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Immunocytochemical localization of the 1,25-dihydroxyvitamin-D3 receptor in target cells

Author(s): Clemens TL, Garrett KP, Zhou XY, Pike JW, Haussler MR, et al.

Abstract

We have used a monoclonal antibody (9A7) against the purified avian 1,25-dihydroxyvitamin D3 receptor to develop an immunocytochemical technique for visualization of the protein in fixed tissues and cultured cells. In Bouin’s-fixed, chick intestine, 1,25-dihydroxyvitamin D3 receptor-like immunoreactivity was localized mainly in nuclei of epithelial cells and was more abundant in the crypt than in the villar cells. Receptor staining was low or undetectable in liver hepatocytes but was present in nuclei of cells lining the hepatic sinusoids. In rat brain, receptor-like immunoreactivity was abundant and widely distributed, but did not always coincide with the presence of vitamin D-dependent calcium-binding protein; 1,25-dihydroxyvitamin D3 receptor was absent from cerebellar Purkinje cells that contained abundant calcium-binding protein. In disaggregated rat bone cells, receptor immunoreactivity was present in mononuclear cells including osteoblasts and fibroblasts but was absent from osteoclasts. Two separate clones of osteoblast-like, rat osteosarcoma cells, shown in previous studies to be either receptor positive (17/2.8) or negative (24.1), demonstrated nuclear immunoreactivity in exact concordance with receptor levels as determined by ligand binding. The phenomenon of hormoneinduced up-regulation of receptor was visualized in receptorpositive 3T6 fibroblasts by demonstration of markedly enhanced nuclear reactivity in cells treated with 10-7 M 1,25-dihydroxyvitamin D3 for 48 h. Our studies demonstrate the feasibility of the immunocytochemical approach to visualize the 1,25-dihydroxyvitamin D3 receptor in target tissues and show that it is predominantly a nuclear protein in the relatively unoccupied and fully activated states. Moreover, the vitamin D-dependent calcium binding is not a universal marker for 1,25-dihydroxyvitamin D3 action. Rather, our observations suggest that the expression of the 1,25-dihydroxyvitamin D3 receptor may be connected with the state of cellular differentiation.

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