Ovary

Progesterone Regulates Granulosa Cell Viability Through a Protein Kinase G-Dependent Mechanism That May Involve 14-3-3

Abstract
Progesterone (P4) inhibits granulosa cell and spontaneously immortalized granulosa cell (SIGC) apoptosis by regulating membrane-initiated events. However, the nature of the signal transduction pathway that is induced by these membrane-initiated events has not been defined. To gain insights into the P4-regulated signal transduction pathway, mouse granulosa cells and SIGCs were cultured with 8-br-cGMP and P4. In culture 8-br-cGMP mimicked P4's anti-apoptotic actions. Since cGMP activates protein kinase G (PKG), the effect of PKG antagonists on P4-regulated SIGC viability was assessed. P4's anti-apoptotic action was attenuated by the PKG inhibitors, Rp-8-pCPT-cGMP, KT5823, the PKG-1-specific inhibitor, DT-3, and a dominant negative PKG-1. Further the type I isoform of PKG was shown to be expressed by SIGCs and activated by P4. P4's anti-apoptotic action was not affected by the PKA inhibitor, KT5720. Collectively, these findings indicate that P4 maintains SIGC viability by activating PKG-1. PKG-1-GFP was shown to localize predominantly to the cytoplasm of SIGCs. To identify potential cytoplasmic targets of PKG-1, SIGCs were cultured for 5 h with P4 in the presence or absence of DT-3. Cell lysates were prepared and subjected to two-dimensional electrophoresis. The resulting gels were sequentially stained with ProQ-Diamond Gel Stain and Coomassie Blue to reveal phosphorylated proteins. The two-dimensional gels revealed one major protein whose phosphorylation status was abrogated by DT-3. Mass spectrometric analysis identified this protein as 14-3-3 with 14-3-3 being phosphorylated on tyrosine 18, serine 28, serine 69, serine 74, threonine 90, threonine 98 and serine 115. Finally difopein, a specific 14-3-3 inhibitor, was shown to induce apoptosis even in the presence of serum. These data suggest that 1) P4 regulates the phosphorylation status of 14-3-3 through a PKG-dependent pathway and 2) 14-3-3 plays a central and essential role in maintaining the viability of SIGCs.

Key words: Cyclic guanosine monophosphate • Granulosa cells • Kinases • Progesterone • Signal transduction

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