EXPRESSION AND CHARACTERIZATION OF RECOMBINANT TYPE-2 3-ALPHA HYDROXYSTEROID DEHYDROGENASE (HSD) FROM HUMAN PROSTATE - DEMONSTRATION OF BIFUNCTIONAL 3-ALPHA 17-BETA-HSD ACTIVITY AND CELLULAR-DISTRIBUTION/

In androgen target tissues, 3 alpha-hydroxysteroid dehydrogenase (3 al pha-HSD) may regulate occupancy of the androgen receptor (AR) by catal yzing the interconversion of 5 alpha-dihydrotestosterone (5 alpha-DHT) (a potent androgen) and 3 alpha-androstanediol (a weak androgen). In this study, a 3 alpha-HSD cDNA (1170 bp) was isolated from a human pro state cDNA library. The human prostatic 3 alpha-HSD cDNA encodes a 323 -amino acid protein with 69.9%, 84.1%, 99.4%, and 87.9% sequence ident ity to rat liver 3 alpha-HSD and human type 1, type 2, and type 3 3 al pha-HSDs, respectively, and is a member of the aldo-keto reductase sup erfamily. The close homology with human type 2 3 alpha-HSD suggests th at it is either identical to this enzyme or a structural allele. Surpr isingly, when the recombinant protein was expressed and purified from Escherichia coli, the enzyme did not oxidize androsterone when measure d spectrophotometrically, an activity previously assigned to recombina nt type 2 3 alpha-HSD using this assay. Complete kinetic characterizat ion of the purified protein using spectrophotometric, fluorometric, an d radiometric assays showed that the catalytic efficiency favored 3 al pha-androstanediol oxidation over 5 alpha-DHT reduction. Using [C-14]- 5 alpha-DHT as substrate, TLC analysis confirmed that the reaction pro duct was [C-14]-3 alpha-androstanediol. However, in the reverse reacti on, [H-3]-3 alpha-androstanediol was oxidized first to [H-3]-androster one and then to [H-3]-androstanedione, revealing that the expressed pr otein possessed both 3 alpha- and 17 beta-HSD activities. The 17 beta- HSD activity accounted for the higher catalytic efficiency observed wi th 3 alpha-androstanediol. These findings indicate that, in the prosta te, type 2 3 alpha-HSD does not interconvert 5 alpha-DHT and 3 alpha-a ndrostanediol but inactivates 5 alpha-DHT through its 3-ketosteroid re ductase activity. Levels of 3 alpha-HSD mRNA were measured in primary cultures of human prostatic cells and were higher in epithelial cells than stromal cells. In addition, elevated levels of 3 alpha-HSD mRNA w ere observed in epithelial cells derived from benign prostatic hyperpl asia and prostate carcinoma tissues. Expression of 3 alpha-HSD was not prostate specific, since high levels of mRNA were also found in liver , small intestine, colon, lung, and kidney. This study is the first co mplete characterization of recombinant type 2 3 alpha-HSD demonstratin g dual activity and cellular distribution in the human prostate.