Analysis of the mechanism of 1(alpha),25-dihydroxyvitamin D(3)-mediated repression of the gene expression of the endogenous inhibitor of cAMP-dependent protein kinase

The primary mechanism by which 1α,25-dihydroxyvitamin D₃ [1α,25(OH)₂D₃] generates biological responses is alteration of the expression of target genes through binding of the ligand-activated vitamin D receptor (VDR). These changes can be regulated both tissue-specifically and developmentally.; Numerous genes have been shown to be regulated by 1α,25(OH) ₂D₃, and the vast majority of 1α,25(OH)₂D ₃-regulated genes which have been studied are up-regulated in response to ligand. However, many genes are down-regulated in response to 1α,25(OH) ₂D₃, including the parathyroid hormone and the cAMP-dependent protein kinase inhibitor (PKI).; 1α,25(OH)₂D₃ decreases PKI activity in primary cultures of chick kidney cells and also decreases steady state levels of PKI mRNA both in vivo and in cell culture. To further study the molecular mechanism of transrepression of the PKI gene, the genomic clone of this gene was previously obtained through library screening with the PKI cDNA.; It is not clear what molecular signals are involved in the processes by which the PKI gene is tissue-specifically down-regulated by 1α,25(OH) ₂D₃, but a putative vitamin D response element (VDRE) was found close to the transcriptional start site. In order to investigate the role of this element in down-regulation by 1α,25(OH)₂D ₃, a functional assay was developed based on the transfection of primary cultures of chick kidney cells. A construct encompassing a region of the PKI promoter linked to the secreted alkaline phosphatase reporter gene was down-regulated by 1α,25(OH)₂D₃. Twenty-six additional constructs with different regions and/or mutations of the PKI promoter were created and used to map the locations and determine the specific sequences required for down-regulation of the PKI gene.; In order to elucidate the tissue-specific nature of the down-regulation of PKI, three complementary techniques were developed to isolate and compare specific proteins from nuclear extracts of chick intestine and kidney tissues. Two of these techniques were based upon proteins binding to the nVDRE sequence and another was based upon proteins binding to VDR ligand-binding domain.; Our current studies show that the sequence encoded by nucleotides −31 to −45 of the PKI promoter encode a novel VDRE whose position immediately adjacent to the proximal TATA box, rather than specific sequence, is responsible for 1α,25(OH)₂D₃-mediated down-regulation.