Regulation of clusterin gene expression by transforming growth factor beta.
Transforming growth factor beta (TGFbeta) induces the expression of a wide variety of genes in many cell types. Our previous studies have shown that TGFbeta stimulates both clusterin mRNA and protein levels, and induces its accumulation in the nucleus of CCL64 cells. To further investigate the molecular mechanism of clusterin mRNA induction by TGFbeta, we created a 1.3-kilobase rat clusterin promoter/luciferase reporter construct. We demonstrate that TGFbeta enhances luciferase activity 2.5-6-fold in transient transfection assays of epithelial, endothelial, and fibroblast cell lines. Deletional analysis reveals that an AP-1-binding site (5'-TGAGTCA) in the minimal promoter region is necessary for initiating transactivation by TGFbeta. A single T to G base mutation in the AP-1 site (5'-TGAGGCA) abolishes TGFbeta-induced clusterin promoter transactivation. In transcription factor decoy experiments, 23-mer oligonucleotides of wild type AP-1 reduce TGFbeta induction of clusterin mRNA levels and promoter transactivation, while an oligonucleotide containing the mutated AP-1 site has no effect. Two specific protein kinase C inhibitors, GF109203X and calphostin C, block TGFbeta-induced clusterin mRNA levels and promoter transactivation. Together these results indicate that TGFbeta regulates clusterin gene expression through an AP-1 site and its cognate transcription factor AP-1, and requires the involvement of protein kinase C.
Jin
G
G
Department of Cell Biology (NC-1), Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.
Howe
P H
PH
D000818:Animals; D002460:Cell Line; D051152:Clusterin; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006023:Glycoproteins / Q000235:genetics*; D006801:Humans; D018832:Molecular Chaperones; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D011493:Protein Kinase C / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D018808:Transcription Factor AP-1 / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000378:metabolism*
C495242:CLU protein, human; D051152:Clusterin; D006023:Glycoproteins; D018832:Molecular Chaperones; D012333:RNA, Messenger; D018808:Transcription Factor AP-1; D016212:Transforming Growth Factor beta; D011493:Protein Kinase C