Transforming growth factor beta regulates clusterin gene expression via modulation of transcription factor c-Fos.
Transforming growth factor-beta (TGFbeta) induces gene expression of the glycoprotein clusterin in a variety of cell types via a consensus AP-1 binding site. Here, we demonstrate, by supershift analysis, that JunB, JunD, Fra1, Fra2, and c-Fos bound to AP-1 but that prior treatment of the cells with TGFbeta reduced dramatically c-Fos binding, suggesting that c-Fos might be playing a negative regulatory role in clusterin gene expression. Transient cotransfection assays in mink lung epithelial (CCL64) cells, using a human c-Fos expressing plasmid together with a clusterin promoter/reporter construct or the artificial TGFbeta-inducible reporter construct 3TPLux, revealed that c-Fos was indeed repressive for TGFbeta-induced promoter transactivation. Further, we demonstrate that in stable c-Fos-overexpressing cell lines, TGFbeta 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 and Ser374) are required for repression of clusterin and 3TPLux transactivation. TGFbeta treatment of CCL64 cells resulted in the induction of c-Fos mRNA but caused no alternation in total c-Fos protein levels. The results suggest that the c-Fos represses clusterin gene expression, maintaining a low basal level in the absence of TGFbeta, and that TGFbeta, presumably through its effects on c-Fos protein synthesis and/or stability, abrogates the repression of c-Fos, thereby resulting in gene expression.
Department of Cell Biology, The Lerner Research Institute, Cleveland Clinic Foundation, OH, USA.
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D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D051152:Clusterin; D004305:Dose-Response Relationship, Drug; D004847:Epithelial Cells; D005786:Gene Expression Regulation; D006023:Glycoproteins / Q000378:metabolism*; D006801:Humans; D008907:Mink / Q000378:metabolism; D008954:Models, Biological; D018832:Molecular Chaperones; D008969:Molecular Sequence Data; D019696:Peptidylprolyl Isomerase / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D016760:Proto-Oncogene Proteins c-fos / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000494:pharmacology / Q000502:physiology*
C495242:CLU protein, human; D051152:Clusterin; D006023:Glycoproteins; D018832:Molecular Chaperones; D016760:Proto-Oncogene Proteins c-fos; D016212:Transforming Growth Factor beta; D019696:Peptidylprolyl Isomerase