NHE1 Function Research Articles

1,25(OH)2 D3 inhibits Lewis lung cancer cell migration via NHE1-sensitive metabolic reprograming.

High prevalence and metastasis rates are characteristics of lung cancer. Glycolysis provides energy for the development and metastasis of cancer cells. The 1,25-dihydroxy vitamin D3 (1,25(OH)2 D3 ) has been linked to reducing cancer risk and regulates various physiological functions. We hypothesized that 1,25(OH)2 D3 could be associated with the expression and activity of Na+ /H+ exchanger isoform 1 (NHE1) of Lewis lung cancer cells, thus regulating glycolysis as well as migration by actin reorganization. Followed by online public data analysis, Vitamin D3 receptor, the receptor of 1,25(OH)2 D3 has been proved to be abundant in lung cancers. We demonstrated that 1,25(OH)2 D3 treatment suppressed transcript levels, protein levels, and activity of NHE1 in LLC cells. Furthermore, 1,25(OH)2 D3 treatment resets the metabolic balance between glycolysis and OXPHOS, mainly including reducing glycolytic enzymes expression and lactate production. In vivo experiments showed the inhibition effects on tumor growth as well. Therefore, we concluded that 1,25(OH)2 D3 could amend the NHE1 function, which leads to metabolic reprogramming and cytoskeleton reconstruction, finally inhibits the cell migration.

Alterations of sodium-hydrogen exchanger 1 function in response to SGLT2 inhibitors: what is the evidence?

This review summarizes and describes the current evidence addressing how sodium-glucose cotransporter 2 (SGLT2) inhibitors alter the function of sodium-hydrogen exchanger 1 (NHE-1), in association with their protective effects against adverse cardiovascular events. In the heart, SGLT2 inhibitors modulate the function of NHE-1 (either by direct inhibition or indirect attenuation of protein expression), which promotes cardiac contraction and an enhanced energy supply, in association with improved mitochondrial function, reduced inflammation/oxidative/endoplasmic reticulum stress, and attenuated fibrosis and apoptotic/autophagic cell death. The vasodilating effect of SGLT2 inhibitors has also been proposed due to NHE-1 inhibition. Moreover, platelet-expressed NHE-1 might serve as a target for SGLT2 inhibitors, since these drugs and selective NHE-1 inhibitors produce comparable activity against adenosine diphosphate-stimulated platelet activation. Overall, it is promising that the modulation of the functions of NHE-1 on the heart, blood vessels, and platelets may act as a contributing pathway for the cardiovascular benefits of SGLT2 inhibitors in diabetes and heart failure.

Inhibition of NHE-1 Increases Smoke-Induced Proliferative Activity of Barrett's Esophageal Cell Line.

Several clinical studies indicate that smoking predisposes its consumers to esophageal inflammatory and malignant diseases, but the cellular mechanism is not clear. Ion transporters protect esophageal epithelial cells by maintaining intracellular pH at normal levels. In this study, we hypothesized that smoking affects the function of ion transporters, thus playing a role in the development of smoking-induced esophageal diseases. Esophageal cell lines were treated with cigarettesmoke extract (CSE), and the viability and proliferation of the cells, as well as the activity, mRNA and protein expression of the Na+/H+ exchanger-1 (NHE-1), were studied. NHE-1 expression was also investigated in human samples. For chronic treatment, guinea pigs were exposed to tobacco smoke, and NHE-1 activity was measured. Silencing of NHE-1 was performed by using specific siRNA. CSE treatment increased the activity and protein expression of NHE-1 in the metaplastic cells and decreased the rate of proliferation in a NHE-1-dependent manner. In contrast, CSE increased the proliferation of dysplastic cells independently of NHE-1. In the normal cells, the expression and activity of NHE-1 decreased due to in vitro and in vivo smoke exposure. Smoking enhances the function of NHE-1 in Barrett’s esophagus, and this is presumably a compensatory mechanism against this toxic agent.

NHE1 exacerbates profibrotic activity in idiopathic pulmonary fibrosis and post‐COVID‐19 infection

