ALDH1A enzymes regulate retinoic acid homeostasis in a tissue specific manner: A model of how novel methods can unravel old unanswered questions

Thesis (Ph.D.)--University of Washington, 2015Retinoic acid (RA), the active form of vitamin A, is an essential signaling molecule in many tissues and the concentrations of RA are spatiotemporally controlled in target tissues. However, the source of RA in each tissue is not clear and the role of specific enzymes in RA synthesis and metabolism has not been described. While the enzymes of the aldehyde dehydrogenase 1A family (ALDH1A) are believed to control the synthesis of RA, general knowledge on the expression pattern and relative contribution of these enzymes to tissue RA formation is unknown. In addition, a direct relationship between altered ALDH1A activity and tissue RA concentrations has never been shown. To characterize the importance of each ALDH1A isoform to RA formation, novel methods were developed to measure tissue RA, ALDH1A protein tissue expression levels, and intrinsic RA formation. These methods were applied to the human testis to demonstrate that the intratesticular concentration of RA is positively associated with RA formation by ALDH1A in the testis. In addition, a distinct localization of ALDH1A was identified in the testis suggesting a specific role for each enzyme in controlling RA formation. To directly test whether inhibition of ALDH1A enzymes decreases RA concentrations in vivo, the potent ALDH1A inhibitor WIN 18,446 was used to inhibit ALDH1A activity in mice. ALDH1A expression levels, RA formation kinetics by ALDH1A, kinetics of ALDH1A inhibition by WIN 18,446, and WIN 18,446 disposition were used to predict the time course and extent of inhibition of RA formation in the mouse testis and liver. While ALDH1A1 and ALDH1A2 were responsible for the majority of RA formation in the mouse testis, ALDH1A1 and aldehyde oxidase contributed to RA formation in the liver. Due to the different complement of enzymes contributing to RA formation in each tissue and the distinct inhibition kinetics of WIN 18,446, WIN 18,446 administration was predicted to generate a tissue specific reduction in atRA concentrations. As predicted, WIN 18,446 decreased liver RA only 50% but testicular RA was decreased over 95%. These data demonstrate that inhibition of ALDH1A enzymes will decrease RA concentrations in a tissue specific manner and selective ALDH1A inhibition could be used to alter RA concentrations in select target tissues. Taken together, the comprehensive novel methodology developed to evaluate RA homeostasis in individual tissues provides insight to how the individual ALDH1A enzymes mediate RA concentrations in tissue and specific cell types