Abstract
Background: The Na-H exchangers (NHEs) are a family of membrane glycoproteins which transport H out of the cell in exchange for Na with a stoichiometry of 1:1. In mammalian cells, the NHE family consists of nine isoforms, NHE-1 to NHE-9. NHE-1, the first one of the isoforms to be cloned, is ubiquitously distributed. Several studies have shown both increased activity and protein of NHE-1 in transformed cells. Apart from its role as a principal regulator of intracellular pH (pHi) and cell volume, NHE-1 has been implicated in cell proliferation, transformation and migration. Cell migration is a multi-step process that requires spatial asymmetry which is stimulated by Rho GTPases, phosphoinositides and actin polymerization. Integrin family of receptors is responsible for cell surface interactions with extracellular matrix (ECM). NHE-1 may contribute to cell migration by: (a) affecting the cell volume, (b) regulating the intracellular pH and thereby the assembly and activity of cytoskeletal elements, (c) anchoring the cytoskeleton to the plasma membrane, and (d) by controlling cell adhesion. Disrupting NHE-1 function leads to impaired polarity of cells and their inability to migrate. Although NHE1 has been shown to affect cell migration through its various functions, a role for the exchanger in cell migration regulated by integrins has not been extensively studied. Fact that NHE1 binds several other proteins in the cytoplasmic regulatory domain, have led to the hypothesis that NHE1 can act as a plasma membrane scaffold that brings together many proteins so they can interact functionally. Thus, it is plausible to hypothesize the possible role NHE-1 might play by direct or indirect structural interaction with the assembly of cell adhesion molecules. Materials and Methods: 1. Inhibition of NHE1 activity (using ethyl-isopropyl-amiloride) and its effect on pHi and cell viability (Measurement of intracellular pH, MTT assay) 2. Effect of NHE1 silencing (using siRNA transfection) and its effect on cell viability (Western blot analysis, MTT assay) 3. Effect of NHE1 silencing and pharmacological inhibition of its activity on cell adhesion and motility. (In vitro cell adhesion assay, In vitro cell migration assay) Results: The data obtained shows that with either EIPA treatment or NHE-1 siRNA transfection, migratory capacity was impaired in MDA-MB231 human breast cancer cells. Interestingly, pharmacological inhibition of NHE-1 did not significantly reduce the integrins-dependent cell adhesion in these cells. However, down-regulation of NHE-1 protein expression had significant effect on integrins-mediated cell adhesion to fibronectin. Conclusion: This study shows that the effect of NHE-1 on integrinsdependent cell adhesion is independent of its activity. However, NHE-1 protein expression seems to be an important upstream event in the functional assembly of integrin receptors and may play an essential role in cancer cell adhesion to the extracellular matrix.
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