INTRACELLULAR REGULATION OF 17-BETA-HYDROXYSTEROID DEHYDROGENASE TYPE-2 CATALYTIC ACTIVITY IN A431 CELLS

There is growing evidence that various isoforms of 17 beta-hydroxyster oid dehydrogenase (17-HSD) are regulated at the level of catalysis in intact cells. A number of investigators have proposed that the NAD(P)/ NAD(P)H ratio may control the direction of reaction. In a previous stu dy, we obtained evidence that A431 cells, derived from an epidermoid c arcinoma of the vulva, are enriched in 17-HSD type 2, a membrane-bound isoform reactive with C-18 and C-19 17 beta-hydroxysteroids and 17-ke tosteroids. The present investigation was undertaken to confirm the pr esence of 17-HSD type 2 in A431 cells and to assess intracellular regu lation of 17-HSD at the level of catalysis by comparing the activity o f homogenates and microsomes with that of cell. monolayers. Northern b lot analysis confirmed the presence of 17-HSD type 2 mRNA, Exposure of cells to epidermal growth factor resulted in an increase in type 2 mR NA and, for microsomes, increases in maximum velocity (V-max) with no change in Michaelis constant (K-m) for testosterone and androstenedion e, resulting in equivalent increases in the V-max/Y-m ratio consistent with the presence of a single enzyme. Initial velocity data and inhib ition patterns were consistent with a highly ordered reaction sequence in vitro in which testosterone and androstenedione bind only to eithe r an enzyme-NAD or an enzyme-NADH complex respectively. Microsomal deh ydrogenase activity with testosterone was 2- to 3-fold higher than red uctase activity with androstenedione. In contrast, although cell monol ayers rapidly converted testosterone to androstenedione, reductase act ivity with androstenedione or dehydroepiandrosterone (DHEA) was barely detectable. Lactate but not glucose, pyruvate or isocitrate stimulate d the conversion of androstenedione to testosterone by monolayers, sug gesting that cytoplasmic NADH may be the cofactor for 17-HSD type 2 re ductase activity with androstenedione, However, lactate did not result in exposure to a significant change in the NAD/NADH ratio of cell mon olayers. It appears that within A431 cells 17-HSD type 2 is regulated at the level of catalysis to function almost exclusively as a dehydrog enase. These findings give further support to the concept that 17-HSD type 2 functions in vivo principally as a dehydrogenase and that its r ole as a reductase in testosterone formation by either the Delta 4 or Delta 5 pathway is limited.