Human Aldo Keto Reductases and Steroid Hormone Action

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DESCRIPTION: (Scanned from the applicant's description): Human Aldo-Keto Reductases (AKR) of the AKR1C sub-family include 3alpha-, 17beta-, and 20alpha-hydroxysteroid dehydrogenases (HSDs). By acting as ketosteroid reductases or hydroxysteroid oxidases they can either convert potent sex hormones (androgens, estrogens and progestins) into their cognate inactive metabolites or they can form potent hormones by catalyzing the reverse reactions. The synthesis of sex hormones in target tissues is known as their intracrine formation. Our hypothesis is that tissue specific expression of AKR isoforms regulates the occupancy and trans-activation of steroid hormone receptors and provides a pre-receptor regulation of steroid hormone action. In vitro, recombinant AKR1C2 (formerly type 3 3alpha-HSD) and AKR1C3 formerly type 2 3alpha-HSD and type 5 17beta-HSD) function as 3-, 17 and 20-ketosteroid reductases and as 3alpha-, 17beta- and 20alpha-hydroxysteroid oxidases. Both isoforms are expressed in human prostate and AKR1C3 is dominantly expressed in human mammary gland. The role of AKR1C2 and AKR1C3 in androgen, estrogen and progestin metabolism will be studied by transient and stable expression of their cDNA's into CHOP and human embryonic kidney cells, respectively. Forced expression of AKR1C2 and AKR1C3 in androgen receptor (AR; +/-ive) prostate cells (LNCaP and PC3) as well as forced expression of AKR1C3 into estrogen receptor (ERalpha; +/-ive) mammary cells (MCF-7 and MDA-MB-435) will elucidate their roles in steroid hormone metabolism and steroid receptor trans-activation. AKR 1 C2- and AKR1C3 expression will be measured in normal, benign prostatic hyperplasia and prostatic carcinoma microdissected sections of radical prostatectomy samples using RT-PCR. Co-localization with the AR will be measured by in situ hybridization and immunohistochemistry. Identical techniques will be used to measure AKR 1 C3 expression and its co-localization with the ER and PR in normal and ductal carcinoma regions of breast biopsy samples. These studies will determine whether AKRs co-localize with steroid receptors and whether AKR expression is associated with disease. Selective AKR1C2 and AKR1C3 inhibitors will be developed from two lead compounds, ursodeoxycholate and N-phenylanthranilic acids (e.g. flufenamic acid). These leads will be used to develop transition-state analogs, tight-binding inhibitors, and mechanism-based inactivators. These inhibitors may provide tools to dissect function and provide routes to the first Selective Intracrine Modulators that target AKRs.