The overall objective of this proposal is to gain a detailed understanding of the molecular regulation of the synthesis of 1,25-dihydroxyvitamin D (1,25D). Although this steroid hormone plays a crucial role in calcium metabolism, bone growth, and tissue differentiation, little is known about the molecular mechanisms of regulation of its synthesis. The key quantitative regulatory step in the synthesis of 1,25D is its 1alpha-hydroxylation from its endogenous precursor, 25-hydroxyvitaminD (25(OH)D), catalyzed by the enzyme 25(OH)D-1alpha-hydroxylase (1-OHase). The 1-OHase is a mitochondrial cytochrome P450 enzyme similar to the steroidogenic enzymes in the adrenal and gonad. Dr. Portale's laboratory has recently cloned the cDNA and gene for the human 1alpha-hydroxylase enzyme, designated P450c1. They now propose to study the molecular mechanisms of regulation of 1,25D production, and specifically how PTH, phosphorus, and 1,25D regulate the synthesis of 1,25D. Production of 1,25D is disordered in acute and chronic renal failure, X-linked hypophosphatemic rickets, autosomal recessive vitamin D dependent rickets Type 1, renal Fanconi syndrome, and with advanced age. The proposed studies of the physiologic regulation of the 1-OHase at the molecular level will provide the basis for subsequent studies of the potential molecular mechanisms by which regulation of this enzyme is altered by aging and renal disease: 1) They will clone a cDNA for rodent P450c1 and raise antibodies to human P450c1 protein; 2) They will examine hormonal regulation of P450c1 mRNA and protein abundance in mice in vivo and in isolated mouse proximal tubules, in vitro, and determine if induced changes are mediated by transcriptional events. 3) Using immortalized human proximal tubule cells, they will study transcriptional regulation using functional assays of promoter/reporter constructs, and will examine protein/DNA interactions in the relevant regions by bandshift assays, UV-crosslinking, and Southwestern blotting to localize specific cis-elements and their cognate DNA binding proteins; and 4) They will determine the tissue distribution of P450c1 mRNA and protein in kidney and will localize P4501 gene expression in microdissected rat nephron segments.
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