This application focuses on the regulation of retinol esterification by LRAT, a microsomal enzyme that specifically recognize retinol bound to CRBP. Prior work from this group has shown that hepatic LRAT activity is highly sensitive to vitamin A nutritional status and is regulated rapidly by the endogenous retinoid hormone, retinoic acid (RA) and well as by exogenous, synthetic retinoids such as 4-(N-hydroxyphenyl) retinamide (4-HPR). The applicant proposes a cell biological approach to better understand 1) the roles of CRBP in retinol esterification by LRAT, and 2) the regulation of LRAT activity by endogenous and exogenous retinoids. The applicant hypothesizes that the regulation of LRAT activity may be important in regulating the availability of retinol for oxidation.
Specific aims 1 and 2 make use of liver cell lines (HepG2 cells stably transfected with cDNA or anti-sense cDNA and newly cloned cells with features of liver stellate cells) to test the hypothesis that CRBP regulates retinol uptake, esterification, oxidation, metabolite formation, and the secretion of RBP.
In aim 3 the applicant will test the hypothesis that the nuclear retinoid receptor RAR-alpha is a key regulator of endogenous LRAT activity by transfecting cells with a dominant negative mutant of RAR-alpha.
In aim 4, the applicant proposes to clone, characterize and express LRAT activity. It is anticipated that the proposed research will improve the understanding of the normal feedback regulation of retinol metabolism by endogenously-produced RA as well as the influence of therapeutic retinoids used in cancer chemoprevention on the metabolism of retinol and the requirement for dietary vitamin A.
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