THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 258, No. 11, Issue of June 10, pp. 7190-7194. 1983 Printed in U.S.A.

The Culture of Chick Embryo Chondrocytes and the Control of Their Differentiated Functions in Vitro TRANSFORMATION BY ROUS SARCOMA VIRUS INDUCES A SWITCH IN THE COLLAGEN TYPE SYNTHESIS AND ENHANCES FIBRONECTIN EXPRESSION*

(Received for publication, November 30, 1982)

Elisa Gionti, Olga Capasso, and Ranieri Cancedda From the Istituto di Bwchimica Cellulare e Molecolare, I f Facolta di Medicina e Chirurgia, Uniuersitir di Napoli, Via Pansini, 5, 80131, Naples, ftaly

Epithelial-like chondrocytes obtained from chick embryo were transformed with Rous sarcoma virus. Cellular transformation was monitored looking at the morphology change, the cell growth, and the expres- sion of plasminogen activator.

Analysis on polyacrylamide gel of intracellular and secreted proteins showed: 1) a disappearance of the specific products of differentiated chondrocytes; 2) a switch in the collagen synthesis from the type 11, the chondrocyte-specific type, to the type I, characteristic of fibroblasts and other cells of mesenchymal origin; 3) an enhancement of fibronectin synthesis.

Analysis of the proteins from chondrocytes infected with Rous-associated virus 1, a virus unable to induce cell transformation in vitro, indicated that the altered expression of the differentiated proteins in Rous sar- coma virus-infected chondrocytes depended upon the action of src gene product.

The interference of viral transformation with the expres- sion of differentiated functions has been studied in several cell systems. The transformation process blocks the expres- sion of differentiation markers in most cells, including melan- oblasts (2) , myoblasts (3, 4), iris epithelial cells (5), and thyroid epithelial cells (6). To date neuroretinal cells trans- formed by RSV’ may represent the only exception to this general rule (7).

Chick embryo chondrocytes are another example of differ- entiated cells that can be infected and transformed by RSV (8,9). In transformed chondrocytes the synthesis of cartilage- specific proteoglycans is stopped (9,lO). Yoshimura et al. have described that, in these cells, transformation reduces the synthesis of cartilage type I1 collagen, but they have failed to demonstrate any conversion from type I1 to type I collagen; the same authors have also observed an increased synthesis and secretion of a protein with an electrophoretic mobility identical with fibronectin (11). More recently Adams et al.

* This work was supported by grants from “Progetto Finalizzato Controllo della Crescita Neoplastica,” and “Progetto finalizzato in- gegneria genetica e basi molecolari delle malattie metaboliche,” Con- siglio Nazionale delle Ricerche, Rome, Italy. This paper is the second of a series. The previous publication is Reference 1. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduer- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

’ The abbreviations used are: RSV, Rous sarcoma virus; m.o.i., multiplicity of infection; SR-RSV, Schmidt-Ruppin strain of Rous sarcoma virus; nd, nondefective; RAV-1, Rous-associated virus of subgroup A.

confirmed some of these data and reported a reduction in the synthesis of several specific polypeptides (including type I1 procollagen) in chondrocytes infected at the permissive tem- perature with a temperature-sensitive mutant of Rous sar- coma virus (12).

In a recent work we have optimized conditions to maintain in culture for several weeks differentiated chondrocytes (1). Chondrocytes in vitro grow in suspension either as floating cells or attached to the dishes with an epithelial-like mor- phology. We have successfully infected and transformed with RSV both cell populations. In this paper we report studies on the synthesis and secretion of the components of the extra- cellular matrix in RSV-transformed chondrocytes. We show that the expression of the src gene blocks the synthesis of some differentiation markers of chondrocytes and, at the same time, enhances the synthesis of the fibronectin. When we investigated the nature of the collagen made, we observed a conversion from the synthesis of type I1 procollagen to the synthesis of type I procollagen.

