Activation of phospholipase A2 by complement C5b-9 in glomerular epithelial cells.
In rat membranous nephropathy, C5b-9 induces glomerular epithelial cell (GEC) injury and proteinuria, which 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 phospholipase A2 (PLA2). To address mechanisms of PLA2 activation, GEC were stably transfected with cDNAs of wild-type cytosolic PLA2 (cPLA2-wt), or group II secretory PLA2, producing overexpression of PLA2 activity. Sublytic C5b-9 markedly increased free [3H]AA in cPLA2-wt-transfected GEC, but only trivial increases were evident in secretory PLA2-transfected, or neo (control) GEC. In cPLA2-wt-transfected GEC, reduction of extracellular free Ca2+ or down-regulation of protein kinase C inhibited [3H]AA release. To further address the regulation of cPLA2, we stably expressed a mutant cPLA2 in which the Ca2+-dependent lipid binding domain was deleted (deltaCaLB). In GEC that express cPLA2-deltaCaLB, the C5b-9-induced increase in free [3H]AA was comparable with neo, despite expression of cPLA2-deltaCaLB at levels similar to cPLA2-wt. We then stably expressed another cPLA2 mutant (cPLA2-srcmyr) in which the CaLB domain was replaced by the N-terminal myristoylation domain of c-Src. cPLA2-srcmyr is permanently membrane associated. At low extracellular free Ca2+, C5b-9 increased free [3H]AA significantly in GEC that express cPLA2-srcmyr, while in neo GEC, the change was negligible. Thus, C5b-9 activates the cPLA2 isoform. Activation is dependent on the CaLB domain, and is mediated by phosphorylation, Ca2+ influx, and membrane association.
Panesar
M
M
Department of Medicine, Royal Victoria Hospital, McGill University, Montreal, Quebec, Canada.
Papillon
J
J
McTavish
A J
AJ
Cybulsky
A V
AV
D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D019556:COS Cells; D002118:Calcium / Q000502:physiology; D002478:Cells, Cultured; D015938:Complement Membrane Attack Complex / Q000502:physiology*; D003600:Cytosol / Q000201:enzymology; D004789:Enzyme Activation / Q000276:immunology; D004847:Epithelial Cells; D004848:Epithelium / Q000201:enzymology / Q000276:immunology; D007678:Kidney Glomerulus / Q000166:cytology / Q000201:enzymology* / Q000276:immunology*; D050356:Lipid Metabolism; D016297:Mutagenesis, Site-Directed; D010741:Phospholipases A / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D054467:Phospholipases A2; D011493:Protein Kinase C / Q000502:physiology; D017434:Protein Structure, Tertiary; D051381:Rats
D015938:Complement Membrane Attack Complex; D016718:Arachidonic Acid; D011493:Protein Kinase C; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium