The human transient receptor potential vanilloid type 6 distal promoter contains multiple vitamin D receptor binding sites that mediate activation by 1,25-dihydroxyvitamin D3 in intestinal cells.
Transient receptor potential vanilloid type 6 (TRPV6) (ECAC2, CaT1) is the major ion channel in intestinal epithelial cell membranes responsible for calcium entry. Its expression is actively regulated at the transcriptional level by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. In this report, we identify mechanisms integral to the regulation of TRPV6 by 1,25-(OH)2D3. Based upon the hormonal responsiveness of a 7-kb TRPV6 promoter fragment in intestinal cell lines, we used a chromatin immunoprecipitation (ChIP) scanning method to search for possible vitamin D receptor (VDR) and retinoid X receptor (RXR) regulatory regions within the TRPV6 locus. VDR/RXR binding was broad, ranging from -1.2 to -5.5 kb relative to the start site of TRPV6 transcription. These results were consistent with an in silico analysis that revealed putative regulatory elements (VDREs) located at -1.2, -2.1, -3.5, -4.3, and -5.5 kb. Despite the ChIP analyses, only regions of the TRPV6 gene that contained putative elements at -2.1 and -4.3 kb transferred 1,25-(OH)2D3 response to a heterologous promoter. Further study revealed that each of these two active regions contained composite VDREs comprised of two separate regulatory elements. Mutagenesis of the VDREs within the -2.1- and -4.3-kb region and the VDRE at -1.2 kb abrogated all response to 1,25-(OH)2D3 when examined within the natural TRPV6 promoter. A final ChIP assay revealed that VDR/RXR heterodimer binding to the TRPV6 gene was accompanied by both the recruitment of steroid receptor coactivator 1 as well as a broad change in histone 4 acetylation. These studies identify a mechanism by which 1,25-(OH)2D3 regulates the expression of TRPV6 in human intestinal cells.
Meyer
Mark B
MB
Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison, Wisconsin 53706, USA.
Watanuki
Makoto
M
Kim
Sungtae
S
Shevde
Nirupama K
NK
Pike
J Wesley
JW
D000107:Acetylation; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D018938:Caco-2 Cells; D002117:Calcitriol / Q000494:pharmacology; D015220:Calcium Channels / Q000235:genetics*; D002460:Cell Line; D004247:DNA / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D004790:Enzyme Induction / Q000187:drug effects; D005786:Gene Expression Regulation / Q000187:drug effects; D051548:Histone Acetyltransferases; D006657:Histones / Q000378:metabolism; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism; D007422:Intestines / Q000187:drug effects; D056920:Nuclear Receptor Coactivator 1; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D034622:RNA Interference; D018167:Receptors, Calcitriol / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D047490:Retinoid X Receptor alpha / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013250:Steroid Hydroxylases / Q000096:biosynthesis; D050916:TRPV Cation Channels / Q000235:genetics*; D014157:Transcription Factors / Q000378:metabolism; D065668:Vitamin D3 24-Hydroxylase
D015220:Calcium Channels; D006657:Histones; D018167:Receptors, Calcitriol; D011994:Recombinant Proteins; D047490:Retinoid X Receptor alpha; D050916:TRPV Cation Channels; C422827:TRPV6 protein, human; D014157:Transcription Factors; D004247:DNA; D013250:Steroid Hydroxylases; D065668:Vitamin D3 24-Hydroxylase; D051548:Histone Acetyltransferases; C538859:NCOA1 protein, human; D056920:Nuclear Receptor Coactivator 1; D002117:Calcitriol