Molecular cloning, characterization, and promoter analysis of the human 25-hydroxyvitamin D3-1α-hydroxylase gene

The human 25-hydroxyvitamin D-3-1 alpha-hydroxylase (1 alpha-OHase) gene has been cloned. It contained nine exons and eight introns spanning approximate to 6.5 kb and a 1.4-kb 5'-flanking region. The 5'-flanking region contains consensus or highly conserved sequences for TATA, Pu, and CCAAT boxes, four cAMP response elements, two activator protein-1 (AP-1) response elements, two AP-2 response elements, three specific protein-1 (Spl) response elements, and four NF-kappa B binding sites, but no vitamin D response element. By using luciferase reporter gene constructs of truncated forms of the 1 alpha-OHase promoter transfected into a modified pig kidney cell line, AOK-B50, we identified regulatory regions of the I. l-kb 1 alpha-OHase promoter for parathyroid hormone 1-34 [PTH(1-34)], forskolin, and 1,25-hydroxyvitamin D-3 [1,25(OH)(2)D-3]. The 1.4-kb 1 alpha-OHase promoter (AN1) modestly (1.7-fold) induced luciferase activity, whereas 1,100-(AN2), 827- (AN3), 672- (AN4), 463-(AN5), and 363-bp (AN6)-truncated promoters greatly stimulated luciferase activity by 494-fold, 18.4-fold, 55.3-fold, 643-fold, and 56.4-fold, respectively. PTH(1-34) and forskolin stimulated the activity of all constructs to varying degrees with significantly greater responsiveness for both compounds on AN2 and AN5, 1,25(OH)(2)D-3 suppressed PTH(1-34)-induced activity on AN2 and AN5 constructs by 58% and 52%, respectively, but had no effect on the other constructs. These studies characterize the regulatory regions of the human 1 alpha-OHase gene and provide insight into the physiologic basis for regulation of the expression of this gene by PTH and 1,25(OH)(2)D-3.