Fibronectin stimulates migration through lipid raft dependent NHE-1 activation in mouse embryonic stem cells: involvement of RhoA, Ca(2+)/CaM, and ERK.

BACKGROUND:Extracellular matrix (ECM) components and intracellular pH (pH(i)) may serve as regulators of cell migration in various cell types. METHODS:The Oris migration assay was used to assess the effect of fibronectin (FN) on cell motility. The Na(+)/H(+) exchanger (NHE)-1 activity was evaluated by measuring pH(i) and [(22)Na(+)] uptake. To examine activated signaling molecules, western blot analysis and immunoprecipitation was performed. RESULTS:ECM components (FN, laminin, fibrinogen, and collagen type I) increased [(22)Na(+)] uptake, pH(i), and cell migration. In addition, FN-induced increase of cell migration was inhibited by NHE-1 inhibitor amiloride or NHE-1-specific siRNA. FN selectively increased the mRNA and protein expression of NHE-1, but not that of NHE-2 or NHE-3. FN binds integrin β1 and subsequently stimulates caveolin-1 phosphorylation and Ca(2+) influx. Then, NHE-1 is phosphorylated by RhoA and Rho kinases, and Ca(2+)/calmodulin (CaM) signaling elicits complex formation with NHE-1, which is enriched in lipid raft/caveolae microdomains of the plasma membrane. Activation of NHE-1 continuously induces an increase of [(22)Na(+)] uptake and pH(i). Finally, NHE-1-dependent extracellular signal-regulated kinase (ERK) 1/2 phosphorylation enhanced matrix metalloproteinase-2 (MMP-2) and filamentous-actin (F-actin) expression, partially contributing to the regulation of embryonic stem cells (ESCs) migration. CONCLUSIONS:FN stimulated mESCs migration and proliferation through NHE-1 activation, which were mediated by lipid raft-associated caveolin-1, RhoA/ROCK, and Ca(2+)/CaM signaling pathways. GENERAL SIGNIFICANCE:The precise role of NHE in the modulation of ECM-related physiological functions such as proliferation and migration remains poorly understood. Thus, this study analyzed the relationship between FN and NHE in regulating the migration of mouse ESCs and their related signaling pathways.

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