The long term goal of these studies is to develop an understanding of the genes responsible for the metabolism of retinoic acid in mammalian tissues. Retinoic acid plays a significant role in pattern formation during vertebrate embryogenesis and exerts profound effects on cell differentiation and on homeostatic properties of diverse tissues in the adult. Although the conversion of dietary retinol to RA has been characterized in some detail, the metabolism of RA, the induction of RA metabolism by RA, and the conversion of RA into other active retinoid metabolites are still poorly understod. The work proposed here is based on the recent discovery of the first putative specific RA metabolizing enzyme, RA-4-hydroxylase (CYP26A1). The investigators will evaluate the hypothesis that this RA-hydroxylase is one member of a related family of enzymes that are responsible for the conversion of RA into active retinoid metabolites in different tissues.
The specific aims are to: (1) identify and clone cDNAs that encode isozymes of RA-4-hydroxylase; (2) characterize this new gene family by sequence analysis of cDNA clones and comparison of the cellular distribution and regulation by dietary RA status of CYP26 mRNA species; and (3) isolate genomic clones encoding this gene family. To accomplish this, they will use degenerate PCR cloning, RT-PCR with RNA from rats fed a vitamin A-deficient diet as well as a vitamin A-deficient but RA-treated diet to examine regulation by dietary RA status, and protein expression studies. Retinoid function, which is critical to vertebrate development and survival, depends upon the relationships among retinoid receptors, retinoid binding proteins, and enzymes that catalyze retinoid synthesis and metabolism. Much is already known of the structures and functions of the receptors, binding proteins, and synthetic enzymes. The work proposed here will provide insight into the function of the last group, enzymes that metabolize RA. An understanding of the regulation of the metabolism of RA in different tissues will advance our knowledge of physiological mechanisms that control steady state levels of active retinoids in cells and target tissues. Since retinoids are crucial regulators of embryonic development, the insight gained from these studies will have particular relevance to morphogenesis and our understanding of both developmental abnormalities and vitamin A deficiency.
