This application proposes the continuation of our investigation of the molecular and biological functions of the vitamin D hormone (1,25(OH)2D3) in cultured cell systems. There will be a new focus on 1,25(OH)2D3 action in osteoblast-like bone cells and epithelial cells of the intestine, where vitamin D exerts its major effects upon bone remodeling and mineral absorption, respectively. In the last grant period we accomplished the molecular cloning, overexpression and immunocytochemical localization of the human vitamin D receptor (hVDR), and elucidated the vitamin D response element (VDRE) in the rat osteocalcin gene. These findings will be extended to probe the phosphorylation of hVDR in relation to its ability to bind to the VDRE and enhance transcription of an osteocalcin promoter/reporter construct in transfected cells. Site-directed mutagenesis will be employed to determine the residues in hVDR that are phosphorylated and stimulation of the relevant protein kinases in intact cells will be effected to test in vivo significance. Next, hVDR will be overexpressed in the insect baculovirus system and in E. coli to generate sufficient quantities for biochemical study. Expressed hVDR will be purified for study of phosphorylation by protein kinase C and casein kinase II, in vitro. Phosphopeptide mapping of hVDR overexpressed in COS-7 cells will be used to elucidate the site of 1,25(OH)2D3-dependent, hVDR phosphorylation. Preliminary data that a nuclear receptor auxiliary factor (RAF) is required for high-affinity VDR binding to the VDRE will be pursued by characterizing, purifying and cloning the major RAF in CV-1 cells. RAF will be cloned by screening an expression vector library with purified hVDR/[32p]VDRE. Ultimately, the above components will be reassembled as purified factors in an in vitro transcription system designed to determine the mechanism whereby 1,25(OH)2D3 stimulates the tissue specific expression of osteocalcin. Finally, a molecular comparison of 1,25(OH)2D3/VDR action on osteocalcin will be made with the effect on calbindin-D28k (CaBP) induction in cultured embryonic duodena. Because it is inhibited by cycloheximide, CaBP regulation by 1,25(OH)2D3 may be unique in that an intermediary induced protein may bind to a non-VDRE enhancer in the CaBP promoter and/or stabilization of CaBP mRNA may occur. In toto, these experiments should define the precise signal transduction pathways whereby vitamin D controls the expression of crucial 'bone and intestinal genes, and these results may provide clues to the molecular basis of clinical vitamin D resistance as well as to the role of vitamin D in the prevention of osteoporosis.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37DK033351-12
Application #
2139035
Study Section
Orthopedics and Musculoskeletal Study Section (ORTH)
Program Officer
Tondravi, Mehrdad M
Project Start
1984-07-01
Project End
1997-06-30
Budget Start
1995-07-01
Budget End
1996-06-30
Support Year
12
Fiscal Year
1995
Total Cost
Indirect Cost
Name
University of Arizona
Department
Biochemistry
Type
Schools of Medicine
DUNS #
City
Tucson
State
AZ
Country
United States
Zip Code
85721
Sabir, Marya S; Haussler, Mark R; Mallick, Sanchita et al. (2018) Optimal vitamin D spurs serotonin: 1,25-dihydroxyvitamin D represses serotonin reuptake transport (SERT) and degradation (MAO-A) gene expression in cultured rat serotonergic neuronal cell lines. Genes Nutr 13:19
Karrys, Amitis; Rady, Islam; Chamcheu, Roxane-Cherille N et al. (2018) Bioactive Dietary VDR Ligands Regulate Genes Encoding Biomarkers of Skin Repair That Are Associated with Risk for Psoriasis. Nutrients 10:
Sabir, Marya S; Khan, Zainab; Hu, Chengcheng et al. (2017) SIRT1 enzymatically potentiates 1,25-dihydroxyvitamin D3 signaling via vitamin D receptor deacetylation. J Steroid Biochem Mol Biol 172:117-129
Haussler, Mark R; Whitfield, G Kerr; Haussler, Carol A et al. (2016) 1,25-Dihydroxyvitamin D and Klotho: A Tale of Two Renal Hormones Coming of Age. Vitam Horm 100:165-230
Dampf Stone, Angelika; Batie, Shane F; Sabir, Marya S et al. (2015) Resveratrol potentiates vitamin D and nuclear receptor signaling. J Cell Biochem 116:1130-43
Kaneko, Ichiro; Saini, Rimpi K; Griffin, Kristin P et al. (2015) FGF23 gene regulation by 1,25-dihydroxyvitamin D: opposing effects in adipocytes and osteocytes. J Endocrinol 226:155-66
Hsieh, Jui-Cheng; Estess, Rudolf C; Kaneko, Ichiro et al. (2014) Vitamin D receptor-mediated control of Soggy, Wise, and Hairless gene expression in keratinocytes. J Endocrinol 220:165-78
Austin, Heather R; Hoss, Elika; Batie, Shane F et al. (2014) Regulation of late cornified envelope genes relevant to psoriasis risk by plant-derived cyanidin. Biochem Biophys Res Commun 443:1275-9
Bartik, Leonid; Whitfield, G Kerr; Kaczmarska, Magdalena et al. (2010) Curcumin: a novel nutritionally derived ligand of the vitamin D receptor with implications for colon cancer chemoprevention. J Nutr Biochem 21:1153-61
Haussler, Mark R; Haussler, Carol A; Whitfield, G Kerr et al. (2010) The nuclear vitamin D receptor controls the expression of genes encoding factors which feed the ""Fountain of Youth"" to mediate healthful aging. J Steroid Biochem Mol Biol 121:88-97

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