EXPERIMENTAL PROCEDURES

Cell Cultures and Vim/ Infection-The preparation of chondrocyte cultures from tibiae of 19-day-old White Leghorn chick embryos (SPF eggs, Lohmann Toerzucht GMBH, Cuxhaven, Germany) as well as the preparation of media and the source of materials have been previously described (1). Fibroblasts were prepared according to Pfefferkorn and Hunter (13) and grown in E 199 medium supple- mented with 4% calf serum, 1% chicken serum, and 10% tryptose phosphate broth (Difco).

Epithelial-like cells grown in the absence of ascorbic acid (5 X lo4 cells per 35 mm dish) were infected in monolayer 24 h after plating with a small volume of virus suspension at a multiplicity of infection of approximately 20 (20 m.0.i.). After 1 h the cultures were given 2 ml of fresh medium; medium was changed after 1, 4, 8, and 12 days. Alternatively the epithelial-like cells were harvested with a mixture of0.25% trypsin and 100 units/ml of collagenase I1 and infected with the virus a t 2 m.0.i. in a small volume for 1 h; infected and mock infected chondrocytes were replated and fresh medium was added. Fibroblasts were always infected in suspension at 2 or 20 m.0.i. A clonal isolate from the SR-RSV of subgroup A served as nd virus; RAV-1 served as lymphoid leukosis virus (14). Both viruses were obtained from Dr. Philippe Vigier, Fondation Curie, Orsay, France.

Infected Cell Assay-The cells were washed twice with phosphate- buffered saline, detached from the dishes with the trypsin-collagenase mixture, and counted. Serial dilutions of the cells were plated on monolayers of chick embryo fibroblasts (3 X IO5 cells per 35-mm dish) and after 1 h the fibroblasts were overlaid with medium containing agar a t 0.75% final concentration. Dishes were incubated a t 37 “C. Fibroblasts cultures were scored for transformation foci under the microscope after 7 days.

Fibrinolysis Assay-Fibrinolytic activity was assayed according to Wigler and Weinstein (15) using ””I-fibrin-coated plastic Linbro multiwella. The assay was performed on cell lysates in phosphate- buffered saline containing 0.1% Triton X-100 and on culture media

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harvested 24 h after the cells were fed. To inactivate acid-sensitive protease inhibitors, the media were acidified with 1 N HCI and neutralized with 1 N NaOH before the assay (16). We assayed each sample in triplicate a t four time points and considered for the final result only the points in the time interval a t which linearity was observed. One unit of activity was defined as the amount of enzyme catalyzing the release of 1 mg of fibrin at 37 “C in 1 h.

p5S]Methionine Labeling, Immunoprecipitation, and Polyacrylam- ide Gel Electr~phoresis-[~S]Methionine labeling, immunoprecipita- tion, and polyacrylamide gel electrophoresis were performed as al- ready described (1). Rabbit serum anti-chick fibronectin was gener- ously provided by Dr. Guido Tarone, University of Turin, Italy; cross- immunoadsorbed anti-chick type I and type I1 collagen antibodies were a gift of Dr. Manfred Wiestner, Max-Plank-Institut fur Bioch- emie, Munchen, Germany. Each serum recognized specifically only one type of collagen (1, 17).

RESULTS

Transformation of Chondrocytes-Epithelial-like chondro- cytes obtained from chick embryo were infected in suspension with SR-RSV A after digestion of the extracellular matrix. Infected cells presented the characteristically bipolar mor- phology of the transformed chondrocytes (Fig. 1). (8,9). The infection and the release of virus was monitored with the infected cell assay. In Table I are reported the results of a typical experiment in which the percentages of infected cells in two parallel cultures of chondrocytes and fibroblasts were determined a t different times. Consistent with their longer doubling time the spreading of infection was slower in chon- drocytes. The number of the chondrocytes with bipolar mor- phology was proportional to the number of the virus-produc- ing cells. When, after 14 days, 100% of chondrocytes released infectious virus, all cells presented a bipolar morphology. In some experiments epithelial-like chondrocytes were infected with a 10-fold higher viral concentration directly as mono- layer, 24 h after plating, without previous digestion with proteolytic enzymes; we did not observe any major difference in the efficiency of transformation when the cultures were infected with the two procedures.

