EFFECT OF THE PROTEIN PHOSPHATASE INHIBITOR OKADAIC ACID ON FSH-INDUCED GRANULOSA-CELL STEROIDOGENESIS

To address a possible role of type 1 and 2A serine/threonine protein p hosphatases (PP1 and PP2A) in regulating granulosa cell hormonal respo nses, we investigated the effects of okadaic acid (OA) on FSH- and cAM P-induced steroidogenesis in these cells. When added alone (0.01-1 nmo l/l), the cell-permeant phosphatase inhibitor did not affect progester one and 3 beta-hydroxysteroid dehydrogenase/Delta(5-4) isomerase (3 be ta-HSD) enzyme activity, whereas when added with FSH it dose-dependent ly augmented (minimal effective dose, 0.1 nmol/l) gonadotropin-stimula ted steroidogenesis in cultured granulosa cells. A similar stimulatory effect of the toxin was observed in cells cultured for 48 h with the cell-permeant analogue dibutyryl cAMP (1 mmol/l), or when granulosa ce lls were stimulated with the cAMP-inducing agents cholera toxin (1 mu g/ml), forskolin (15 mu mol/l) or 1-methyl-3-isobutyl-xanthine (0.1 mm ol/l). The observed effect of OA on FSH-supported granulosa cell stero idogenesis was not a consequence of increased cAMP generation, and tim e course experiments also revealed that a minimal time period of 12 h was necessary for OA (0.1 and 1 nmol/l) to significantly enhance FSH-i nduced progesterone and 3 beta-HSD enzyme activity. Since OA also inhi bits the dephosphorylation of protein kinase C (PKC) substrates, we al so compared the effect of OA and the PKC activator 12-O-tetradecanoylp horbol-13-acetate (TPA) on FSH-induced granulosa cell steroidogenic ac tivity. While activation of the PKC pathway with the tumor promoter TP A (10 nmol/l) inhibited progesterone and cAMP accumulation in FSH-stim ulated granulosa cells, treatment with OA augmented steroidogenesis an d did not affect gonadotropin-induced cAMP generation. Collectively th ese results suggest that PP1 and PP2A may be important in regulating t he phosphorylation state of proteins implicated in the cAMP-protein ki nase A-stimulated steroidogenic activity of these cells.