RhoA activation in mesangial cells by mechanical strain depends on caveolae and caveolin-1 interaction.
Increased intraglomerular pressure is an important hemodynamic determinant of glomerulosclerosis and can be modeled in vitro by exposing mesangial cells to cyclic mechanical strain. A previous study showed that RhoA mediates strain-induced production of fibronectin; herein is investigated the role of caveolae in RhoA activation. Cyclodextrin and filipin, agents that disrupt caveolae, abrogated strain-induced RhoA activation in mesangial cells. Caveolin-1 (cav-1), the defining protein of caveolae, was Y14 phosphorylated by strain, and this was inhibited by PP1, showing Src dependence. Strain also induced c-SrcY416 phosphorylation and hence activation. Strain increased RhoA association with cav-1, which was blocked by PP1. Cyclodextrin and filipin inhibited the strain-induced RhoA/cav-1 association, indicating dependence on caveolar structural integrity. Restoration of caveolae by coincubation of cyclodextrin with cholesterol rescued both RhoA activation and RhoA/cav-1 association in response to strain. Sucrose gradient detected a significant portion of RhoA in caveolae, with Src located exclusively in these domains. Finally, in cells that were infected with retrovirus that encodes the nonphosphorylatable cav-1 Y14A, RhoA/cav-1 association, RhoA activation, and fibronectin secretion in response to strain were abrogated. It is concluded that strain-induced RhoA activation depends on the integrity of caveolae and on physical association of cav-1 and RhoA. The phosphorylation of cav-1 at Y14 by Src kinases is required for this to occur. These studies define a novel function for cav-1 and caveolae as positive effectors of RhoA activation. Targeting caveolae thus may provide a new therapeutic option for glomerular sclerosis that is associated with elevated intraglomerular pressure.
Peng
Fangfang
F
Division of Nephrology, McMaster University, Ontario, Canada.
Wu
Dongcheng
D
Ingram
Alistair J
AJ
Zhang
Baifang
B
Gao
Bo
B
Krepinsky
Joan C
JC
D019943:Amino Acid Substitution; D000818:Animals; D021941:Caveolae / Q000378:metabolism*; D051242:Caveolin 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D004789:Enzyme Activation; D005353:Fibronectins / Q000378:metabolism; D005920:Glomerular Mesangium / Q000166:cytology / Q000378:metabolism*; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation; D051381:Rats; D013314:Stress, Mechanical; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*; D019061:src-Family Kinases / Q000378:metabolism
D051242:Caveolin 1; D005353:Fibronectins; D019061:src-Family Kinases; D010749:Phosphoprotein Phosphatases; D020742:rhoA GTP-Binding Protein