Regulation of c-fos gene induction and mitogenic effect of transforming growth factor-beta1 in rat articular chondrocyte

• Published in: Endocrine journal 1999, Vol.46 (2), p.253-261
• Main Authors: Osaki, M, Tsukazaki, T, Yonekura, A, Miyazaki, Y, Iwasaki, K, Shindo, H, Yamashita, S
• Format: Article
• Language: English
• Subjects: Animals; Cartilage, Articular - cytology; Cartilage, Articular - drug effects; Cartilage, Articular - physiology; Cell Division - drug effects; Cells, Cultured; Chloramphenicol O-Acetyltransferase - metabolism; Chondrocytes - drug effects; Chondrocytes - physiology; DNA - biosynthesis; Electrophoresis; Gene Expression; Gene Expression Regulation; Genes, fos; Mitogens - pharmacology; Rats; Rats, Sprague-Dawley; Space life sciences; Transcriptional Activation; Transforming Growth Factor beta - pharmacology
• ISSN: 0918-8959; 1348-4540
• DOI: 10.1507/endocrj.46.253

We have previously reported that type I transforming growth factor beta (TGF-beta1) is a potent stimulator of cell growth in articular chondrocytes. In this study, we examined the mechanism of TGF-beta1 induced cellular proliferation by using cultured rat articular chondrocytes (CRAC). A time-course study of [3H]thymidine incorporation upon TGF-beta1 (1 ng/mL) or 10% fetal bovine serum stimulation revealed that TGF-beta1 directly stimulates DNA synthesis in CRAC. Pretreatment with H7, an inhibitor for protein kinase C (PKC), completely blocks TGF-beta1-induced proliferation. Since TGF-beta1 has been shown to transduce signals through MAP kinase cascades, we investigated the induction of several protooncogenes by Northern blotting. TGF-beta1 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-beta1 signalling and PKC is involved in this c-fos induction. To refine our understanding of TGF-beta1 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-beta1 responsive element in a region between -403 and -329 bp 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-beta1 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.