The NHE family of ion exchangers includes six isoforms (NHE1-NHE6) that function in an electroneutral exchange of intracellular H(+) for extracellular Na(+). This review focuses on the only ubiquitously expressed isoform, NHE1, which is localized at the plasma membrane where it plays a critical role in intracellular pH (pHi) and cell volume homeostasis. All NHE isoforms share a similar topology: an N-terminus of 12 transmembrane (TM) alpha-helices that collectively function in ion exchange, and a C-terminal cytoplasmic regulatory domain that modulates transport activity by the TM domain. Extracellular signals, mediated by diverse classes of cell-surface receptors, regulate NHE1 activity through distinct signaling networks that converge to directly modify the C-terminal regulatory domain. Modifications in the C-terminus, including phosphorylation and the binding of regulatory proteins, control transport activity by altering the affinity of the TM domain for intracellular H(+). Recently, it was determined that NHE1 also functions as a membrane anchor for the actin-based cytoskeleton, independently of its role in ion translocation. Through its effects on pHi homeostasis, cell volume, and the actin cortical network, NHE1 regulates a number of cell behaviors, including adhesion, shape determination, migration, and proliferation.
It is well established that activation of the Na-H exchanger NHE1 and increases in intracellular pH (pH i ) are early and universal responses to mitogens and have permissive effects in promoting cell proliferation. Despite this evidence, a specific role for NHE1 or pH i in cell cycle progression remains undetermined. We now show that NHE1 activity and pH i regulate the timing of G 2 /M entry and transition. Prior to G 2 /M entry there is a rapid and transient increase in NHE1 activity and pH i , but in fibroblasts expressing a mutant NHE1 that lacks ion translocation activity, this increase in pH i is attenuated, S phase is delayed, and G 2 /M transition is impaired. In the absence of ion translocation by NHE1, expression of cyclin B1 and the kinase activity of Cdc2 are decreased and Wee1 kinase expression increases. Increasing pH i in the absence of NHE1 activity, however, is sufficient to restore Cdc2 activity and cyclin B1 expression and to promote G 2 /M entry and transition. These data indicate that a transient increase in pH i induced by NHE1 promotes the timing of G 2 /M, and they suggest that increases in pH i at the completion of S phase may constitute a previously unrecognized checkpoint for progression to G 2 and mitosis.The ubiquitously expressed plasma membrane Na-H exchanger NHE1 1 regulates intracellular pH (pH i ) homeostasis and has a permissive effect in promoting cell proliferation. Activation of NHE1 and increased pH i are early and universal responses to mitogenic stimulation (1). Growth factor-dependent cell proliferation is attenuated in NHE1-deficient cells (2, 3) in cells treated with pharmacological inhibitors of NHE1 (4 -6) and in cells expressing a mutant NHE1 that is deficient in ion translocation (7). Additionally, retrovirus-induced transformation requires an NHE1-dependent increase in pH ii , and clamping pH i to prevent alkalinization inhibits proliferation and a transformed phenotype (8). Despite an established role for NHE1 in mitogeninduced cell proliferation, the mechanisms whereby NHE1 activity and increased pH i promote cell proliferation are not well understood. We therefore investigated cell cycle progression in fibroblasts expressing a mutant NHE1 that selectively lacks ion translocation activity and is unable to regulate pH i . EXPERIMENTAL PROCEDURES Cell Preparation-LtkϪ and LAP1 fibroblasts were obtained from J. Pouyssegur (University of Nice, France) (9). LAP1 cells stably expressing wild-type and NHE1-266I were obtained by co-transfection of pRSV-neo (1.0 g) with NHE1 plasmids (10 g of pCMV-NHE1) as described previously (7). Cells were maintained in DMEM supplemented with 10% FCS (growth medium).NHE1 Activity and pH i -NHE1 activity was determined as described previously (10) in cells loaded with the acetoxy-methyl ester derivative of the pH-sensitive dye 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF, Molecular Probes). NHE1 activity was determined by measuring the rate of pH i recovery (dpH i /dt) from an NH 4 Cl-induced acid load by evaluating the derivative...
Background: In mammalian cells changes in intracellular pH (pH i ), which are predominantly controlled by activity of plasma membrane ion exchangers, regulate a diverse range of normal and pathological cellular processes. How changes in pH i affect distinct cellular processes has primarily been determined by evaluating protein activities and we know little about how pH i regulates gene expression.
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