Parathyroid hormone activation of the 25-hydroxyvitamin D3-1alpha-hydroxylase gene promoter

Abstract

The DNA flanking the 5' sequence of the mouse 1alpha-hydroxylase gene has been cloned and sequenced. A TATA box has been located at -30 bp and aCCAAT box has been located at -79 bp. The gene's promoter activity has been demonstrated by using a luciferase reporter gene construct transfected into a modified pig kidney cell line, AOK-B50. Parathyroid hormone stimulates this promoter-directed synthesis of luciferase by 17-fold, whereas forskolin stimulates it by 3-fold. The action of parathyroid hormone is concentration-dependent. 1,25-Dihydroxyvitamin D3 does not suppress basal promoter activity and marginally suppresses parathyroid hormone-driven luciferase reporter activity. The promoter has three potential cAMP-responsive element sites, and two perfect and one imperfect AP-1 sites, while no DR-3 was detected. These results indicate that parathyroid hormone stimulates 25-hydroxyvitamin D3-1alpha-hydroxylase by acting on the promoter of the 1alpha-hydroxylase gene.

Figure 1

Nucleotide sequence of the 5′ flanking region of the mouse 1α-hydroxylase gene. The transcriptional start site is designated as +1. Putative cis regulatory elements are underlined. The translational start codon is indicated in bold. An asterisk (∗) indicates the end of the published mouse 1α-hydroxylase cDNA.

Figure 2

Primer extension analysis of mouse kidney 1α-hydroxylase mRNA population. Primer extension has been performed with op/op mouse kidney mRNA (+ lanes) by using five different labeled primers: m1a1, m1a2, m1a3, m1a4, and m1a5 (see Materials and Methods section for specific sequences). Control lanes (−) represent primer extensions performed with the primer alone. Primer extension products have been fractionated on a sequencing gel. Arrows represent specific 1α-hydroxylase cDNAs. Molecular weight markers (MWM) are indicated in nucleotide bases.

Figure 3

Transcriptional activity of the mouse 1α-hydroxylase 1.7-kb 5′ flanking sequence. AOK-B50 cells were transiently transfected with the promoter luciferase reporter construct, pGL2–1αPr, or empty pGL2b by using lipofectin. Transfected cells were treated for 24 h with 100 nM PTH or vehicle (water). Luciferase activity was normalized to protein content for each sample. Experiments were repeated three times in duplicate. Values are mean ± SD.

Figure 4

Dose-dependent stimulation of mouse 1α-hydroxylase gene promoter by PTH. AOK-B50 cells were transfected with pGL2–1αPr. Transfected cells were treated for 24 h with the indicated concentrations of PTH. The induction is relative to luciferase activity detected in untreated transfected cells. Experiments were repeated three times in duplicate.

Figure 5

Activation of mouse 1α-hydroxylase promoter by forskolin. AOK-B50 cells were transfected with pGL2–1αPr. Transfected cells were treated for 24 h with 10 μM forskolin or vehicle (ethanol). Data are presented as fold activation relative to vehicle-treated transfected cells. Values were obtained from three independent experiments performed in duplicate.

Figure 6

1,25-(OH)₂D₃ suppression of PTH-stimulated luciferase activity in AOK-B50 cells. Cells were transfected with pGL2–1αPr. Transfected cells were treated with 10 nM 1,25-(OH)₂D₃, 100 nM PTH, and both 1,25-(OH)₂D₃ and PTH or vehicle (water and/or ethanol) for 24 h. Luciferase activity is corrected for protein. Experiments were performed at least two times in duplicate. Values are mean ± SD.