SV40-immortalization of rabbit articular chondrocytes: alteration of differentiated functions.

Cell lines were established from rabbit articular chondrocytes following transfection with a plasmid encoding SV40 early function genes. This resulted in cell immortalization (130 passages have been completed for the oldest cell line) with acquisition of characteristics of partial transformation such as reduced serum requirements for normal and clonal growth. The immortalized chondrocytes, called SVRAC, did not form multilayer foci when maintained in postconfluent culture. Their ability to form colonies in soft agar was not increased in comparison with normal chondrocytes, but they were weakly tumorigenic in nude mice. SVRAC lost the ability to synthesize type II collagen and Alcian blue-stainable matrix, which are markers of the differentiated chondrocyte phenotype, and synthesized predominantly type I collagen. Studies of collagen gene expression showed that pro alpha 1 (II) mRNA was undetectable, whereas pro alpha 1 (I) collagen mRNA was expressed even in late passage cultures. Unlike normal dedifferentiated chondrocytes, SVRAC were unable to re-express the differentiated phenotype in response to tridimensional culture or microfilament depolymerization. Cell lines obtained from chondrocytes transfected either in primary culture or just after release of cells from cartilage displayed the same behaviour. Thus SV40 early genes were able to immortalize rabbit articular chondrocytes, but the resulting cell lines displayed an apparently irreversibly dedifferentiated phenotype. These cell lines can be used as models to identify regulatory pathways that are required for the maintenance or reexpression of differentiated function in chondrocytes.     

Methylation of type II and type I collagen genes in differentiated and dedifferentiated chondrocytes

The methyl-sensitive restriction endonucleases HpaII and HhaI as well as the methyl-insensitive enzyme MspI were used to examine the methylation status of the pro-alpha 1(II) collagen gene of cartilage. Five different cell types with varying abilities to express type II collagen were studied. Chick embryo chondrocytes express type II collagen, while 5-bromodeoxyuridine-treated chondrocytes, retinoic acid-treated chondrocytes, chick embryo fibroblasts, and erythrocytes do not synthesize type II collagen. Both cDNA and genomic probes for the pro-alpha 1(II) collagen gene were used, covering the complete 3' end of the gene and its flanking sequences. The pro-alpha 1(II) collagen DNA was undermethylated in chondrocytes, compared to either fibroblasts or erythrocytes. However, the methylation of the 5-bromodeoxyuridine-treated and retinoic acid-treated chondrocytes was identical to that of control chondrocytes. The methylation pattern of two regions of the gene of the pro-alpha 2(I) collagen chain was identical in all cell types tested, whether or not the gene was expressed. Our results indicate that genes for these collagen chains differ in their methylation pattern. The type II collagen gene shows reduced methylation in expressing cartilage, but does not acquire an increase in methylation in "dedifferentiated" chondrocytes. The changes in DNA methylation that occur during cell differentiation do not appear to be sufficient to explain gene activation and deactivation.     

Microfilament modification by dihydrocytochalasin B causes retinoic acid-modulated chondrocytes to reexpress the differentiated collagen phenotype without a change in shape

Primary monolayers of rabbit articular chondrocytes synthesize high levels of type II collagen and proteoglycan. This capacity was used as a marker for the expression of the differentiated phenotype. Such cells were treated with 1 microgram/ml retinoic acid (RA) for 10 d to produce a modulated collagen phenotype devoid of type II and consisting of predominantly type I trimer and type III collagen. After transfer to secondary culture in the presence of RA, the stability of the RA-modulated phenotype was investigated by culture in the absence of RA. Little reexpression of type II collagen synthesis occurred in this period unless cultures were treated with 3 X 10(-6) M dihydrocytochalasin B to modify microfilament structures. Reexpression of the differentiated phenotype began between days 6-8 and was essentially complete by day 14. Substantial reexpression occurred by day 8 without a detectable increase in cell rounding. Colony formation, characteristic of primary chondrocytes, was infrequent even after reexpression was complete. These data suggest that the integrity of microfilament cytoskeletal structures can be a source of regulatory signals that mechanistically appear to be more proximal to phenotypic change than the overt changes in cell shape that accompany reexpression of subculture-modulated chondrocytes in agarose culture.     