Transformation by RSV stimulated cell growth; in one experiment the doubling times of normal and infected chon- drocytes were 2.8 and 1.5 days, respectively. In addition the final level of cell density in cultures of transformed chondro- cytes was always at least 3 times higher than in cultures of mock infected cells. Transformed chondrocytes secreted plas- minogen activator to an extent comparable to the one ob- served in transformed fibroblasts (Table 11). We did not check the growth in soft agar of transformed cells since normal chondrocytes have already a relatively high colony-forming efficiency (data not shown).

Protein Synthesis of Transformed Chondrocytes-We have recently analyzed and partially characterized by collagenase digestion, immunoprecipitation, and pulse-chase experiments some of the proteins synthesized in large amount by cultured chondrocytes (1). Arrows in Fig. 2 refer to four of the proteins that we consider as specific differentiation markers; from top to bottom: 1) a high molecular weight protein, not recognized by antibodies raised against type I collagen, type I1 collagen, and fibronectin; 2) the pro-cul (11) collagen; 3) a -75,000 protein comigrating with chondronectin (18, 19) specifically immunoprecipitated from media’; 4) a 64,000 collagenase- sensitive polypeptide.

[3sS]Methionine-labeled intracellular proteins from normal and transformed chondrocytes were compared with proteins from normal and transformed fibroblasts (Fig. 2). Viral trans- formation resulted in the disappearance of the above recalled differentiation markers. In some experiments (see, for exam-

’ 0. Capasso, unpublished data.

FIG. 1. Normal and RSV transformed chondrocytes. a, nor- mal epithelial-like chondrocytes. X 160. b, RSV-transformed epithe- lial-like chondrocytes. X 160. Cells were infected in suspension at 2 m.0.i.; the picture was taken 14 days after infection.

TABLE I Infection of chick embryo chondrocytes and fibroblasts with Rous

sarcoma virus Epithelial-like chondrocytes and fibroblasts were infected in sus-

pension at 2 m.0.i. The infected cell assay is described under “Exper- imental Procedures.” Each number represents the average of three indeDendent determinations.

Days after infection

2 I 14

% of infected ceUs

Chondrocytes 5 47 100 Fibroblasts 55 100

ple, lune 3 of Figdd) the disappearance of pro-cyl(I1) chain, induced by RSV transformation, is accompanied by the ap- pearance of a polypeptide clearly detectable in the collagen region of the gel; since it comigrated with the major collagen- ase-sensitive polypeptide synthesized by cultured fibroblasts (arrowhead in Fig. 2) it was tentatively identified as pro-cul(1) collagen.

In vivo chondrocytes do not synthesize fibronectin (20,21). In vitro the enzymatic dissociation of the extracellular matrix favors the synthesis of this protein (1, 20). The amount of fibronectin made by cultured chondrocytes can vary from undetectable to considerable levels. When chondrocytes that did not synthesize fibronectin were infected, the viral trans- formation resulted in the appearance on the gel of the 220,000 fibronectin band (Fig. 3, lunes 3 and 4 and Fig. 5d, lunes 1

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TABLE I1 Induction of plasminogen activator by Rous sarcoma virus in chick

embryo chondrocytes and fibroblasts Epithelial-like chondrocytes were infected as monolayer a t 20 m.0.i.

and assayed for plasminogen activator 14 days after infection. Fibro- blasts were infected in suspension at 20 m.0.i. and assayed 4 days after infection. The fibrinolysis assay is described under "Experimen- t a l Procedures." The amount of proteins in each lysate was deter- mined and the weight of cells was deduced on the assumption that proteins represent 10% of the total cellular mass.

