in aliquots of medium, 4 M guanidine. HCl extract, and papain-digested residue was then determined. Each value represents the mean and SD for 10 culture plates. See Table I, Footnote a and asterisk footnotes, for an explanation of the P values.

152

LOHMANDER,

MADSEN,

secretion in addition to inhibiting proteoglycan synthesis [Table III; see also Ref. (lo)]. Treatment of the cells with a combination of colchicine (10-j M) and cytochalasin B (1 pg/ml) resulted in a greater inhibition than when either drug was used separately. This finding suggests that the modes of action of each drug differ (Table III). P-DXylosides stimulate the incorporation of labeled precursors into glycosaminoglycan chains in various cell types by acting as exogenous acceptors at the second glycosyl transfer step (21, 22, 35, 36). Incubation of chondrocytes with p-nitrophenyl-P-Dxylopyranoside at concentrations of from 5 x 10e6to 2.5 x 10m3M increasingly stimulated glycosaminoglycan synthesis (Fig. 1). The majority of material synthesized by the cells in the presence of ,6-D-xyloside was recovered in the medium (Fig. l), a finding that is consistent with preferential incorporation of label into free chondroitin sulfate chains which rapidly diffuse into the medium. With 5 x 10e4 M P-D-xyloside, a maximal stimulation was achieved with a 30-min preincubation with /3-D-xyloside followed by a 30-min incubation with P-Dxyloside in the presence of isotope. Extension of the preincubation period up to 6 h did not alter the degree of stimulation. Appar500,

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AND HINEK

ently, a maximal response is reached within 60 min and the cells can maintain this maximal level of synthesis for at least 6 h under the culture conditions used in the present study. The response of the guinea pig chondrocytes is considerably faster than that of chick sternal chondrocytes (22). Under identical culture conditions, a higher degree of stimulation was reached with chondrocytes from fetal guinea pig cartilage (Fig. 1A) than with chondrocytes from ear cartilage of young rabbits (Fig. 1B). The dramatic stimulation of glycosaminoglycan synthesis with P-D-xyloside indicates that the availability of xylosylated core protein is a rate-limiting step in proteoglycan synthesis in cultured chondrocytes. Incubation with P-D-xyloside at a concentration of 5 x lop4 M inhibited the synthesis of noncollagenous protein and collagen in chondrocytes to about the same extent (Table IV), in contrast to fibroblast cultures in which collagen synthesis was preferentially inhibited (37). The general decrease in protein synthesis during xyloside treatment could be attributable to a decreased availability of metabolic energy for purposes other than glycosaminoglycan synthesis. Treatment of xyloside-stimulated chondrocytes with colchicine resulted in a relative 500,

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,

,

,

,

/

,

,

,

,

/

-5.3 4.9 4.6 4.3 -3.9 -3.6 -3.3 -2.9 -2.6 LOG DOSE XYLOSIDE

(Ml

-5.3 4.9 LOG DOSE XYLOSIDE

(MI

FIG. 1. Relation between dose of p-nitrophenyl+n-xylopyranoside and degree of stimulation of WO, into glycosaminoglycans. Cells were preincubated with P-D-xyloside for 90 min, 10 &i added per milliliter of medium, and the incubation was continued for 4 h. A, chondrocytes from cartilage; B, chondrocytes from rabbit ear cartilage; 0 ~ 0, total incorporation in medium; between incorporation in medium and that in cell layer.

of incorporation of Na,WO, was fetal guinea pig A - - - A, ratio

SECRETION

OF PROTEOGLYCANS TABLE

153

BY CHONDROCYTES IV

INHIBITION OF COLLAGEN AND PROTEIN SYNTHESIS BY XYLOSIDW Cell layer Praline hydroxyproline

Hydroxyproline cpm Control Xyloside,

5 x 1O-4 M

SD

Medium

cpm

A%

SD

A%

5527 401 3447 304 -3ts***

1357 120 959 176 -29*

Hydroxyproline cpm

SD

AQ

2160 125 llt53 384 -47***

Praline hydroxyproline cpm

SD

A%’

2990 266 1688 <529 -44**

a Guinea pig chondrocytes were preincubated with or without xyloside for 90 min, 10 pCi of [‘Hlprolineiml of medium was added, and the incubation was continued for 4 h. The incorporation of radioactivity into nondiffusible hydroxyproline and praline was then determined. A labeling ratio of 1:l of tritiated praline to tritiated hydroxyproline in collagen was assumed (3,4). Subtraction of the radioactivity in hydroxyproline from that in praline gives the incorporation of tritiated praline in noncollagenous protein. Each value represents the mean and SD for five culture plates. See Table I, Footnote a and asterisk footnotes, for an explanation of the P values.

