Abstract
In experimental membranous nephropathy, complement C5b-9-induces glomerular epithelial cell (GEC) injury and proteinuria. The effects of C5b-9 are mediated via signaling pathways, including calcium-independent phospholipase A(2)γ (iPLA(2)γ), and mitogen-activated protein kinases (MAPKs) such as extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38. The iPLA(2)γ pathway is cytoprotective. This study addresses the mechanisms of iPLA(2)γ activation. iPLA(2)γ activity was monitored by quantifying prostaglandin E(2) (PGE(2)) production. In GECs, iPLA(2)γ localized at the endoplasmic reticulum and mitochondria. Complement-mediated production of PGE(2) was amplified in GECs that overexpress iPLA(2)γ, compared with control cells, and was blocked by the iPLA(2)γ inhibitor bromoenol lactone in both iPLA(2)γ-overexpressing and control GECs. In GECs that overexpress iPLA(2)γ, complement-mediated PGE(2) production was reduced by inhibitors of MAP/ERK kinase 1 (MEK1) and p38 but not JNK. In COS-1 cells that overexpress iPLA(2)γ and cyclooxygenase-1, PGE(2) production was induced by co-expression of constitutively active MEK1 or MAPK-interacting kinase 1 (MNK1) as well as by stimulation with epidermal growth factor (EGF) + ionomycin. Complement- and EGF + ionomycin-stimulated iPLA(2)γ activity was attenuated by the S511A/S515A double mutation. Moreover, complement and EGF + ionomycin enhanced phosphorylation of Ser-511. Thus, complement-mediated activation of iPLA(2)γ is mediated via ERK and p38 pathways, and phosphorylation of Ser-511 and/or Ser-515 plays a key role in the catalytic activity and signaling of iPLA(2)γ. Defining the mechanisms by which complement activates iPLA(2)γ provides opportunities for development of novel therapeutic approaches to GEC injury and proteinuria.
Highlights
Calcium-independent phospholipase A2␥ is a mediator of complement-induced glomerular injury
When M1 green fluorescent protein (GFP)-independent PLA2 (iPLA2)␥ wild type (WT) or untagged iPLA2␥ was transfected in COS-1 cells together with COX1, prostaglandin E2 (PGE2) production was markedly increased compared with untransfected control (Fig. 1B) or cells transfected with COX1 alone (Fig. 1C)
We examined the localization of M1 GFP-iPLA2␥ WT in stably transfected glomerular epithelial cell (GEC)
Summary
Calcium-independent phospholipase A2␥ (iPLA2␥) is a mediator of complement-induced glomerular injury. Significance: Understanding the regulation of iPLA2␥ activity is essential for developing novel therapeutic approaches to glomerular injury and proteinuria. The effects of C5b-9 are mediated via signaling pathways, including calcium-independent phospholipase A2␥ (iPLA2␥), and mitogen-activated protein kinases (MAPKs) such as extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38. In COS-1 cells that overexpress iPLA2␥ and cyclooxygenase-1, PGE2 production was induced by co-expression of constitutively active MEK1 or MAPK-interacting kinase 1 (MNK1) as well as by stimulation with epidermal growth factor (EGF) ؉ ionomycin. Complement-mediated activation of iPLA2␥ is mediated via ERK and p38 pathways, and phosphorylation of Ser-511 and/or Ser-515 plays a key role in the catalytic activity and signaling of iPLA2␥. Defining the mechanisms by which complement activates iPLA2␥ provides opportunities for development of novel therapeutic approaches to GEC injury and proteinuria
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