Regulation of c-fos gene induction and mitogenic effect of transforming growth factor-beta 1 in rat articular chondrocyte

We have previously reported that type I transforming growth factor beta (TG F-beta 1) is a potent stimulator of cell growth in articular chondrocytes. In this study, we examined the mechanism of TGF-beta 1 induced cellular pro liferation by using cultured rat articular chondrocytes (CRAC). A time-cour se study of [H-3]thymidine incorporation upon TGF-beta 1 (1 ng/mL) or 10% f etal bovine serum stimulation revealed that TGF-beta 1 directly stimulates DNA synthesis in CRAC. Pretreatment with H7, an inhibitor for protein kinas e C (PKC), completely blocks TGF-beta 1-induced proliferation. Since TGF-be ta 1 has been shown to transduce signals through MAP kinase cascades, we in vestigated the induction of several protooncogenes by Northern blotting. TG F-beta 1 addition causes an immediate and transient induction of c-fos but not myc or jun mRNA. Furthermore, this c-fos expression is not inhibited by cycloheximide, but is completely abolished by pretreatment with TPA, so th at the c-fos gene is a direct target of TGF-beta 1 signalling and PKC is in volved in this c-fos induction. To refine our understanding of TGF-beta 1 r egulation of the c-fos promoter region, we performed chloramphenicol acetyl transferase (CAT) assays. A serial deletion analysis of the c-fos promoter region reveals a TGF-beta 1 responsive element in a region between -403 and -329 bp upstream of the transcription initiation site. We attempted gel sh ift assays on this response element with CRAC nuclear extracts. Although th is region contains a sis-inducible binding element, we fail to detect speci fic DNA-protein complexes. Our results, however, suggest that TGF-beta 1 ac ts as a primary mitogen in CRAC and this mitogenic activity requires PKC ac tivation. Finally, the subsequent induction of c-fos expression occurs thro ugh an as yet unidentified transactivation mechanism.