The Action of Phorbol Ester on Steroidogenesis in Cultured Human Fetal Adrenal Cells

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Abstract
The potent mitogen and tumor promoter, phorbol 12-myristate 13-acetate (PMA), has a primary action via activation of calciumdependent protein kinase C. The treatment of monolayer cultures of human fetal adrenal neocortex (HFA) cells with PMA (50–250 nM) stimulated basal dehydroepiandrosterone sulfate (DS) secretion 2–3 fold. ACTH-treated HFA cells secreted amounts of DS and cortisol (F) 10–50 fold greater than basal secretions. PMA (250 nM) addition with ACTH to HFA cells decreased DS and F secretions at least 75% on days 2 and 3 of treatment. Treatment of HFA cells with 4α-phorbol, which does not activate calcium-dependent protein kinase C, did not inhibit steroidogenesis. The attenuated rates of steroidogenesis after PMA treatment correlated with the decreased amounts of steroid 11β, 17α- and 21-hydroxylase cholesterol side-chain cleavage steroid dehydrogenase and sulfotransferase activities. The decrease of steroid 17α-hydroxylase activity correlated with the decreased amount of cytochrome P-45017α as determined after protein immunoblotting of NaDodSO4 cell lysates. After PMA treatment the ACTH-promoted increases of hydroxysteroid sulfotransferase and dehydrogenase activities of HFA cells were suppressed. PMA (50 nM) inhibited cAMP accumulation in ACTH-treated HFA cells, while 4α-phorbol had no effect. Importantly, dibutyryl cAMP (0.2 mM) treatment of HFA cells did not reverse phorbol ester-promoted attenuation of steroidogenesis. We conclude that, in the presence of ACTH, phorbol ester chronically inhibits both cAMP synthesis and cAMP-dependent protein kinase action with resultant decreased steroidogenic enzyme synthesis and steroid production. This may be a consequence of activation, migration and a slow degradation of protein kinase C activity. These multifaceted actions of phorbol ester and associated protein kinase C activation may have critical effects on the ontogeny of fetal adrenal function.

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