Units of activ- Units of activity secreted in the

ity in 1 g of medium in 24 h bv 1 e of cells cells

~Y

Mock infected chondrocytes 0.54 0.43 Infected chondrocytes 0.98 6.71 Mock infected fibroblasts 0.01 co.01 Infected fibroblasts 2.27 8.62

1 " - - 9 3 - - - 6 8 " -

IC "

- 45

- 30

FIG. 2. Intracellular proteins from normal and RSV-trans- formed chondrocytes and fibroblasts. 1, normal chondrocytes; 2, RSV-transformed chondrocytes; 3, normal fibroblasts, 4, RSV-trans- formed fibroblasts. Cells were labeled when massively transformed, i.e. 14 days after infection for the chondrocytes and 5 days for the fibroblasts. Middle arrow refers to the pro-al(I1) collagen. Arrows refer to the specific proteins of the differentiated chondrocytes (see text). Arrowhead refers to the pro-al(1) collagen. The numbers on the right refer to kilodaltons of molecular weight markers. The polyac- rylamide concentration of the gel was 9%. All samples were reduced and alkylated before the electrophoresis.

and 3); when chondrocytes already synthesizing fibronectin were transformed, the cells maintained and enhanced their fibronectin production (Fig. 2, lanes 1 and 2).

To prove the nature of the proteins synthesized by trans- formed chondrocytes, cell lysates were immunoprecipitated with antibodies against chicken fibronectin and type I and type I1 chicken collagens (Fig. 3). The data clearly indicated a block of the type I1 collagen synthesis and an induction of the synthesis of both type I collagen a1 chain and fibronectin in transformed chondrocytes. The presence of a2(I) chains was not observed in this experiment. A t the moment we cannot distinguish whether this is due to a low concentration of a2(I) chains in the lysates or to a low affinity of the antibodies for this polypeptide.

As already observed (22-25) the synthesis of fibronectin and type I collagen was strongly inhibited in transformed fibroblasts (Figs. 2, 3).

1 2 3 4 5 6 7 8 9 10 n 12 13 14

T o t a l Fn. A b . o < I A b . W z A b . FIG. 3. Immunoprecipitation of intracellular proteins from

normal and RSV-transformed chondrocytes and fibroblasts. 1,5,9, proteins from normal fibroblasts. 2,6,10, proteins from RSV- transformed fibroblasts; 3, 7, 11, 13, proteins from normal chondro- cytes; 4,8,12,14, proteins from RSV-transformed chondrocytes. The immunoprecipitation was performed with antibodies from rabbit im- munized with chicken fibronectin (Fn. Ab.), chicken type I collagen (a1 Ab.), and chicken type I1 collagen (alZAb.). The lower molecular weight protein in lane 8 recognized by these antibodies is most probably a specific degradation product of the fibronectin, due to proteolytic activity present in the cell lysates. The polyacrylamide concentration of the gel was 7.5%. All samples were reduced and alkylated before the electrophoresis.

Collagen Secretion by Transformed Chondrocytes-The electrophoretic pattern of proteins secreted by control fibro- blasts (Fig. 4, lane 3) revealed the presence of several poly- peptides migrating in the collagen region of the gel. To iden- tify these polypeptides the secreted proteins were immuno- precipitated with type I collagen antibodies (not shown) and the relative electrophoretic migrations of the immunoprecip- itated polypeptides (i.e. all type I-related collagen chains named in the figure) were compared to the reported migra- tions of the type I procollagen and PC collagen chains (26, 27). In addition to type I collagen, cultured chicken fibroblasts synthesize and secrete a relatively large amount of type I11 collagen (28). The pro-al(II1) chain has an electrophoretic migration intermediate between the migrations of the pro- al(1) and pC-al(1) chains (29). Purified type I collagen anti- bodies do not cross-react with type I11 collagen (30); the band, not recognized by type I antibodies, was, therefore, tentatively identified as pro-al(II1) collagen (full dot in Fig. 4).

