Transforming growth factor beta regulates clusterin gene expression via modulation of transcription factor c-Fos

Transforming growth factor-beta (TGF beta) induces gene expression of the g lycoprotein clusterin in a variety of cell types via a consensus AP-1 bindi ng site. Here, we demonstrate, by supershift analysis, that JunB, JunD, Fra 1, Fra2, and c-Fos bound to AP-1 but that prior treatment of the cells with TGF beta reduced dramatically c-Fos binding, suggesting that c-Fos might b e playing a negative regulatory role in clusterin gene expression. Transien t cotransfection assays in mink lung epithelial (CCL64) cells, using a huma n c-Fos expressing plasmid together with a clusterin promoter/reporter cons truct or the artificial TGF beta-inducible reporter construct 3TPLux, revea led that c-Fos was indeed repressive for TGF beta-induced promoter transact ivation. Further, we demonstrate that in stable c-Fos-overexpressing cell l ines, TGF beta induction of-endogenous clusterin mRNA, as well as clusterin promoter transactivation are blocked. Co-transfection with c-Fos deletion constructs revealed that the C-terminal region, including the homologue box 2 motif and the extreme C-terminal serine phosphorylation sites (Ser362 an d Ser374) are required for repression of clusterin and 3TPLux transactivati on. TGF beta treatment of CCL64 cells resulted in the induction of c-Fos mR NA but caused no alternation in total c-Fos protein levels. The results sug gest that the c-Fos represses clusterin gene expression,, maintaining a low basal level in the absence of TGF beta, and that TGF beta, presumably thro ugh its effects on c-Fos protein synthesis and/or stability, abrogates the repression of c-Fos, thereby resulting in gene expression.