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 phosphatases (PP1 and PP2A) in regulating granulosa cell hormonal responses, we investigated the effects of okadaic acid (OA) on FSH- and cAMP-induced steroidogenesis in these cells. When added alone (0.01-1 nmol/l), the cell-permeant phosphatase inhibitor did not affect progesterone and 3 beta-hydroxysteroid dehydrogenase/delta 5-4 isomerase (3 beta-HSD) enzyme activity, whereas when added with FSH it dose-dependently augmented (minimal effective dose, 0.1 nmol/l) gonadotropin-stimulated 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 cells were stimulated with the cAMP-inducing agents cholera toxin (1 microgram/ml), forskolin (15 mumol/l) or 1-methyl-3-isobutyl-xanthine (0.1 mmol/l). The observed effect of OA on FSH-supported granulosa cell steroidogenesis was not a consequence of increased cAMP generation, and time 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-induced progesterone and 3 beta-HSD enzyme activity. Since OA also inhibits the dephosphorylation of protein kinase C (PKC) substrates, we also compared the effect of OA and the PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA) on FSH-induced granulosa cell steroidogenic activity. While activation of the PKC pathway with the tumor promoter TPA (10 nmol/l) inhibited progesterone and cAMP accumulation in FSH-stimulated granulosa cells, treatment with OA augmented steroidogenesis and did not affect gonadotropin-induced cAMP generation. Collectively these results suggest that PP1 and PP2A may be important in regulating the phosphorylation state of proteins implicated in the cAMP-protein kinase A-stimulated steroidogenic activity of these cells.
González Reyes
J
J
Departamento de Endocrinología Celular y Molecular, Facultad de Medicina, Universidad de Las Palmas de Gran Canaria, Spain.
Santana
P
P
González Robaina
I
I
Cabrera Oliva
J
J
Estévez
F
F
Hernández
I
I
López Blanco
F
F
Quintana Aguiar
J
J
Fanjul
L F
LF
Ruiz de Galarreta
C M
CM
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D015096:3-Hydroxysteroid Dehydrogenases / Q000378:metabolism; D000818:Animals; D003994:Bucladesine / Q000494:pharmacology; D002478:Cells, Cultured; D002772:Cholera Toxin / Q000494:pharmacology; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism / Q000494:pharmacology; D004789:Enzyme Activation; D005260:Female; D005640:Follicle Stimulating Hormone / Q000494:pharmacology*; D006107:Granulosa Cells / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D019319:Okadaic Acid / Q000494:pharmacology*; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors*; D011374:Progesterone / Q000096:biosynthesis*; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D005576:Colforsin; D019319:Okadaic Acid; D011374:Progesterone; D003994:Bucladesine; D005640:Follicle Stimulating Hormone; D002772:Cholera Toxin; D000242:Cyclic AMP; D015096:3-Hydroxysteroid Dehydrogenases; D011493:Protein Kinase C; D010749:Phosphoprotein Phosphatases; D013755:Tetradecanoylphorbol Acetate; D015056:1-Methyl-3-isobutylxanthine