This renewal application proposes to continue the investigation of 1,25-dihydroxy-vitamin D3 (1,25(OH)2D3) hormone action in cell culture. We plan to expand on published observations made during the first three years of this grant in mouse fibroblasts (3T6), rat osteosarcoma (ROS 17/2.8) osteoblast-like cells, porcine (LLC- PK1) and monkey (LLC-MK2) kidney cells, human leukemia (HL- 60) cells and human fibroblasts from patients with vitamin D- dependent rickets type II. Biological responses that will be monitored include 1,25(OH)2D3 receptor upregulation and modification via phosphorylation, calbindin D28k (CaBP) induction, 25(OH)vitamin D3-240Hase catabolic enzyme activity, and cell growth and differentiation. The hypothesis to be tested is that the nuclear receptor for 1,25(OH)2D3, in its occupied and phosphorylated form, binds enhancer sequences in DNA and this association triggers the transcription of vitamin D induced genes which ultimately code for proteins affecting the myriad of bioresponses. We have recently screened an expression vector library with our receptor monoclonal antibody to obtain a cDNA to the avian 1,25(OH)2D3 receptor and propose to exploit this to select for mammalian 1,25(OH)2D3 receptor cDNAs via nucleic acid hybridization screening of appropriate libraries. If successful, we propose to use mammalian 1,25(OH)2D3 receptor cDNA to screen mouse, rat and human genomic libraries for the respective natural receptor genes. These reagents, if obtained even in part, would facilitate the following novel experiments in cultured cells. i) Characterization of 1,25(OH)2D3 receptor autoregulation by identifying enhancer regions in the natural receptor gene. ii) Elucidation of the role of receptor phosphorylation through transfection with receptor cDNAs possessing various deletions. iii) Transfection of CaBP negative but receptor-rich 3T6 cells with CaBP-promoter constructs. iv) Transfection of 1,25(OH)2D3 receptor DNA into receptor deficient LLC-MK2 kidney cells and fibroblasts from patients with resistance to 1,25(OH)2D3 to determine if bioresponsiveness is restored. v) Probe the mechanism of HL-60 cell differentiation by transfecting resistant HL-60 blast cells with the human 1,25(OH)2D3 receptor. Finally, antisense mRNA to the receptor will be incorporated to determine its effect on 1,25(OH)2D3 action. These studies should not only provide the final test for the nuclear receptor hypothesis of vitamin D action, but should add to our insight into the basic mechanisms of clinical vitamin D resistance and the potential anticancer effect of 1,25(OH)2D3.
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