EFFECTS OF PHORBOL ESTER AND STAUROSPORINE ON THE ACTIONS OF INSULIN-LIKE GROWTH-FACTOR-I ON RAT OVARIAN GRANULOSA-CELLS

Insulin-like growth factor I (ICF-I) is able to stimulate ovarian gran ulosa cell steroidogenesis induced by gonadotopins. This gonadotropin- induced potentiation of ICF-I action appears to be due, at least in pa rt, to a gonadotropin-induced increase in membrane-bound ICF-I recepto r number and/or decrease in extracellular IGF binding proteins (IGFBPs ). Protein kinase C (PKC) has recently been reported to inhibit gonado tropin-induced steroidogenesis in rat ovarian granulosa cells. The rol e of PKC in the effects of IGF-I on gonadotropin action, however, is u nknown. In this study, the effects of phorbol 12-myristate 13-acetate (PMA, a PKC activator) and staurosporine (ST, a PKC inhibitor) on ICF- I action were studied using immature rat ovarian granulosa cells. Acti vation of PKC by PMA did not affect steroidogenesis or cAMP secretion in cells treated with or without IGF-I. On the other hand, inhibition of PKC by ST alone (10(-9)-10(-7) M) led to an increase in progesteron e production in a dose- and time-dependent manner without affecting cA MP secretion. In the presence, but not absence, of ST, ICF-I was able to stimulate progesterone production in the absence of any gonadotropi n. PMA decreased ST-induced steroidogenesis and essentially abolished ST-potentiated IGF-I stimulation of steroidogenesis, suggesting the ef fects of ST on ICF-I action involved a PKC-dependent mechanism. Unlike gonadotropin, ST did not change ICF-I receptor binding. However, ST s ignificantly decreased a major ICF binding protein (IGFBP, similar to 30 kDa) which is likely to be IGFBP-5, whereas it increased a minor IG FBP (similar to 24 kDa) which is likely to be IGFBP-4. Both effects of ST were dose- and time-dependent. Furthermore, ST inhibited the expre ssion of mRNA for IGFBP-5 suggesting that ST decreased IGFBP-5 levels by inhibiting its transcription and/or decreasing the stability of its mRNA. Interestingly, ST also decreased mRNA levels of IGFBP-4 despite a significant increase in secreted IGFBP-4 levels. The mechanisms inv olved are not known. Activation of PKC by PMA had no acute effect on t hese IGFBPs. The regulation by ST of IGFBPs was not antagonized by PMA , and was not affected by PKC-down regulation. Thus, it is likely that ST induces granulosa cell steroidogenesis, potentiates the ICF-I stim ulation of steroidogenesis and regulates IGFBP via both PKC-dependent and -independent pathways.