When we compared the patterns of proteins secreted by normal and transformed chondrocytes (Fig. 4, lanes 1 and 2) to that of control fibroblasts, we observed that transformation of chondrocytes resulted in a dramatic decrease of the pro- al(I1) chain and in the appearance of the pro-al(1) chain and of its maturation product PC-al(1). In overexposed autoradi- ographies, in the lane of proteins secreted by transformed chondrocytes we also observed polypeptides with the same electrophoretic migration of the pro-a2(1) and pC-a2(1) chains; however, the amount of radioactivity in these poly- peptides was too small to allow any further analysis.

Infection of Chondrocytes with RA V-1"We performed some

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1 2 3

FIG. 4. Secreted protein form normal and RSV-trans- formed chondrocytes and fibroblasts. I, normal chondrocytes; 2, RSV-transformed chondrocytes; 3, normal fibroblasts. Transformed chondrocytes were labeled 14 days after infection. Arrows refer to specific proteins of the differentiated chondrocytes. The protein marked with full dot was tentatively identified as pro-al(II1) collagen (see text). The polyacrylamide concentration of the gel was 9%. All samples were reduced and alkylated before the electrophoresis. Since the same amount of trichloroacetic acid-precipitable counts was lay- ered on each slot, each sample represented labeled proteins released in the medium by a different number of cells.

1 2 3

"

FIG. 5. RAV-1-infected chondrocytes. a, epithelial-like chon- drocytes infected in suspension with RAV-1 a t approximately 2 m.0.i. The picture was taken 10 days after infection X 256. b, RAV-1- infected chondrocytes passaged and superinfected after 7 days with SR-RSV A. The picture was taken 7 days after SR-RSV A superin- fection. X 256. c, control chondrocytes 7 days after infection with SR- RSV A. X 256. d, intracellular proteins of 1 , normal; 2, RAV-1- infected chondrocytes; 3, SR-RSV A-transformed chondrocytes. Cells were labeled 14 days after infection. The polyacrylamide concentra- tion of the gel was 9%. All samples were reduced and alkylated before the electrophoresis.

Transformed Chondrocytes 7193 experiments with two temperature-sensitive transformation mutants of Rous sarcoma virus (ts-T PA3 and ts-T PA6 of Reference 31). At the nonpermissive temperature the large majority of the infected cells did not present the characteristic morphology of the transformed chondrocytes. Moreover we could oscillate between the normal and transformed pheno- types by simply shifting the incubation temperatures of the infected cells. In this condition the morphological reversion was obtained within 2-3 days. Nevertheless, due to the leak- iness of these temperature-sensitive mutants, we always ob- served some residual focal areas of bipolar cells. We, therefore, decided to utilize the lymphoid leukosis virus RAV-1. This virus is able to synthesize infectious progeny virus but unable to induce cell transformation in fibroblast cultures or to induce sarcomas in the animal (14). Its genome is smaller than that of nd RSV and lacks a specific portion near the 3' end including the src gene (32). We infected chondrocytes with this virus to determine whether the altered expression of differentiated proteins in transformed chondrocytes de- pended upon the action of src gene product. Half of the cultures were used to control the efficiency of the infection looking at the resistance of the cells to a superinfection with nd SR-RSV A (Fig. 5, a, b, and c ) (33); the other half was labeled with [35S]methionine. The analysis on polyacrylamide gel of the proteins showed an identical pattern for RAV-1 infected and normal chondrocytes; in chondrocytes trans- formed with nd SR-RSV A the expected enhancement of the fibronectin synthesis and the switch of the collagens were observed (Fig. 5d).

