Cyclic stretch-induced cPLA2 mediates ERK 1/2 signaling in rabbit proximal tubule cells

Abstract

Recent evidence from this laboratory have demonstrated a critical role of phospholipase A2 (PLA2) and arachidonic acid in angiotensin II type 2 (AT2) receptor-mediated kinase activation in renal epithelium independent of phosphoinositide- specific phospholipase C (PLC) and without the necessity of eicosanoid biosynthesis. In the present study, we investigated whether cyclic stress phosphorylates and activates the mitogen-activated protein kinase (MAPK) pathway and whether PLA2 activation mediates mechanotransduction in renal epithelial cells. The rational for studying kidney epithelial cells relates to their similarity to podocytes, which undergo mechanical stretch related to changes in intraglomerular pressure. To produce strain or stretch, primary cultures of rabbit proximal tubular cell cells are grown in tissue culture wells having a collagen-coated Silastic deformable membrane bottoms and applying vacuum to the well to generate alternating cycles of stretch and relaxation (30 cycles/min). We found that cyclic stretching of rabbit proximal tubular cells caused a time- and intensity-dependent activation of extracellular signal-regulated kinases 1 and 2 (ERK 1/2) in proximal tubular cells as detected by its phosphorylation. In addition, mechanical stretch induced PLA2 activation and a subsequent rapid release of arachidonic acid. Inhibition of PLA2 by mepacrine and methyl arachidonyl fluorophosphonate ketone (AACOCF3) attenuated both arachidonic acid release and ERK 1/2 activation by cyclic stretch, supporting the importance of PLA2 as a mediator of mechanotransduction in renal proximal tubular cells. A requirement for extracellular Ca2+ and stretch-activated Ca2+ channels was also documented. Complete inhibition of ERK 1/2 by PD98059, a MAPK kinase (MEK) inhibitor, did not suppress stretch- induced PLA2 activation and arachidonic acid release, suggesting the later events were upstream of ERK 1/2. Cyclic stretch also caused rapid phosphorylation of the EGF receptor kinase and c-Src. Furthermore, arachidonic acid itself induced time- and dose-dependent phosphorylation of c-Src. In addition, the c-Src inhibitor PP2 and selective EGF receptor kinase inhibitor AG1478 attenuated both ERK 1/2 and EGF receptor phosphorylation by cyclic stretch. PLA2 dependence for ERK 1/2 activation in response to cyclic stretch in proximal tubular epithelial cells was established in this report. In addition, these findings indicate cyclic stretch increased the tyrosine phosphorylation of the EGF receptor and c-Src and that c-Src acts upstream of the EGF receptor to mediate its phosphorylation, whereby both are critical for stretch- induced ERK 1/2 activation in rabbit proximal tubular cells. These observations documents for the first time a mechanism of mechanical stretch-induced kinase activation mediated by stretch activated Ca2+ channels and PLA2-dependent release of arachidonic acid.