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.

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 expression 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 II, 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 sarcoma virus-infected chondrocytes depended upon the action of src gene product.

The interference of viral transformation with the expression of differentiated functions has been studied in several cell systems. The transformation process blocks the expression of differentiation markers in most cells, including melanoblasts (2), myoblasts (3, 4), iris epithelial cells (5), and thyroid epithelial cells (6). To date neuroretinal cells transformed by RSV' may represent the only exception to this general rule (7).
Chick embryo chondrocytes are another example of differentiated cells that can be infected and transformed by RSV (8,9). In transformed chondrocytes the synthesis of cartilagespecific 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 ingegneria genetica e basi molecolari delle malattie metaboliche," Consiglio 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 "aduertisement" in accordance with 18  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 temperature with a temperature-sensitive mutant of Rous sarcoma 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 morphology. 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 extracellular 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. 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 phosphatebuffered saline containing 0.1% Triton X-100 and on culture media 7190 by guest on March 24, 2020 http://www.jbc.org/ Downloaded from 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 at four time points and considered for the final result only the points in the time interval at 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 Polyacrylamide Gel Electr~phoresis-[~S]Methionine labeling, immunoprecipitation, and polyacrylamide gel electrophoresis were performed as already described (1). Rabbit serum anti-chick fibronectin was generously provided by Dr. Guido Tarone, University of Turin, Italy; crossimmunoadsorbed anti-chick type I and type I1 collagen antibodies were a gift of Dr. Manfred Wiestner, Max-Plank-Institut fur Biochemie, Munchen, Germany. Each serum recognized specifically only one type of collagen (1, 17).

RESULTS
Transformation of Chondrocytes-Epithelial-like chondrocytes obtained from chick embryo were infected in suspension with SR-RSV A after digestion of the extracellular matrix. Infected cells presented the characteristically bipolar morphology 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 at different times. Consistent with their longer doubling time the spreading of infection was slower in chondrocytes. The number of the chondrocytes with bipolar morphology was proportional to the number of the virus-producing 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 monolayer, 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 chondrocytes were 2.8 and 1.5 days, respectively. In addition the final level of cell density in cultures of transformed chondrocytes was always at least 3 times higher than in cultures of mock infected cells. Transformed chondrocytes secreted plasminogen activator to an extent comparable to the one observed 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 collagenasesensitive polypeptide.
[3sS]Methionine-labeled intracellular proteins from normal and transformed chondrocytes were compared with proteins from normal and transformed fibroblasts (Fig. 2). Viral transformation resulted in the disappearance of the above recalled differentiation markers. In some experiments (see, for exam-  ple, lune 3 of Figdd) the disappearance of pro-cyl(I1) chain, induced by RSV transformation, is accompanied by the appearance of a polypeptide clearly detectable in the collagen region of the gel; since it comigrated with the major collagenase-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 transformation resulted in the appearance on the gel of the 220,000 fibronectin band (Fig. 3, lunes 3 and 4 and Fig. 5d, lunes 1   . Arrowhead refers to the pro-al(1) collagen. The numbers on the right refer to kilodaltons of molecular weight markers. The polyacrylamide 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).

Collagen and Fibronectin in Transformed Chondrocytes
To prove the nature of the proteins synthesized by transformed 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. At 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)(23)(24)(25) the synthesis of fibronectin and type I collagen was strongly inhibited in transformed fibroblasts (Figs. 2, 3). Collagen Secretion by Transformed Chondrocytes-The electrophoretic pattern of proteins secreted by control fibroblasts (Fig. 4, lane 3) revealed the presence of several polypeptides migrating in the collagen region of the gel. To identify these polypeptides the secreted proteins were immunoprecipitated with type I collagen antibodies (not shown) and the relative electrophoretic migrations of the immunoprecipitated polypeptides (i.e. all type I-related collagen chains named in the figure) were compared to the reported migrations 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 proal(1) and pC-al(1) chains (29). Purified type I collagen antibodies 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 proal(I1) chain and in the appearance of the pro-al(1) chain and of its maturation product PC-al (1). In overexposed autoradiographies, 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 polypeptides was too small to allow any further analysis.
Infection of Chondrocytes with RA V-1"We performed some  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 phenotypes 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 leakiness of these temperature-sensitive mutants, we always observed 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 depended 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 transformed 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 chondroc y t e~ 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 identification of the type I a 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 chondrocytes one can observe polypeptides with the same electrophoretic mobilities of the aB(1) chains, we were unable to distinguish whether RSV transformation induces in the chondrocytes 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 transformed chondrocytes.
Yoshimura et al. (11) have reported that transformation of chondrocytes resulted in a decreased amount of type I1 collagen 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 chromatography 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 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, synthesize and secrete this protein in an extremely variable amount. We found that, after RSV transformation, chondrocytes 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 enhancement 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][23][24][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 fibronectin and type I collagen was detected by immunofluorescence in the cartilage blastema before the deposition of the extracellular 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 chondrocytes incorporate fibronectin into their extracellular matrix, a t variance with RSV-transformed fibroblasts (35). Understanding 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.