inhibition of secretion comparable to that obtained when nonstimulated cells were incubated with colchicine (Table III). The degree of inhibition was not appreciably altered if the cells were preincubated first with colchicine and then stimulated with xyloside. These results suggest that the inhibitory effect of colchicine on synthesis and secretion of proteoglycan probably occurs after protein synthesis and xylosyl transfer. The results also show that the nonmicrotubule-dependent pathways of secretion of glycosaminoglycans in the chondroGml

A

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cl

I

cyte have a large capacity. Furthermore, the Golgi apparatus, in spite of structural alterations due to colchicine treatment (9), seems to retain most of its synthetic and secretory capacities. An alternative interpretation would be to question the obligatory role of the Golgi apparatus in synthesis and secretion of proteoglycan in chondrocytes [see Ref. (38)]. Such a suggestion would, however, contradict the present body of evidence (39, 40). Chondrocytes treated with cytochalasin B could be stimulated with p-D-xyloside to synthesize free chondroitin sulfate chains to the same relative degree as cells with intact microfilaments. Thus, xyloside is to a large extent able to overcome the synthetic block caused by cytochalasin B. Qualitative EfJects of P-D-Xyloside on Synthesis of Glycosaminoglycans and Proteoglycans

FIG. 2. Chromatography on Sephadex G-200 of Al fractions isolated from media of cultures treated with increasing levels of /3-D-xyloside. Incubation conditions were as in Fig. 1. A, control; B, 5 x lo-” M pnitrophenyl-P-D-xylopyranoside; C, 5 x lo-” M p-nitrophenyl-/3-rr-xylopyranoside; D, 5 x 10m4M p-nitrophenyl-/3-D-xylopyranoside.

Density gradients were run under associative conditions on culture media and guanidine. HCl extracts of cell layers. Al fractions prepared from media of cultures incubated with /3-D-xyloside and 35S0, were chromatographed on Sephadex G-ZOO(Fig. 2). In the absence of P-D-xyloside, all incorporated radioactivity was excluded from the gel, indicating that only proteoglycans and no free glycosaminoglycan chains were produced under control conditions (10, 33).

154

LOHMANDER.

MADSEN,

With increasing levels of /3-D-xyloside, a retarded peak of increasing area appeared, while the area of the excluded peak decreased. At 5 X 1O-4M /3-D-xyloside, incorporation of radioactivity into excluded material was inhibited by about 80%. Material included in the column was progressively more retarded as levels of P-Dxyloside increased. Material isolated from extracts of the cell layers or from fractions A2 and A3 showed different ratios of excluded to included material. All samples, however, showed increasing ratios of included to excluded material with increasing levels of P-D-xyloside. In a separate experiment with 5 x 1O-4M P-D-xyloside, materials excluded and included by Sephadex G-200 were recovered, digested with papain, and rerun on Sephadex G-200. The unchanged position of the retarded peak indicated that it contained single glycosaminoglycan chains. By contrast, the elution position of excluded material shifted, indicating that the glycosaminoglycan chains had been bound to protein core. After papain digestion, the elution positions of the two peaks were the same, a fact which shows that the molecular weight of glycosaminoglycan chains synthesized both on the xyloside and on the core-protein I

I

Grn

25 20 h; 2 x 2 k

15 10 5

ELUTION VOLUME hnl)

FIG. 3. Chromatography on Sephadex G-ZOO. Fractions corresponding to excluded and retarded peaks in Fig. 2D were pooled, dialyzed, lyophilized, and digested with papain, and aliquots were rechromatographed on Sephadex G-200. -, excluded peak; - - - -, retarded peak.

