Complement C5b-9 activates cytosolic phospholipase A2 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. By analogy, sublytic C5b-9 injures plasma membranes and releases arachidonic acid (AA) and eicosanoids in cultured rat GEC. In this study, we demonstrate that, in GEC, sublytic C5b-9 stably increased the activity of a high-molecular-mass cytosolic phospholipase A2 (PLA2), which we identified as "cPLA2." This increase was abolished with inhibitors of protein kinase C. C5b-9 did not affect low-molecular-mass membrane-associated or secretory PLA2 activities. In GEC that stably overexpress cPLA2 activity and protein (produced by transfection of cPLA2 cDNA), immunoblot analysis showed that sublytic C5b-9 induced a decreased mobility of cPLA2, consistent with cPLA2 phosphorylation. Incubation of cPLA2-transfected GEC with sublytic C5b-9 significantly increased production of free AA and prostaglandin E2, whereas, in control GEC, the C5b-9-induced changes in free AA and prostaglandin E2 were small. Furthermore, both C5b-9-dependent sublytic cytotoxicity and cytolysis were enhanced in GEC overexpressing cPLA2, compared with control cells. Thus C5b-9 increased cPLA2 activity, probably via phosphorylation involving a protein kinase C-dependent pathway. Phospholipid hydrolysis by cPLA2 resulted in release of substrate for eicosanoid synthesis and in enhancement of C5b-9-dependent GEC injury. Both processes may facilitate glomerular damage in membranous nephropathy.

Complement C5b-9 Membrane Attack Complex Increases Expression of Endoplasmic Reticulum Stress Proteins in Glomerular Epithelial Cells

“…Stimuli that raise [Ca 2ϩ ] i in the submicromolar range may induce translocation of cPLA 2 from cytosol to an intracellular membrane, where cPLA 2 would bind via its N-terminal Ca 2ϩ -dependent lipid binding or C2 domain, gaining access to phospholipid substrate. We have demonstrated that cPLA 2 is the major PLA 2 in GEC and that complement enhances cPLA 2 phosphorylation and catalytic activity (16). Moreover, C5b-9 increases free AA in GEC, and release of AA is amplified by overexpression of cPLA 2 (16,17).…”

“…We have demonstrated that cPLA 2 is the major PLA 2 in GEC and that complement enhances cPLA 2 phosphorylation and catalytic activity (16). Moreover, C5b-9 increases free AA in GEC, and release of AA is amplified by overexpression of cPLA 2 (16,17). Recently, we demonstrated that cPLA 2 localizes and hydrolyzes phospholipids at the plasma membrane in GEC, the membrane of the endoplasmic reticulum (ER), and the nuclear envelope but not at mitochondria or Golgi (29).…”

“…First, arachidonic acid (AA) released by cPLA 2 is metabolized in GEC via cyclooxygenases-1 and -2 to prostaglandin E 2 and thromboxane A 2 (20), and inhibition of prostanoid production reduces proteinuria in PHN (21)(22)(23)(24)(25) and in human membranous nephropathy (26). Second, cPLA 2 may mediate GEC injury more directly (16). cPLA 2 (group IV PLA 2 ) is regulated by [Ca 2ϩ ] i and phosphorylation (27,28).…”

Podocyte Biology and Response to Injury

“…These observations suggest that podocytes can undergo mitosis but that there is either an abnormality in the completion of mitosis and/or in cytokinesis (cell division). When cultured podocytes are exposed to sublytic C5b-9 attack, a variety of signaling pathways are activated, including JNK, phospholipases, calcium, and MAPK cascades (143)(144)(145). Sublytic C5b-9 attack also causes cells to engage the cell cycle in vitro and in vivo.…”

Experimental membranous nephropathy redux

“…In GEC in culture and in vivo, a major endogenous PLA 2 isoform is cytosolic PLA 2 -␣ (cPLA 2 ; group IV) (27,49), and activation of cPLA 2 has been linked with C5b-9 (16,24,98). As in other cells, cPLA 2 activation in GEC is regulated by changes in cytosolic [Ca 2ϩ ] and phosphorylation, the latter being dependent on PLC activation, production of 1,2-diacylglycerol, and activation of the protein kinase C (PKC) pathway (16,98). In the GEC, cPLA 2 localizes and hydrolyzes phospholipids at the plasma membrane, the membrane of the endoplasmic reticulum (ER), and the nuclear envelope, but not at mitochondria or Golgi (72).…”