Abstract
We have previously reported that type I transforming growth factor beta (TGF-β1) is a potent stimulator of cell growth in articular chondrocytes. In this study, we examined the mechanism of TGF-β1 induced cellular proliferation by using cultured rat articular chondrocytes (CRAC). A time-course study of [3H]thymidine incorporation upon TGF-β1 (1ng/mL) or 10% fetal bovine serum stimulation revealed that TGF-β1 directly stimulates DNA synthesis in CRAG. Pretreatment with H7, an inhibitor for protein kinase C (PKC), completely blocks TGF-β1-induced proliferation. Since TGF-β1 has been shown to transduce signals through MAP kinase cascades, we investigated the induction of several protooncogenes by Northern blotting. TGF-β1 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 that the c-fos gene is a direct target of TGF-β1 signalling and PKC is involved in this c-fos induction. To refine our understanding of TGF-β1 regulation of the c-fos promoter region, we performed chloramphenicol acetyltransferase (CAT) assays. A serial deletion analysis of the c-fos promoter region reveals a TGF-β1 responsive element in a region between -403 and - 329bp upstream of the transcription initiation site. We attempted gel shift assays on this response element with CRAC nuclear extracts. Although this region contains a sis-inducible binding element, we fail to detect specific DNA- protein complexes. Our results, however, suggest that TGF-β1 acts as a primary mitogen in CRAC and this mitogenic activity requires PKC activation. Finally, the subsequent induction of c-fos expression occurs through an as yet unidentified transactivation mechanism.
