The purpose of this research project is to investigate the role of retinoids and specific retinoid-binding proteins in ocular tissues. In studies on the vitiligo mouse model of retinal degeneration we determined the levels of retinoic acid (RA) and its distribution between neural retina and retinal pigment epithelium (RPE). The level of RA in the RPE was the same for both affected and control mice at 6 weeks of age, but a marked elevation in RA was noted in the neural retina of affected mice as compared to controls at 6 weeks of age that continued through at least 10 weeks of age. The expression of the RA-generating enzyme, class 1 aldehyde dehydrogenase (retinaldehyde dehydrogenase (RALDH) ),was evaluated by immunohistochemistry of affected and age-matched control mouse eyes at post-natal ages 2, 4, and 10 weeks. Expression of RALDH was consistently detected in three ocular tissues: the epithelium of the ciliary body; a single layer of cells on the anterior surface of lens; and the dorsal retina. The level of RALDH expression was similar at 2 and 4 weeks of age for both affected and control mice. At 10 weeks, however, a marked increase in RALDH expression was noted in the dorsal neural retina. Since retinoic acid is a potent mediator of several biologic processes through control of the transcriptional properties of the RAR and RXR nuclear receptor families, elevation of retinoic acid and its metabolites in the mutant mouse neural retina could exert profound effects on other metabolic pathways, possibly contributing to the retinal degeneration observed in the mutant. Drosophila retinoid- and fatty acid- binding glycoprotein (RFABG) is highly expressed in the Semper (cone) cells of the fly compound eye. Both RFABG protein and message are reduced to negligible levels in retinoid-deprived flies and retinoic acid supplementation restores both gene and protein expression to normal levels. We have now cloned and sequenced the 5'- flanking region of the RFABG gene in order to characterize the promoter region and examine its transcriptional regulation by retinoic acid. An RFABG promoter construct was cloned into a luciferase reporter vector and transiently transfected into D. melanogaster SL2 and COS-7 cells then different concentrations of all-trans retinoic acid were added to the cells in culture. An all-trans retinoic acid-dependent increase in reporter activity was observed that supports the concept that the RFABG gene is transcriptionally regulated by retinoic acid.
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