Inhibition of chondrocyte differentiation in vitro by constitutive and inducible overexpression of the c-fos proto-oncogene

We have investigated the role of c-Fos in chondrocyte differentiation in vi tro using both constitutive and inducible overexpression approaches in ATDC 5 chondrogenic cells, which undergo a well-defined sequence of differentiat ion from chondroprogenitors to fully differentiated hypertrophic chondrocyt es. Initially, we constitutively overexpressed exogenous c-fos in ATDC5 cel ls. Several stable clones expressing high levels of exogenous c-fos were is olated and those also expressing the cartilage marker type II collagen show ed a marked decrease in cartilage nodule formation. To investigate further whether c-Fos directly regulates cartilage differentiation independently of potential clonal variation, we generated additional clones in which exogen ous c-fos expression was tightly controlled by a tetracycline-regulatable p romoter. Two clones, DT7.1 and DT12.4 were capable of nodule formation in t he absence of c-fos, However, upon induction of exogenous c-fos, differenti ation was markedly reduced in DT7.1 cells and was virtually abolished in cl one DT12.4. Pulse experiments indicated that induction of c-fos only at ear ly stages of proliferation/differentiation inhibited nodule formation, and limiting dilution studies suggested that overexpression of c-fos decreased the frequency of chondroprogenitor cells within the clonal population. Inte restingly, rates of proliferation and apoptosis were unaffected by c-fos ov erexpression under standard conditions, suggesting that these processes do not contribute to the observed inhibition of differentiation. Finally, gene expression analyses demonstrated that the expression of the cartilage mark ers type II collagen and PTH/PTHrP receptor were downregulated in the prese nce of exogenous c-Fos and correlated well with the differentiation status. Moreover, induction of c-fos resulted in the concomitant increase in the e xpression of fra-1 and c-jun, further highlighting the importance of AP-1 t ranscription factors in chondrocyte differentiation. These data demonstrate : that c-fos overexpression directly inhibits chondrocyte differentiation i n vitro, and therefore these cell lines provide very useful tools for ident ifying novel c-Fos-responsive genes that regulate the differentiation and a ctivity of chondrocytes.