The long term goals of this project are to determine the biological significance of 9cRA (9-cis-retinoic acid) in the broader context of retinoid homeostasis. 9cRA has been proposed as an endogenous retinoid that activates RXR. RXRs regulate growth, development and metabolism by inducing gene expression, and by modulating the effects of type II nuclear receptors, such as RAR and PPAR. Determining whether or not 9cRA serves as an endogenous RXR ligand would provide new insight into regulation of the biological functions controlled by these receptors. We hypothesize that a quantitatively important route of 9cRA biosynthesis in vivo involves conversion of 9-cis-retinol into 9-cis-retinal, catalyzed by short-chain dehydrogenases/reductases, followed by conversion of 9-cis-retinal into 9cRA, catalyzed by retinal dehydrogenases. Implications of this hypothesis include: 1) all-trans-retinoic acid does not serve as an obligatory precursor of 9cRA; 2) 9cRA biosynthesis can be regulated independently of atRA biosynthesis. We also hypothesize that mitochondria and peroxisomes, organelles not previously connected with retinoid metabolism, contribute significantly to retinoid homeostasis, and may form 9cRA.
The specific aims are to determine: 1) phenotypes of CRAD1 and CRAD3 null mice; 2) expression patterns of mRNAs encoded by the Rdh6 (CRAD1) and Rdh9 (CRAD3) genes; 3) expression loci of the enzymes CRAD1 and 3; 4) the contribution of mitochondria to 9-cis-retinoid biosynthesis and RA catabolism; 5) the contribution of PPARalpha and peroxisomes to retinoid metabolism. These studies will rely on a combination of techniques/approaches, including molecular genetics (knock-out, transgenic and knock-in mice), analytical biochemistry (LC/MS, hplc), cell biology (in situ hybridization, fluorescence microscopy), and biochemistry (metabolic studies in vivo and in vitro). This work will provide new insight into retinoid metabolism/ function.
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