Vertebrate alcohol dehydrogenase (ADH) is encoded by a family of genes falling into seven classes with unique patterns of tissue-specific and developmental expression. Class I ADH oxidizes ethanol efficiently, while all other classes of ADH are inefficient with ethanol suggesting they have evolved to catalyze the oxidation of other alcohols. Class IV ADH is most efficient in catalyzing the oxidation of retinol (vitamin A). This is the rate-limiting step in the synthesis of retinoic acid, a hormone that regulates embryogenesis, spermatogenesis, and epithelial differentiation by functioning as a transcriptional regulatory ligand for a nuclear receptor signaling pathway. Our understanding of how retinol is physiologically activated in the correct spatiotemporal pattern to produce the ligand for this pathway is very limited. To unravel this layer of biological control in retinoid signaling, metabolic enzymes and their genes must be characterized. In this regard, studies on class IV ADH gene regulation are likely to provide much- needed information. Expression of this gene in mouse embryos coincided spatiotemporally with sites of retinoic acid detection, suggesting class IV ADH plays a role in regulating the initial turn-on of retinoic acid synthesis during embryogenesis. Also, expression coincides with sites of retinoic acid synthesis in several adult retinoid target tissues such as testis, epididymis, and epidermis. Since ethanol acts as a competitive inhibitor of ADH-catalyzed retinol oxidation, we have hypothesized that retinoic acid synthesis may be decreased by excess ethanol consumption. For alcohol use during pregnancy, this may be a contributing factor in fetal alcohol syndrome, characterized by malformations of craniofacial tissues known to require retinoic acid for proper development. Studies on class IV ADH gene expression in vivo are needed to complement the enzymatic studies done in vitro in order to fully understand the physiological role of this enzyme in retinoic acid synthesis, fetal alcohol syndrome, and perhaps other alcohol-related disorders. Proposed goals: (1) Locate regulatory elements for mouse and human class IV ADH gene expression in embryonic and adult tissues in vivo using promoter-lacZ fusions in transgenic mice. (2) Use ADH antibodies to study the pattern of ADH localization in mouse embryos and adult tissues by immunohistochemistry. (3) Initiate a molecular genetic analysis of class IV ADH in the frog Xenopus whose embryogenesis is easily studied; mRNA, protein, and promoter expression will be examined, and sites conserved from frog to mouse will help identify cells where this enzyme performs an important function. (4) Perform in vivo functional studies on retinoic acid synthesis by injection of frog embryos with mRNAs for ADHs and other retinoid metabolic enzymes (gain-of-function) or with antibodies to inhibit endogenous class IV ADH activity (loss-of- function).
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