Complement activates phospholipases and protein kinases in glomerular epithelial cells

Abstract

In rat membranous nephropathy, complement C5b-9 induces glomerular epithelial cell (GEC) injury and proteinuria, which in some models is partially mediated by eicosanoids. In cultured rat GEC, sublytic C5b-9 injures plasma membranes and releases arachidonic acid (AA) and eicosanoids, due to activation of cytosolic phospholipase A2 (cPLA2). In this study, we address the role of protein kinases in cPLA2 activation. GEC were stably transfected with cDNAs of wild-type (wt) cPLA2, and serine505-->alanine mutant (cPLA2-SA505), which lacks the mitogen-activated protein kinase (MAPK) phosphorylation site. Complement stimulated protein kinase C (PKC) activity in GEC, and activated p42 (but not p38) MAPK. Overexpression of either cPLA2-wt or cPLA2-SA505 markedly amplified the release of [3H]AA by C5b-9. Depletion of PKC blocked the complement-dependent activation of cPLA2-wt or cPLA2-SA505, but inhibition of the p42 MAPK pathway had no effect. Epidermal growth factor was a strong activator of p42 MAPK, but stimulated PKC activity weakly. Unlike complement, activation of cPLA2-wt by epidermal growth factor was dependent on PKC, and was augmented significantly by p42 MAPK. Stable overexpression of phospholipase C-gamma 1 in GEC amplified C5b-9-induced production of [3H]inositol phosphates and [3H]diacylglycerol, an endogenous activator of PKC, and complement stimulated tyrosine phosphorylation of phospholipase C-gamma 1. C5b-9 induces activation of cPLA2 that is dependent on the diacylglycerol-PKC pathway. The role of p42 MAPK in cPLA2 activation becomes redundant in the presence of relatively potent PKC activation.