DISCUSSION

In this paper we report that in RSV transformed chondro- cy te~ the synthesis of specific proteins of the differentiated status is repressed; in particular we observed the shut-off of type I1 collagen and of a 64,000 collagenase-sensitive protein we have recently identified (1). The shut-off of these proteins was accompanied by a switch on of the synthesis and secretion of type I collagen (namely of the al(1) chain). The identifi- cation of the type Ia l chain was based on its electrophoretic comigration with the collagen synthesized by cultured chicken fibroblasts and on its specific immunoprecipitation. Although in overexposed gels of proteins secreted by transformed chon- drocytes one can observe polypeptides with the same electro- phoretic mobilities of the aB(1) chains, we were unable to distinguish whether RSV transformation induces in the chon- drocytes the only synthesis of al(1) chain or whether the al(1) chain is expressed preferably in comparison to the a2(I) chain. We favor the second hypothesis; in fact, making use of a DNA probe (kindly provided by Drs. B. de Crombrugghe and I. Pastan, National Institutes of Health, Bethesda, MD) we obtained by dot blot hybridization preliminary data showing the appearance of the mRNA for the a2(I) chain in trans- formed chondrocytes.

Yoshimura et al. (11) have reported that transformation of chondrocytes resulted in a decreased amount of type I1 colla- gen synthesized but they failed to demonstrate any type I collagen chain in the proteins associated to the cells and secreted in the media. The carboxymethylcellulose chroma- tography they used to demonstrate the absence of &!(I) chains in the medium did not clearly separate al(1) and al(I1) chains; a secretion of type I collagen a1 chain accompanied by a decrease in the synthesis of type I1 collagen could, therefore, have gone undetected. Adams et al. (12) have also found a reduction in the synthesis of type I1 collagen and, although they were able to show the presence of type I collagen mRNAs, they stated they were not able to detect any type I collagen

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7194 Collagen and Fibronectin in Transformed Chondrocytes

synthesis in transformed chondrocytes. The authors did not report any attempt to reveal the presence of type I collagen in the lysates making use of specific antibodies or to analyze the proteins secreted in the medium.

In vivo chondrocytes do not express fibronectin. Cultured chondrocytes, after removal of the extracellular matrix, syn- thesize and secrete this protein in an extremely variable amount. We found that, after RSV transformation, chondro- cytes always synthesized and released in the medium large quantities of fibronectin. The enhancement of the synthesis of fibronectin that we observed is in good agreement with the results obtained by the other groups (11, 12).

The switch between the collagen types and the enhance- ment of the fibronectin synthesis depended upon the action of src gene product; when we infected the cells with the lymphoid leukosis virus RAV-1 we failed to observe both phenomena. It must be noted that the effect of src gene in chicken chondrocytes is completely different from the effect of the same gene in chicken fibroblasts. In the latter cells the synthesis of fibronectin and type I collagen (and their mRNAs) is in fact strongly inhibited (Figs. 2 and 3; Refs. 22- 25).

The block of the synthesis of specific products and the production of type I collagen and fibronectin in transformed chondrocytes could be interpreted as a consequence of a dedifferentiation process. On the other hand, it must be recalled that in developing limb buds an increase of fibronec- tin and type I collagen was detected by immunofluorescence in the cartilage blastema before the deposition of the extra- cellular cartilage matrix. These proteins disappeared in the mature cartilage when the presence of type I1 collagen was detectable (34). Given that, it is tempting to speculate that, as a consequence of the expression of src gene, cultured chondrocytes revert to a predifferentiated state.

In preliminary experiments, by immunofluorescence with antibodies against fibronectin, we observed a we11 developed extracellular network of fibronectin fibers in transformed chondrocytes. These data suggest that transformed chondro- cytes incorporate fibronectin into their extracellular matrix, a t variance with RSV-transformed fibroblasts (35) . Under- standing the reasons for this difference would greatly help to clarify the regulation of the synthesis and the role of the extracellular matrix in different types of cells.

Acknowledgments- We thank Gianfranco Pontarelli for technical assistance and Drs. Guido Tarone and Manfred Wiestner for the specific antibodies.

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E Gionti, O Capasso and R Canceddacollagen type synthesis and enhances fibronectin expression.

functions in vitro. Transformation by rous sarcoma virus induces a switch in the The culture of chick embryo chondrocytes and the control of their differentiated

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