Mechanical strain-induced RhoA activation requires NADPH oxidase-mediated ROS generation in caveolae.
Increased intraglomerular pressure leads to kidney fibrosis, and can be modeled by exposing glomerular mesangial cells (MC) to mechanical strain. We previously showed that RhoA mediates strain-induced matrix production. Here we investigate whether reactive oxygen species (ROS) are required for RhoA activation. Maximal RhoA activation (1 min) was inhibited by ROS scavenge or NADPH oxidase inhibition. Strain activated NADPH oxidase, with Rac1, p47(phox), and p67(phox) membrane translocation, and Rac1 activation, observed within 30 sec. Epidermal growth factor receptor (EGFR) inhibition blocked RhoA and Rac1 activation, p67(phox) membrane translocation, and ROS generation. However, EGFR activation was unaffected by ROS inhibitors, placing it upstream of ROS generation. We previously showed, using chemical disruption, that caveolae mediate strain-induced EGFR and RhoA activation. In MC from caveolin-1 knockout mice, which lack caveolae, RhoA and Rac1 activation, p67(phox) membrane translocation, and ROS generation were absent. These were rescued by caveolin-1 re-expression. ROS generation, Rac1 activation, and p67(phox) membrane translocation were also prevented by Src inhibition. They were absent in MC stably infected with caveolin-1 Y14A, a mutant resistant to Src phosphorylation. In MC, caveolae are thus important mediators of strain-induced ROS generation through NADPH oxidase, mediating a signaling cascade which results in RhoA activation.
Zhang
Ying
Y
Division of Nephrology, McMaster University, Hamilton, Canada.
Peng
Fangfang
F
Gao
Bo
B
Ingram
Alistair J
AJ
Krepinsky
Joan C
JC
D000818:Animals; D021941:Caveolae / Q000378:metabolism*; D051242:Caveolin 1 / Q000378:metabolism; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000378:metabolism; D050527:Mesangial Cells / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015398:Signal Transduction; D013314:Stress, Mechanical; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*; D019061:src-Family Kinases / Q000378:metabolism
D051242:Caveolin 1; D010750:Phosphoproteins; D017382:Reactive Oxygen Species; C079789:neutrophil cytosol factor 67K; D019255:NADPH Oxidases; D066246:ErbB Receptors; D019061:src-Family Kinases; D020742:rhoA GTP-Binding Protein