Na-H Exchange-dependent Increase in Intracellular pH Times G2/M Entry and Transition

Abstract

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...

Oxidative repression of NHE1 gene expression involves iron-mediated caspase activity

“…This could lead to a long-term decrease in NHE1 expression without the need for constant production of ROS. Chronic decrease in NHE1 levels could impair cell growth through accumulation in the G2/M phase of the cell cycle 9 or induce acidification of the intracellular milieu. 8 To that regard, exposure of L6 1.1 kb cells to nontoxic concentrations of H 2 O 2 induced cell growth arrest; however, the growth arrest was reversible along with the reactivation of NHE1 promoter upon incubation of the cells in medium containing 10% FBS following incubation with H 2 O 2 .…”

“…8 In mammalian cells, the NHE family consists of nine isoforms, NHE1 to NHE-9. Apart from its role as a principal regulator of pHi and cell volume, the ubiquitously expressed subtype NHE1 has been implicated in cell proliferation and transformation (for a review, see Putney et al 9 ). Owing to the historical focus on gene induction, most reports on the regulation of transcription factors by oxidative stress have described the activation of stress-response genes by factors such as activator protein 1 (AP-1) or nuclear factor kB (NF-kB).…”

The acidic microenvironment as a possible niche of dormant tumor cells

“…This is a quite common mechanism for cell proliferation control in both normal and cancer cells that was demonstrated several years ago [29]. When internal pH drops following a sustained extracellular acidosis, also G2/M entry is limited by a reduced cyclin-dependent kinase 1-cyclin B1 activity [30].…”

The chemistry, physiology and pathology of pH in cancer

“…The pH sensors involved in cancer disease progression have proved to be more challenging to identify, possibly because of difficulties in distinguishing bona fide pH sensors from the plethora of proteins that bind H þ ions [2]. Cells sensing an alkaline pH i have been shown to proliferate [61], enter the cell cycle [62,63], differentiate [64], migrate [65,66], reduce apoptosis [67] and clastogenesis [68], and undergo malignant transformation [69,70]-events that are critical in cancer formation and metastasis. Considering the complexity of these processes, the observed pH i sensitivity may involve a number of H þ -binding molecular switches.…”