title: Effects of phorbol ester and staurosporine on the actions of insulin-like growth factor-I on rat ovarian granulosa cells.
abstract: Insulin-like growth factor I (IGF-I) is able to stimulate ovarian granulosa cell steroidogenesis induced by gonadotopins. This gonadotropin-induced potentiation of IGF-I action appears to be due, at least in part, to a gonadotropin-induced increase in membrane-bound IGF-I receptor number and/or decrease in extracellular IGF binding proteins (IGFBPs). Protein kinase C (PKC) has recently been reported to inhibit gonadotropin-induced steroidogenesis in rat ovarian granulosa cells. The role of PKC in the effects of IGF-I on gonadotropin action, however, is unknown. In this study, the effects of phorbol 12-myristate 13-acetate (PMA, a PKC activator) and staurosporine (ST, a PKC inhibitor) on IGF-I action were studied using immature rat ovarian granulosa cells. Activation 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 progesterone production in a dose- and time-dependent manner without affecting cAMP secretion. In the presence, but not absence, of ST, IGF-I was able to stimulate progesterone production in the absence of any gonadotropin. PMA decreased ST-induced steroidogenesis and essentially abolished ST-potentiated IGF-I stimulation of steroidogenesis, suggesting the effects of ST on IGF-I action involved a PKC-dependent mechanism. Unlike gonadotropin, ST did not change IGF-I receptor binding. However, ST significantly decreased a major IGF binding protein (IGFBP, ∼30kDa) which is likely to be IGFBP-5, whereas it increased a minor IGFBP (∼24kDa) which is likely to be IGFBP-4. Both effects of ST were dose- and time-dependent. Furthermore, ST inhibited the expression 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 involved are not known. Activation of PKC by PMA had no acute effect on these 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 IGF-I stimulation of steroidogenesis and regulates IGFBP via both PKC-dependent and -independent pathways.
authors.affiliation: Centre for Hormone Research and Department of Clinical Biochemistry, Royal Children's Hospital Parkville, 3052, Victoria, Australia.
authors.forename: H
authors.initials: H
authors.lastname: He
authors.forename: A C
authors.initials: AC
authors.lastname: Herington
authors.forename: P
authors.initials: P
authors.lastname: Roupas