Different roles of cAMP/PKA and PKC signaling in regulating progesterone and PGE2 levels in immortalized rat granulosa cell cultures

Follicular cells from various species secrete steroids and prostaglandins, which are crucial for reproduction, in response to gonadotropins. Here, we examined prostaglandin E-2 (PGE(2)) secretion from immortalized rat granulosa cells derived from preovulaotry follicles expressing the rat follicle stimulating hormone receptor (denoted as FSHR cells) that produce progesterone in response to gonadotropins. The cells were stimulated with a) pregnant mare's serum gonadotropin (PMSG; a rat FSH receptor agonist), b) activators of the protein kinase A (PKA) pathway (forskolin and a cell permeable cAMP analog Dibutyryl-cAMP (DB-cAMP)) and c) protein kinase C (PKC) (12-O-tetradecanoylphorbol 13-acetate; TPA), alone and in combination for 24 h. Thereafter, PGE(2) and progesterone levels in the culture media were determined. In accordance with previous studies, while PMSG and the PICA pathway activators induced progesterone accumulation in the media, TPA did not. In contrast, our data indicate that TPA, but neither PMSG, forskolin and DB-cAMP evoked PGE(2) accumulation in the media. Western Blot analysis of cell lysate showed a drastic TPA induced increase of COX-2 levels, which was not seen with neither PMSG nor forskolin treatment. This association between the COX-2 and PGE(2) levels suggests that the enzyme activity is the likely factor that determines the synthesis and levels of the prostaglandin in the culture media of the granulosa-derived cells. The addition of the PKA inhibitor H-89 to the FSHR cultures suppressed the gonadotropin and forskolin induction of progesterone secretion. Incubation in the presence of GF109203X (a PKC inhibitor) attenuated the TPA induced PGE(2) accumulation in the culture media of the cells (a dose dependent reduction of 40-70%). In addition, while TPA inhibited the PMSG and forskolin induced-accumulation of progesterone in the media, the gonadotropin and forskolin inhibited the elevation of PGE(2) levels evoked by TPA (a dose dependent decrease of 35-55%). These data suggest that cAMP/PKA and PKC signaling have opposite effects on PGE(2) and progesterone synthesis in FSHR cells. We propose that this PKA and PKC interplay on progesterone and PGE(2) may be advantageous for the coordination of these key mediators for successful ovulation and luteinization.