There is growing evidence that various
isoforms of 17 beta-
hydroxysteroid 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 study, we obtained evidence that A431 cells, derived from an
epidermoid carcinoma of the vulva, are enriched in 17-HSD type 2, a membrane-bound
isoform reactive with C18 and C19 17 beta-
hydroxysteroids and
17-ketosteroids. The present investigation was undertaken to confirm the presence of 17-HSD type 2 in A431 cells and to assess intracellular regulation of 17-HSD at the level of catalysis by comparing the activity of homogenates and microsomes with that of cell monolayers. Northern blot analysis confirmed the presence of 17-HSD type 2
mRNA. Exposure of cells to
epidermal growth factor resulted in an increase in type 2
mRNA and, for microsomes, increases in maximum velocity (Vmax) with no change in Michaelis constant (Km) for
testosterone and
androstenedione, resulting in equivalent increases in the Vmax/Km ratio consistent with the presence of a single
enzyme. Initial velocity data and inhibition patterns were consistent with a highly ordered reaction sequence in vitro in which
testosterone and
androstenedione bind only to either an
enzyme-
NAD or an
enzyme-
NADH complex respectively. Microsomal
dehydrogenase activity with
testosterone was 2- to 3-fold higher than
reductase activity with
androstenedione. In contrast, although cell monolayers rapidly converted
testosterone to
androstenedione,
reductase activity with
androstenedione or
dehydroepiandrosterone (
DHEA) was barely detectable.
lactate but not
glucose,
pyruvate or
isocitrate stimulated the conversion of
androstenedione to
testosterone by monolayers, suggesting that cytoplasmic
NADH may be the cofactor for 17-HSD type 2
reductase activity with
androstenedione. However, exposure to
lactate did not result in a significant change in the
NAD/
NADH ratio of cell monolayers. It appears that within A431 cells 17-HSD type 2 is regulated at the level of catalysis to function almost exclusively as a
dehydrogenase. These findings give further support to the concept that 17-HSD type 2 functions in vivo principally as a
dehydrogenase and that its role as a
reductase in
testosterone formation by either the delta 4 or delta 5 pathway is limited.