The renal mesangial cell plays an important role in glomerular physiology and pathophysiology. Mesangial cell proliferation is associated with many glomerular diseases. The long term objective of this proposal is to understand the cellular factors involved in the regulation of the Ca2+ signal and phospholipases C and A2, components of the intracellular signalling pathways important for the understanding of mesangial cell activation and proliferation.
One specific aim of the proposal is: To define the role of gelsolin in cellular polyphosphatidylinositol lipid turnover, evaluate the consequences of modulation of phospholipase C activity on the Ca2+ signal, tyrosine protein phosphorylation, mitogenesis, and phospholipase A2 activation, and better define intracellular Ca2+ storage pools. It is proposed that cytoskeletal elements, in particular, gelsolin, regulate Ca2+ signalling and mitogenesis by modulating phospholipase C activity through an interaction with phosphatidylinositol bisphosphate (PI-4,5-P2). This hypothesis will be tested in fibroblast cell lines which have been stably transfected with the gelsolin cDNA and express varying amounts of gelsolin, as well as mesangial cells transfected with the gelsolin cDNA controlled by an inducible promoter. Experiments will be designed to test the hypothesis that the Golgi represents an important storage site for Ca2+ and responds to noncyclooxygenase products of arachidonic acid by releasing Ca2+. A second specific aim is: To further characterize the 110 kDa form of phospholipase A2 and to evaluate the interactions of this protein with other components of the signalling cascade involved in the mesangial cell response to vasopressin and endothelin. The role of phosphorylation in regulation of the enzyme's activity, and the interaction of this form of PLA2 with other signalling molecules and with the cytoskeleton, as well as with the endothelin receptor, will be explored. The 110 kDa PLA2 will be overexpressed in mesangial cells and experiments will be performed to evaluate whether enhanced cellular PLA2 results in increased eicosanoid production, spontaneous Ca2+ oscillations, and higher basal Ca2+. The subcellular localization of the enzyme will be determined using immunocytochemical techniques in both normal cells and cells hyperexpressing the enzyme.
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