Promoter Characterization of the Rat Gene for Ca-binding Protein Regucalcin

To understand the mechanism underlying the regulation of the Ca-binding protein regucalcin gene expression, we characterized the 5*-flanking region of the rat regucalcin gene. The transcriptional start site of the rat regucalcin gene was determined by the cap site hunting method with rat liver cap site cDNA. The 5*-flanking region of the rat regucalcin gene ligated to a luciferase reporter gene possessed functional promoter activity in rat H4-II-E hepatoma cells. 3*and 5*-deletion analyses indicated the sequence required for basal functional promoter activity of the rat regucalcin gene. The promoter activity of the rat regucalcin gene was enhanced by treatment with Bay K 8644, dibutyryl cAMP, phorbol esters, insulin, and dexamethasone. Using gel mobility shift assays, we found that nuclear proteins from H4II-E cells specifically bind to the 5*-flanking region of the rat regucalcin gene. Moreover, gel mobility shift assays revealed that Bay K 8644, dibutyryl cAMP, phorbol esters, and insulin stimulated the binding of nuclear factors to the 5*-flanking region of the rat regucalcin gene in H4-II-E cells. These results suggest that Bay K 8644-, dibutyryl cAMP-, phorbol ester-, and insulin-inducible nuclear factors mediate the stimulatory effect of each regulator on promoter activity of the rat regucalcin gene.