Regulation of c-fos gene induction and mitogenic effect of transforming growth factor-beta1 in rat articular chondrocyte.
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.
Osaki
M
M
Department of Orthopaedic Surgery, Nagasaki University School of Medicine, Japan.
Tsukazaki
T
T
Yonekura
A
A
Miyazaki
Y
Y
Iwasaki
K
K
Shindo
H
H
Yamashita
S
S
D000818:Animals; D002358:Cartilage, Articular / Q000166:cytology* / Q000187:drug effects / Q000502:physiology*; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D019902:Chondrocytes / Q000187:drug effects / Q000502:physiology*; D004247:DNA / Q000096:biosynthesis; D004586:Electrophoresis; D015870:Gene Expression; D005786:Gene Expression Regulation; D016762:Genes, fos; D008934:Mitogens / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015533:Transcriptional Activation; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
D008934:Mitogens; D016212:Transforming Growth Factor beta; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase