STRUCTURE, FUNCTION AND TISSUE-SPECIFIC GENE-EXPRESSION OF 3-BETA-HYDROXYSTEROID DEHYDROGENASE 5-ENE-4-ENE ISOMERASE ENZYMES IN CLASSICAL AND PERIPHERAL INTRACRINE STEROIDOGENIC TISSUES

The membrane-bound enzyme 3beta-hydroxysteroid dehydrogenase/5-ene-4-ene isomerase (3beta-HSD) catalyses an essential step in the transformation of all 5-pregnen-3beta-ol and 5-androsten-3beta-ol steroids into the corresponding 3-keto-4-ene-steroids, namely progesterone as well as all the precursors of androgens, estrogens, glucocorticoids and mineralocorticoids. We have recently characterized two types of human 3beta-HSD cDNA clones and the corresponding genes which encode type I and II 3beta-HSD isoenzymes of 372 and 371 amino acids, respectively, and share 93.5% homology. The human 3beta-HSD genes containing 4 exons were assigned by in situ hybridization to the p11-p13 region of the short arm of chromosome 1. Human type I 3beta-HSD is the almost exclusive mRNA species present in the placenta and skin while the human type II is the predominant mRNA species in the adrenals, ovaries and testes. The type I protein possesses higher 3beta-HSD activity than type II. We elucidated the structures of three types of rat 3beta-HSD cDNAs as well that of one type of 3beta-HSD from bovine and macaque ovary lambdagt11 cDNA libraries, which all encode a 372 amino acid protein. The rat type I and II 3beta-HSD proteins expressed in the adrenals, gonads and adipose tissue share 93.8% homology. Transient expression of human type I and II as well as rat type I and II 3beta-HSD cDNAs in HeLa human cervical carcinoma cells reveals that 3beta-ol dehydrogenase and 5-ene-4-ene isomerase activities reside within a single protein. These expressed 3beta-HSD proteins convert 3beta-hydroxy-5-ene-steroids into 3-keto-4-ene derivatives and catalyze the interconversion of 3beta-hydroxy and 3-keto-5alpha-androstane steroids. By site-directed mutagenesis, we demonstrated that the lower activity of expressed rat type II compared to rat type I 3beta-HSD is due to a change of four residues probably involved in a membrane-spanning domain. When homogenates from cells transfected with a plasmid vector containing mt type I 3beta-HSD is incubated in the presence of dihydrotestosterone (DHT) using NAD+ as co-factor, 5alpha-androstanedione was formed (A-dione), indicating an intrinsic androgenic 17beta-hydroxysteroid dehydrogenase (17beta-HSD) activity of this 3beta-HSD. We cloned a third type of rat cDNA encoding a predicted type III 3beta-HSD specifically expressed in the rat liver, which shares 80% similarity with the two other isoenzymes. Transient expression in human HeLa cells reveals that the type III isoenzyme does not display oxidative activity for the classical substrates of 3beta-HSD. However, in common with the type I enzyme, it converts A-dione and DHT to the corresponding 3beta-hydroxysteroids, thus showing an exclusive 3-ketosteroid reductase activity. When NADPH is used as co-factor, the affinity for DHT of the type III enzyme becomes 10-fold higher than that of the type I. Rat type III mRNA was below the detection limit in intact female liver. Following hypophysectomy, its concentration increased to 55% of the values measured in intact or hypophysectomized male rats, an increase which can be blocked by administration of ovine prolactin (oPRL). Treatment with oPRL for 10 days starting 15 days after hypophysectomy markedly decreased ovarian 3beta-HSD mRNA accumulation accompanied by a similar decrease in 3beta-HSD activity and protein levels. Treatment with the gonadotropin hCG reversed the potent inhibitory effect of oPRL on these parameters and stimulated 3beta-HSD mRNA levels in ovarian interstitial cells. These data indicate that the presence of multiple 3beta-HSD isoenzymes offers the possibility of tissue-specific expression and regulation of this enzymatic activity that plays an essential role in the biosynthesis of all hormonal steroids in classical as well as peripheral intracrine steroidogenic tissues.