An established rat cell line expressing chondrocyte properties

Chondrocytes express a well-characterized set of marker proteins making these cells useful for studies on differentiation and regulation of gene expression. Because of the inherent instability of primary rat chondrocytes in culture, and because several rat chondrocyte genes have been cloned and characterized (including the collagen II promoter and enhancer), a rat chondrocyte cell line would be especially useful. To obtain this line we infected primary fetal rat costal chondrocytes with a recombinant retrovirus (NIH/J-2) carrying the myc and raf oncogenes, which have been shown to have an "immortalizing" function. Following infection, a rapidly proliferating clonal line was isolated that maintained a stable phenotype through 45 passages (11/2 year in culture). This line, termed IRC, grows in suspension culture as multicellular aggregates and in monolayer culture as polygonal cells which accumulate an alcian blue-stainable matrix. IRC cells synthesize high levels of cartilage proteoglycan core protein, and link protein, but show reduced collagen II expression. In addition, the cells express virally derived myc mRNA and protein, but do not express v-raf. Retinoic acid, which is a known modulator of chondrocyte phenotype, down-regulates expression of chondrocyte marker proteins, while stimulating v-myc expression by IRC cells. These data suggest that v-myc expression by chondrocytes results in rapid cell division and maintenance of many aspects of the differentiated phenotype. These "immortalized" cells, however, remain responsive to agents such as retinoic acid which modulate cell phenotype. The potential exists for development of chondrocyte cell lines from diseased cartilage, as well as from human cartilage.     

Modulation of the rabbit chondrocyte phenotype by retinoic acid terminates type II collagen synthesis without inducing type I collagen: the modulated phenotype differs from that produced by subculture

The differentiated phenotype of rabbit articular chondrocytes can be characterized by the synthesis of high levels of cartilage specific proteoglycan and collagen (type II). Treatment of these cells in primary monolayer culture for periods of up to 18 days with 0.03 to 3.0 micrograms/ml retinoic acid (RA) resulted in suppression of colony formation, altered morphology, and decreased (eightfold) proteoglycan and collagen synthesis. With the exception of collagen synthesis, these changes were complete with all doses after 4 days of treatment. Collagen synthesis declined more slowly; it was dose dependent after 4 days and maximally inhibited by all doses by 9 days. Detailed analysis of the collagen phenotype was performed using SDS-PAGE of intact chains and 2-D CNBr peptide analysis. RA caused cessation of type II synthesis, and transient stimulation of type III and type I trimer collagen synthesis, without induction of type I collagen. Essentially identical results were obtained with retinol. The resultant collagen phenotype differed significantly from the type I-containing phenotype induced by subculture. Thus, suppression of this differentiated program did not elicit a common modulated phenotype. The results are discussed in the context of direct and indirect mechanisms of RA-dependent modulation of chondrocyte gene expression.     

Further evidence for the dispersion of the human fibrillar collagen genes.

Recombinant DNA probes specific for the human pro alpha 1(II) and pro alpha 1(III) collagen chains have been used for the chromosomal localization of the two genes. Restriction endonuclease analysis of DNA from human-rodent hybrid cell lines in conjunction with in situ hybridization of human metaphasic chromosomes have shown that the gene coding for the pro alpha 1 chain of type II collagen (COL2A1) is located on chromosome 12 in the segment 12q131----12q132. Likewise, the gene coding for the pro alpha 1 chain of type III collagen (COL3A1) was assigned to the segment 2q31----2q323 of chromosome 2.     

Effect of retinoic acid on the enhancing effect of acetaldehyde on mouse type I collagen expression

Acetaldehyde alone and retinoic acid alone have been shown to increase and decrease, respectively, collagen production by stellate cells in culture. In this study the effects of retinoic acid on alpha(1)(I) and alpha(2)(I) collagen expression and its influence on the enhancing effects of acetaldehyde were determined. Retinoic acid decreased the activation of the alpha(2)(I) collagen promoter and decreased the message of alpha(2)(I) collagen in cultured stellate cells, but had no effect on either the activation of the alpha(1)(I) collagen promoter or on the alpha(1)(I) collagen message. This depressant effect of retinoic acid was also evident in the transfected alpha(2)(I) collagen promoter mutated at the retinoic acid response element (RARE). The activation of the alpha(2)(I) collagen promoter by acetaldehyde was not decreased significantly by retinoic acid, but was suppressed by the retinoic acid receptor (RAR) selective retinoid SRI-6751-84. Retinoic acid, however, decreased the acetaldehyde-induced enhancement of the alpha(1)(I) and alpha(2)(I) collagen messages. Acetaldehyde also resulted in a decrease in RAR beta message and RARbeta protein. This study shows that retinoic acid depresses alpha(2)(I) collagen gene expression but that this effect is less pronounced when the expression of this collagen is enhanced by acetaldehyde, which also decreases RARbeta message and protein. Furthermore, the action of retinoic acid in inhibiting alpha(2)(I) collagen gene expression occurs at sites other than the RARE site.