AND HINEK I

/

10

20

G-X0

ELWION VOLUME Iml)

FIG. 4. Chromatography on Sephadex G-200 of glycosaminoglycans isolated from control cultures and from cultures treated with P-D-xyloside. Incubation conditions were as in Fig. 1. -, chondroitin sulfate from bovine nasal septum, used as carrier; - - - -, control; ‘, 5 x 1O-4 M P-n-xyloside.

acceptor was the same in cells maximally stimulated with P-D-xyloside (Fig. 3). Chromatography of glycosaminoglycans isolated after papain digestion of media from control and xyloside-stimulated cultures (5 x 10P4M) on calibrated columns of Sephadex G-200 (41) gave molecular weights of 21,000 and 17,000, respectively (Fig. 4). Thus, the free glycosaminoglycan chains synthesized in the presence of P-D-xyloside are shorter than those synthesized on proteoglycan core-protein acceptor in the absence of ,6-D-xyloside (35, 36). When glycosaminoglycans were isolated after papain digestion of media (10) from cultures stimulated with increasing levels of P-D-xyloside, the molecular weight of the chains was inversely related to the degree of stimulation (Fig. 5). The data in Fig. 5 were calculated from the total glycosaminoglycan fraction from the medium, that is, both xyloside- and peptidebound chains. The data in Fig. 3 and the shift with increasing dose of xyloside in the position of the retarded peak in Fig. 2 suggest that the decrease in size of glycosaminoglycans with increasing stimulation was not a result of a decreasing ratio of peptidebound to xyloside-bound chains but was the result of a general decrease in length of all synthesized chains with increasing levels of available acceptor.

SECRETIONOFPROTEOGLYCANSBYCHONDROCYTES

155

the differences in chain size and in the quantity of chondroitin sulfate synthesized with and without xyloside treatment as observed in the present study with 35S04as label cannot be attributed to differences in degree of sulfation. In conclusion, treatment of chondrocyte cultures with p-nitrophenyl-p-D-xyloside resulted in a dramatic increase in the synthesis of chondroitin sulfate as free chains, in agreement with previous reports (21,22). Concomitantly, the synthesis of chondroitin sulfate on endogenous proteoglycan core protein was strongly inhibited. These observations point to a competition between exogenous and endogenous acceptor for the first galactosyltransferase in the synthesis of chondroitin sulfate (43). Furthermore, 4.6 4.3 -3.9 -3.6 -3.3 -2.9 ~2.6 the rate-limiting step in the synthesis of proteoglycan is located before the first gaLOG DOSE XYLOSIDE CM) lactosyltransferase. The molecular weights FIG. 5. Chromatography on Sephadex G-200 of glyof the chondroitin sulfate chains synthesized cosaminoglycans isolated from cultures of rabbit chondrocytes with increasing levels of P-D-xyloside. Col- both on the xyloside and on the core-protein umns were calibrated and&f,,. and &f, were calculated acceptor in maximally stimulated cells were as described in Ref. (41). Incubation conditions were similar and significantly shorter than in proteoglycans synthesized by cells in the abas in Fig. 1. 0 ~ 0, i@,V;0 0, B,. sence of P-D-xyloside. The presence of colchicine (10e5M) or cytochalasin B (1 Fg/ml) during treatment of GENERAL DISCUSSION cultures with P-D-xyloside did not affect the size of the chondroitin sulfate chains as comThe present data, together with those of pared with cultures treated only with P-D- earlier biochemical (6, 10) and morphologixyloside (data not shown). Somewhat larger cal (9) studies, show that colchicine retards proportions of 6-sulfated disaccharide were the secretion of proteoglycan from chondrorecovered from glycosaminoglycans isolated cytes. It should, however, be stressed that from cultures treated with 5 x lop4 M P-D- the inhibitory effect on secretion is not draxyloside (53%) than from control cultures matic and that it is superimposed on an in(46%). After digestion with chondroitinase hibition of the synthesis of these molecules. AC II or ABC, similar amounts of material It should also be pointed out that even in remained at the origin of the chromato- the absence of functional microtubules the grams, suggesting the absence of dermatan cells are able to continue their synthetic and sulfate in these cultures. The amount of gly- secretory activities with only minor struccosaminoglycan resistant to digestion with tural changes in the proteoglycan molecules chondroitinase (10%) was the same in stim- (10). Furthermore, the data of the present ulated and nonstimulated cultures. In all study show that chondrocytes treated with cultures, the ratio of 6-sulfated to 4-sulfated colchicine can still be stimulated to about disaccharide was higher in chondroitin sul- the same relative degree with P-Dxylosides fate from cell layer (1.5:l) than from me- to synthesize free chondroitin sulfate chains dium (1:l) and was in agreement with ear- as chondrocytes with intact microtubules. lier reports (42). Together, these data suggest that microtuA previous investigation (35) showed that bules might have a facilitatory rather than the degree of sulfation of chondroitin sulfate obligatory role in the intracellular translodid not change significantly as a result of cation and secretion of proteoglycan in treatment with /3-D-xyloside. Therefore, chondrocytes. I

