The object of this proposal is to obtain a better understanding of the multiple actions of the vitamin D endocrine system, as well as to obtain a better understanding of the molecule interaction of 1,25- dihydroxyvitamin D3 (1,25(OH)2D3) and the vitamin D receptor (VDR) with other steroid-retinoic acid receptor complexes, by studying the function and regulation of mammalian vitamin D dependent calcium binding protein (calbindin-D28k). In an effort to establish the functional significance of calbindin, investigations will focus on effects of calbindin on transport differentiation. Specifically concerning transport, experiments will examine the effects of incorporation of calbindin into basolateral and luminal membrane vesicles of the distal tubule on calcium uptake. Efforts will also be made to examine the effects of the introduction of calbindin by microinjection into immortalized MDCT 210 distal tubular cells (endogenous calbindin is undetectable in these cells) on intracellular Ca++ and on PTH-dependent Ca++ transport. Since PTH-stimulated calcium and calbindin have been demonstrated in the distal tubule, it is indeed possible that there is a concerted action of PTH and calbindin in mediating Ca++ transport in the distal tubule. The studies would suggest, for the first time, a role for calbindin in the kidney. Using overexpression and, if indicated, antisense strategies, the applicants will determine effects of calbindin on: 1) differentiation and insulin secretion in a pancreatic beta cell line and 2) neuronal differentiation in response to retinoic acid. In addition, they will examine the role of calbindin in protecting against Ca++-mediated cell death. Significant findings in the above areas (renal function, growth/differentiation, insulin secretion and protection against cell degeneration) would represent important, major advances concerning the functional significance of calbindin. In addition, efforts are proposed to study the regulation of calbindin by 1,25(OH)2D3, and factors other than or in addition to 1,25(OH)2D3, using renal, pancreatic beta and neuronal cells. These studies will complement the studies concerning the functional significance of calbindin. Finally, the applicants will also study the regulation of calbindin using transfected cells. Since they have found that 1,25(OH)2D3, retinoic acid, estrogen and glucocorticoids can all regulate calbindin-D28k, the regulation of calbindin will be used as a model system in which to investigate the molecular mechanisms of hormonal interaction. It is suggested that an understanding of the molecular mechanisms involved in these interactions will play an increasingly important role in the elucidation of the mechanism of control of calcium homeostasis. Implicit in this study is facilitation of a more detailed understanding of how 1,25(OH)2D3 and other hormones may be involved in the many disease processes associated with abnormalities in calcium homeostasis, such as osteoporosis and perturbations of parathyroid function.
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