In androgen target tissues 3a-hydroxysteroid dehydrogenases (3a-HSDs) may regulate the occupancy of androgen receptor (AR) by interconverting 5a-dihydrotestosterone (5a-DHT, a potent androgen) with 5a-androstane-3a,17b-diol (3a-diol, a weak androgen). We have obtained type 2 and type 3 3a-HSD cDNAs expressed in human prostate and overexpressed the enzymes in E. coli. Kinetic studies of these recombinant enzymes show that type 3 3a-HSD functions as both a 3a-and 17b-HSD to inactivate active androgens, whereas, type 3 3a-HSD interconverts 5a-DHT with 3a- diol. Levels of 3a-HSD mRNA were higher in primary cultures of prostatic epithelial cells than stromal cells; and elevated levels of 3a-HSD mRNA were observed in primary cultures of epithelial cells derived from benign prostatic hyperplasia and prostatic carcinoma tissues. Expression of steady state levels of 3a-HSD mRNA is up-regulated by epidermal growth (EGF) in human prostatic cell lines, LNCaP (androgen sensitive) and PC3 (androgen insensitive). The focus of this proposal is to examine the physiological functions of type 2 and type 3 3a-HSD in regulating androgen metabolism and their activities in modulating prostatic cell proliferation will be investigated. This will be accomplished by stably transfecting type 2 and type 3 3a-HSD cDNAs into these cells. Second, levels of endogenous type 2 and type 3 3a-HSD transcripts will be examined in RNA extracted from the cell lines and normal prostate using ribonuclease protection assay (RPA). Third, EGF-regulated type 2 and type 3 3a-HSD mRNA levels will be examined using RPA. Changes in 3a-HSD expression mediated by EGF will be examined in cell lysates by immunotitration of the enzyme activity. Fourth, to understand the constitutive and EGF- regulated type 2 and type 3 3a-HSD expression in prostatic cells, the 5'-flanking regions of the 3a-HSD genes will be sequenced and cis-acting elements responsible for transcription regulation of two isoforms will be identified.
