Na-H exchange acts downstream of RhoA to regulate integrin-induced cell adhesion and spreading.
The ubiquitously expressed Na-H exchanger NHE1 functions in regulating intracellular pH and cell volume. NHE1 activity is stimulated by hormones, growth factors, and activation of integrin receptors. We recently determined that NHE1 activity is also stimulated by activation of the low molecular weight GTPase RhoA and that increases in NHE1 activity are necessary for RhoA-induced formation of actin stress fibers. We now show that NHE1 acts downstream of RhoA to modulate initial steps in integrin signaling for the assembly of focal adhesions. Adhesion of CCL39 fibroblasts on fibronectin was markedly delayed in the presence of the NHE inhibitor ethylisopropylamiloride. In mutant PS120 cells, derived from CCL39 fibroblasts but lacking NHE1, adhesion was also delayed but was rescued in PS120 cells stably expressing NHE1. In the absence of NHE1 activity, cell spreading was inhibited, and the accumulation of integrins, paxillin, and vinculin at focal contacts was impaired. Additionally, tyrosine phosphorylation of p125(FAK) induced by integrin clustering was also impaired. Inactivation of RhoA with C3 transferase and inhibition of the Rho-kinase p160ROCK with the pyridine derivative Y-27632 completely abolished activation of NHE1 by integrins but not by platelet-derived growth factor. These findings indicate that NHE1 acts downstream of RhoA to contribute a previously unrecognized critical signal to proximal events in integrin-induced cytoskeletal reorganization.
Department of Stomatology, University of California, San Francisco, California 94143, USA.
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D000818:Animals; D002448:Cell Adhesion / Q000502:physiology; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002460:Cell Line; D002465:Cell Movement / Q000502:physiology; D006224:Cricetinae; D005347:Fibroblasts; D051416:Focal Adhesion Protein-Tyrosine Kinases; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D016023:Integrins / Q000502:physiology*; D007700:Kinetics; D008168:Lung; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D015398:Signal Transduction; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein
D015815:Cell Adhesion Molecules; D016023:Integrins; D017923:Sodium-Hydrogen Exchangers; C069139:growth factor-activatable Na-H exchanger NHE-1; D011505:Protein-Tyrosine Kinases; D051416:Focal Adhesion Protein-Tyrosine Kinases; D019204:GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein