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.

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To clarify the role of nitric oxide (NO) in regulation of bone metabolism in response to skeletal loading, we examined inducible NO synthase (iNOS) gene knockout mice in the tail-suspension model. Histomorphometric analyses of proximal tibias revealed that 7 days of tail suspension decreased the bone volume (BV/TV) and bone formation rate (BFR/BS) and increased the osteoclast surface (Oc.S/BS) in mice with all iNOS genotypes. Both iNOS؉/؉ and iNOS ؉/؊ mice responded to subsequent 14-day reloading, with increases in BV/TV and BFR/BS and a decrease in Oc.S/BS, whereas these responses were abolished in iNOS ؊/؊ mice. The osteoblasts flattened after tail suspension appeared cuboidal during subsequent reloading. Immunoreactivity for iNOS was detected in these osteoblasts and osteocytes by immunohistochemistry. These defective responses after reloading were rescued in iNOS

1, (1,25(OH) 2 D 3 ) functions as a systemic signal in vertebrate organisms to control the expression of genes whose products are vital to the maintenance of calcium and phosphorus homeostasis. This regulatory capability is mediated by the vitamin D receptor (VDR) which localizes at DNA sites adjacent to the promoter regions of target genes and initiates the complex events necessary for transcriptional modulation. Recent investigations using chromatin immunoprecipitation techniques combined with various gene scanning methodologies have revealed new insights into the location, structure and function of these regulatory regions. In the studies reported here, we utilized the above techniques to identify key enhancer regions that mediate the actions of vitamin D on the calcium ion channel gene TRPV6, the catabolic calcium-mobilizing factor gene RankL and the anabolic Wnt signaling pathway co-receptor gene LRP5. We also resolve the mechanism whereby 1,25(OH) 2 D 3 autoregulates the expression of its own receptor. The results identify new features of vitamin D-regulated enhancers, including their locations at gene loci, the structure of the VDR binding sites located within, their modular nature and their functional activity. Our studies suggest that vitamin D enhancers regulate the expression of key target genes by facilitating the recruitment of both the basal transcriptional machinery as well as the protein complexes necessary for altered gene expression. KeywordsChIP scanning; ChIP-chip scanning; enhancer modules; VDR/RXR DNA binding; distal transcriptional regulation; chromatin looping; histone acetylation; RNA polymerase II recruitment Introductory backgroundTwo sequential hydroxylations of vitamin D 3 in the liver and kidney lead to the formation of 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ), a hormone whose primary function is to control mineral homeostasis in higher organisms [1]. Like other hormones in this class, the biological effects of 1,25(OH) 2 D 3 are achieved through the regulation of gene expression [2]. These activities are mediated by the vitamin D receptor (VDR), a member of the steroid receptor family of genes that control the diverse biological actions of numerous small molecule hormones [3]. The DNA targets of the VDR can be found in a number of induced genes including those for osteocalcin, osteopontin, several p450-containing genes and p21 as well as *To whom correspondence should be addressed: Department of Biochemistry, University of 433 Babcock Drive, Madison, WI 53706. Tel: (608) 262-8229; fax: (608) 263-7609; email: pike@biochem.wisc.edu.. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and a...

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