| Summary: | 碩士 === 國立陽明大學 === 生理學研究所 === 99 === Activation of G protein-coupled receptors (GPCRs) plays an important role in ovarian physiology. When GPCRs are bound by specific ligands, such as hormones, a series of signaling pathways can be initiated, leading to a variety of physiological events. In fact, GPCRs-mediated activation of protein kinase A (PKA) as well as protein kinase C (PKC) signaling pathways have been recognized in the regulation of proliferation, differentiation and cell survival in ovarian tissue, including granulosa cells. Meanwhile, the regulator of G protein signaling (RGS) can regulate GPCRs signaling by accelerating the deactivation of heterotrimeric G proteins via enhancing the intrinsic GTPase activity of G? subunit, resulting in hydrolysis of GTP to GDP to cease the signaling. Recent literatures have shown that cAMP could increase RGS2 expression in human astrocytoma cells and PKC could decrease RGS2 expression in macrophages. Our previous studies also delineated that RGS2 expression was increased by human chorionic gonadotropin (hCG)-induced GPCR activation in human and mouse granulosa cells, in addition, we also found that cloprostenol-induced GPCR activation could result in translocation of RGS2 from nuclear to cytosol. However, the detailed mechanisms in PKA- and PKC-regulated RGS2 expression as well as translocation in ovary remain mostly uncharacterized. Thus in this study, we aimed to unveil the impacts of PKA and PKC activation in RGS2 expression and cellular localization in NT-1 mouse granulosa cells. We first demonstrated that cAMP and phorbol-12, 13-didecanoate (PDD), a PKC activator were able to elevate both RGS2 protein and mRNA expression by western blotting and reverse transcriptase-PCR assays. To further our understanding of PKA and PKC regulation of RGS2 transcriptional activation, transient transfection with the plasmids carrying a full-length or various sizes in length of mouse RGS2 promoter sequence into NT-1 cells allowed us to discover that both PKA and PKC could result in RGS2 promoter activation and a short 78 bp fragment of this promoter sequence still retained a full-inducibility by cAMP and PDD. In addition, two consensus Sp1 sites within the 78 promoter sequence was mutated individually or in combination to identify that Sp1 element was an essential component to mediate cAMP- and PDD-induced promoter activation. Finally, we also monitored RGS2 protein expression in total cell lysates, and in nuclear or cytosolic compartments and the result indicated an upregulation in total and in nuclear and cytosolic expression by cAMP and PDD. Similar results were also observed by transfecting a FLAG-RGS2 fusion plasmid into COS7 cells. Notably we observed that PDD induced a translocation of nuclear FLAG-RGS2 fusion protein into cytosol at 1 hour. Putting together, our findings suggest that both RGS2 protein and mRNA could elevate via PKA-dependent or PKC-dependent pathway and the Sp1 element within the RGS2 promoter sequence may be an important player to mediate PKA- and PKC-induced RGS2 transcription, PKC activation can induce a translocation of RGS2 from nuclear to cytosol.
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