REGULATION OF CLUSTERIN GENE-EXPRESSION BY TRANSFORMING-GROWTH-FACTOR-BETA

Transforming growth factor beta (TGF beta) induces the expression of a wide variety of genes in many cell types. Our previous studies have s hown that TGF beta 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 TG F beta, we created a 1.3-kilobase rat clusterin promoter/luciferase re porter construct. We demonstrate that TGF beta enhances luciferase act ivity 2.5-6fold in transient transfection assays of epithelial, endoth elial, and fibroblast cell lines. Deletional analysis reveals that an AP-1-binding site (5'-TGAGTCA) in the minimal promoter region is neces sary for initiating transactivation by TGF beta. A single T to G base mutation in the AP-1 site (5'-TGAGGCA) abolishes TGF beta-induced clus terin promoter transactivation. In transcription factor decoy experime nts, 23-mer oligonucleotides of wild type AP-1 reduce TGF beta inducti on of clusterin mRNA levels and promoter transactivation, while all ol igonucleotide containing the mutated AP-1 site has no effect. Two spec ific protein kinase C inhibitors, GF109203X and calphostin C, block TG F beta-induced clusterin mRNA levels and promoter transactivation. Tog ether these results indicate that TGF beta regulates clusterin gene ex pression through an AP-1 site and its cognate transcription factor AP- 1, and requires the involvement of protein kinase C.