t

1

I

I

I

,

156

LOHMANDER,

MADSEN,

The intracellular location of the colchicine action on proteoglycan secretion remains to be determined. However,. the similar relative inhibitions of colchicme on proteoglycans (in nonstimulated cells) and on glycosaminoglycans (in xyloside-stimulated cells) suggest that at least part of the effect of colchicine is located at or after the site of glycosaminoglycan synthesis. It seems unlikely that colchicine inhibits uptake of precursors since colchicine-treated cells can be stimulated with xyloside to double the rate of glycosaminoglycan synthesis. The present body of evidence indicates that the Golgi complex plays an important role in the synthesis and secretion of proteoglycans (39, 40). In the light of recent observations on the disruptive effect of antimicrotubular drugs on the Golgi complex in chondrocytes (9), it may be suggested that interference by colchicine with the facilitatory function of microtubules on secretion of proteoglycans observed in this study may be mediated in the Golgi complex. The role of microfilaments in intracellular transport is unclear. Our data indicate that cytochalasin B, a compound which affects microfilaments, does not have any specific effect on secretion but generally inhibits synthesis of proteoglycans. Possibly, protein synthesis (16) or uptake of precursors necessary for synthesis of proteoglycan core protein could be affected (l&20). However, the uptake of precursors for chondroitin sulfate is obviously sufficient to support even a stimulated synthesis of these molecules in spite of the reported inhibition of glucose and glucosamine uptake by cytochalasin B (18-20). Either the precursors for polysaccharide synthesis are derived from intracellular sources (20) or chondrocytes differ from fibroblasts in their sensitivity to cytochalasin B. P-D-Xylosides were used in this study as a tool to locate the effect of compounds affecting the function of microtubules and microfilaments and to determine the capacity of pathways not dependent on these organelles. As observed previously in various cell types, p-D-xylosides act as exogenous acceptors for the first galactosyltransferase and markedly increase the synthesis of chondroitin sulfate as free chains. The data presented here show that the molecular

AND HINEK

weights of the chondroitin sulfate chains synthesized both on the xyloside and on the core-protein acceptor in maximally stimulated cells were similar and significantly lower than in proteoglycan synthesized in the absenceof xyloside. Furthermore, the decrease in chain length is correlated with the degree of stimulation of synthesis of chondroitin sulfate. At similar concentrations, p-D-xylosides with increasingly aliphatic aglycone moieties increasingly stimulate chondroitin sulfate synthesis and concomitantly give progressively shorter chain length of chondroitin sulfate synthesized on the xyloside (35). It was suggested (36) that this effect might result from differences in rate of transport of xylosides through cell membranes or from differences in the affinity of the xylosides for the enzymes of chondroitin sulfate synthesis. Furthermore, it was proposed (36) that the decrease in size of chondroitin sulfate chains synthesized on ,8-D-xyloside reflects a different behavior of the polymerizing system, depending on the absence or presence of the protein core. The presence of the protein core would have the effect of prolonging the time available for polymerization of glycosaminoglycan chains. On the basis of data presented previously (35) and in this study, an alternative suggestion can be made: The chain length of chondroitin sulfate synthesized in the presence of ,8-D-xyloside, whether on proteincore acceptor or as free chains on P-Dxyloside, is inversely related to the degree of stimulation of synthesis. An increasing degree of stimulation can be the result either of increased levels of xyloside or of xylosides of decreasing polarity. The decrease in chain length with increasing stimulation could be caused by an increased ratio between available acceptor and chain-lengthening enzymes, precursors, or energy suppl~.~ The mechanisms that determine chain length in chondroitin sulfate biosynthesis 4 After submission of this article for publication, Kato et al. published a paper describing experiments with P-D-xylosides and cycloheximide in cultures of chick chondrocytes (Y. Kato, K. Kimata, K. Ito, K. Karasawa, and S. Suzuki, J. Biol. Chem., 19’78, 253, 2784-2789). These authors reached essentially the same conclusions for the variations in chain length of chondroitin sulfate observed at different degrees of stimulation.

SECRETION

OF PROTEOGLYCANS

are as yet unknown (45). The results of the present study, however, suggest that the presence or absence of proteoglycan protein core is not a critical factor in chain termination. ACKNOWLEDGMENTS Financial support was provided by the Swedish Medical Research Council (Project No. 12X-3355), by the King Gustaf V 80th Birthday Fund. and from the funds of Karolinska Institutet.

21. 22.

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