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

Abstract

The human 25-hydroxyvitamin D3-1alpha-hydroxylase (1alpha-OHase) gene has been cloned. It contained nine exons and eight introns spanning approximately 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 (Sp1) response elements, and four NF-kappaB binding sites, but no vitamin D response element. By using luciferase reporter gene constructs of truncated forms of the 1alpha-OHase promoter transfected into a modified pig kidney cell line, AOK-B50, we identified regulatory regions of the 1.4-kb 1alpha-OHase promoter for parathyroid hormone 1-34 [PTH(1-34)], forskolin, and 1,25-hydroxyvitamin D3 [1,25(OH)2D3]. The 1.4-kb 1alpha-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)2D3 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 1alpha-OHase gene and provide insight into the physiologic basis for regulation of the expression of this gene by PTH and 1,25(OH)2D3.

Figure 1

Structure of the human 1α-OHase promoter. A 13-kb genomic clone of the human 1α-OHase gene was sequenced, mapped, and analyzed for consensus sequences to known mammalian transcription factors. (A) The nucleotide sequence for the promoter region is given. Underlined sequences correspond to conservative homologous matches to the noted transcription factor or promoter element. The alternating blocked and unblocked sections indicate the truncated promoters AN1–6. (B) A map of the 1α-OHase promoter showing the relative locations of the putative cAMP-inducible transcription factor response elements.

Figure 2

Activity of the human 1α-OHase promoter. A 1.4-kb clone and several truncated forms of the human 1α-OHase promoter were inserted into a luciferase reporter expression vector, transfected into AOK-B50 kidney cells, grown for 24 hr with and without factors, and then assayed for luciferase activity. (Bottom) The length of the various truncated promoter constructs. The graphs represent the luciferase activity of the constructs relative to that of the promoterless vector. Bars represent the mean ± SEM of three independent determinations. (A) Basal activity of the human 1α-OHase promoter. Transfected cells were assayed for luciferase activity. ∗, P < 0.01 vs. promoterless vector; ^†, P < 0.001 vs. AN2 and AN5. (B) PTH induction of the activity of the human 1α-OHase promoter. Transfected cells were treated with vehicle alone (filled bar) or 100 nM PTH (–34) (open bar) for 24 hr and assayed for luciferase activity. ∗, P < 0.01 vs. vehicle alone. (C) Effect of 1,25(OH)₂D₃ on the activity of the human 1α-OHase promoter. Transfected cells were treated with vehicle alone (filled bar) or with 10 nM 1,25(OH)₂D₃ (open bar). (D) Effect of 1,25(OH)₂D₃ on the PTH (–34)-inducible activity of the human 1α-OHase promoter. Transfected cells were treated with 100 nM PTH (–34) with vehicle alone (filled bar) or with 10 nM 1,25(OH)₂D₃ (open bar). ^§, P < 0.01 vs. the same construct in the absence of 1,25(OH)₂D₃.

Figure 3

Dose-dependent induction of the AN2 and AN5 truncated forms of the human 1α-OHase promoter by PTH (–34). AN2- (1,100 bp, filled bar) and AN5- (463 bp, open bar) truncated forms of the human 1α-OHase promoter were inserted into a luciferase reporter expression vector, transfected into AOK-B50 kidney cells, and treated with increasing concentrations of PTH (–34) for 24 hr. The AOK-B50 cells were then assayed for luciferase activity. Each bar represents the mean fold induction in luciferase activity relative to that of the promoterless construct in the absence of PTH ± SEM of three independent determinations. ∗, P < 0.01 vs. the same construct in the absence of PTH.