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Molecular Endocrinology 11 (13): 1971-1984
Copyright © 1997 by The Endocrine Society

Expression and Characterization of Recombinant Type 2 3{alpha}-Hydroxysteroid Dehydrogenase (HSD) from Human Prostate: Demonstration of Bifunctional 3{alpha}/17ß-HSD Activity and Cellular Distribution

Hsueh-Kung Lin1, Joseph M. Jez1, Brian P. Schlegel, Donna M. Peehl, Jonathan A. Pachter and Trevor M. Penning

Departments of Pharmacology, Biochemistry, and Biophysics (H.K.L., J.M.J., B.P.S., T.M.P) University of Pennsylvania School of Medicine Philadelphia, Pennsylvania 19104
Department of Urology (D.M.P) Stanford University Medical School Stanford, California 94305
Schering-Plough Research Institute (J.A.P.) Kenilworth, New Jersey 07033

In androgen target tissues, 3{alpha}-hydroxysteroid dehydrogenase (3{alpha}-HSD) may regulate occupancy of the androgen receptor (AR) by catalyzing 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 prostate 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 identity to rat liver 3{alpha}-HSD and human type 1, type 2, and type 3 3{alpha}-HSDs, respectively, and is a member of the aldo-keto reductase superfamily. The close homology with human type 2 3{alpha}-HSD suggests that it is either identical to this enzyme or a structural allele. Surprisingly, when the recombinant protein was expressed and purified from Escherichia coli, the enzyme did not oxidize androsterone when measured spectrophotometrically, an activity previously assigned to recombinant type 2 3{alpha}-HSD using this assay. Complete kinetic characterization of the purified protein using spectrophotometric, fluorometric, and radiometric assays showed that the catalytic efficiency favored 3{alpha}-androstanediol oxidation over 5{alpha}-DHT reduction. Using [14C]-5{alpha}-DHT as substrate, TLC analysis confirmed that the reaction product was [14C]-3{alpha}-androstanediol. However, in the reverse reaction, [3H]-3{alpha}-androstanediol was oxidized first to [3H]-androsterone and then to [3H]-androstanedione, revealing that the expressed protein possessed both 3{alpha}- and 17ß-HSD activities. The 17ß-HSD activity accounted for the higher catalytic efficiency observed with 3{alpha}-androstanediol. These findings indicate that, in the prostate, type 2 3{alpha}-HSD does not interconvert 5{alpha}-DHT and 3{alpha}-androstanediol but inactivates 5{alpha}-DHT through its 3-ketosteroid reductase 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 were observed in epithelial cells derived from benign prostatic hyperplasia 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 complete characterization of recombinant type 2 3{alpha}-HSD demonstrating dual activity and cellular distribution in the human prostate.




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