Idiopathic pulmonary fibrosis (IPF) is a destructive, aging-related pulmonary disease characterized by formation of lung scar tissue. Like other coronaviruses, SARS-COV-2 leads to long term lung damage in some patients in the form of lung fibrosis. In either disease, aggregation of fibroblasts, cell differentiation and excessive tissue remodeling causes the lung tissue to lose lung compliance and oxygen exchange.Sodium hydrogen exchanger isoform 1 (NHE1) is a membrane bound transport protein that functions to support cell movement, regulate cell activity, and balance intracellular pH. A multitude of signaling pathways involved in the regulating of NHE1 function are also involved in the profibrotic activity of fibroblasts and myofibroblasts; therefore we investigated if NHE1 is involved in profibrotic activity and tissue damage associated with IPF and COVID-19 infection. Because NHE1 is key in the remodeling of actin stress fibers and is involved in stiffness of both disorders, we first investigated the impact of NHE1 on stress fiber formation using agonists involved with both fibrosis and COVID response. Lung fibroblasts were treated with three agonists: transforming growth factor-β (TGF-β), serotonin (5HT), and lysophosphatidic acid (LPA) in the presence and absence of an NHE1 inhibitor, ethyl-isopropyl amiloride (EIPA). LPA induced stress fiber formation 65.40+/-3.3 percent over the control, and both 5HT and TGF-β also stimulated the formation of stress fibers by 50.27+/−5.7 and 50.91+/−1.4 percent over the untreated cells. In each case, inhibiting NHE1 with EIPA blocked agonist-activated stress fiber formation, delineating that all three profibrotic factors need NHE1 activity for cytoskeletal remodeling. Another indicator of fibrosis is the fibroblast to myofibroblast transdifferentiation. Myofibroblasts are apoptosis-resistant cells that excessively deposit extracellular matrix proteins; the build up of these cells further compromises the structural integrity of the alveoli and contributes to lung dysfunction. TGF- β, a proinflammatory cytokine, significantly contributes to profibrotic activity by stimulating both fibroblast-myofibroblast transition and excess collagen production. Cells treated with TGF-β to induce cell differentiation showed delayed or significantly reduced myofibroblast transformation in the presence of pharmacological inhibitors of NHE1. Specifically, expression of α-smooth muscle actin (α-SMA), the marker for myofibroblast differentiation, was diminished in each CCL39, LL29, and WI38 cell lines by at least 30 percent and a several day delay of the marker's expression. This study demonstrates the potential for NHE1 as a novel target to fight IPF and lung fibrosis in post-COVID-19 infection.

Evaluating the Inhibition of the Na + ‐H + exchanger Isoform 1 (NHE1) and Kinases that Activate NHE1 as a Potential Combination Therapies for the Treatment of Ovarian Cancer with Paclitaxel

Every year in the United States there are over 22,000 newly diagnosed cases of ovarian cancer leading to over 14,000 deaths. Ovarian Cancer is the fifth leading cause of cancerous deaths among women. Due to that advanced stage of cancer progression at the time of diagnosis treatment is typically challenging and frequently accompanied by drug resistance and disease recurrence. Increased expression and activation of NHE1 has been shown to regulate cell proliferation in cancer cells from ovarian cancer patients. NHE1 is an 815 amino acid transmembrane protein that regulates intracellular pH. NHE1 hyperactivity is involved in the pH inversion which creates an optimal environment for cancer cell proliferation and invasion. In an effort to combat this disease recurrence and drug resistance, it is hypothesized that combining the chemotherapeutic paclitaxel with inhibitors that alter NHE1 function there will be a synergistic effect that slows ovarian cancer progression. Paclitaxel is a secondary treatment for ovarian cancer. It is a taxane based chemotherapeutic that acts as a microtubule stabilizer that affects the cells in the G2 and M phases of cell growth. In these experiments a direct inhibitor of NHE1, Ethylisopropylamiloride, is used. Additionally, we evaluate BI-D1870, the inhibitor of Ribosomal S6 Kinase (Rsk). Rsk is activated in ovarian cancer and has the ability to stimulate NHE1 activity. The ability to develop a synergistic effect was assessed using a proliferation assay in two ovarian cancer cell lines CAOV-3 and SKOV-3. Proliferation assays were run with paclitaxel alone and then with both paclitaxel and one of the inhibitors. The IC50 values were then determined for paclitaxel alone and in combination with the inhibitor. For example, in SKOV-3 Cells, the IC50 for paclitaxel was 0.086 µM. When combined with BI-D 1870 the IC50 for paclitaxel decreased to 0.12 µM, an 86% reduction. These data indicate that inhibitors of NHE1 or NHE1 activation may be combined with paclitaxel to improve the efficacy of ovarian cancer treatment.

Reduced Nhe1 (Na+-H+ Exchanger-1) Function Protects ApoE-Deficient Mice From Ang II (Angiotensin II)-Induced Abdominal Aortic Aneurysms.

IgE-mediated activation of Nhe1 (Na+-H+ exchanger-1) induces aortic cell extracellular acidification and promotes cell apoptosis. A pH-sensitive probe pHrodo identified acidic regions at positions of macrophage accumulation, IgE expression, and cell apoptosis in human and mouse abdominal aortic aneurysm (AAA) lesions. Ang II (angiotensin II)-induced AAA in Nhe1-insufficient Apoe-/-Nhe1+/- mice and Apoe-/-Nhe1+/+ littermates tested Nhe1 activity in experimental AAA, because Nhe1-/- mice develop ataxia and epileptic-like seizures and die early. Nhe1 insufficiency reduced AAA incidence and size, lesion macrophage and T-cell accumulation, collagen deposition, elastin fragmentation, cell apoptosis, smooth muscle cell loss, and MMP (matrix metalloproteinase) activity. Nhe1 insufficiency also reduced blood pressure and the plasma apoptosis marker TCTP (translationally controlled tumor protein) but did not affect plasma IgE. While pHrodo localized the acidic regions to macrophage clusters, IgE expression, and cell apoptosis in AAA lesions from Apoe-/-Nhe1+/+ mice, such acidic areas were much smaller in lesions from Apoe-/-Nhe1+/- mice. Nhe1-FcεR1 colocalization in macrophages from AAA lesions support a role of IgE-mediated Nhe1 activation. Gelatin zymography, immunoblot, and real-time polymerase chain reaction analyses demonstrated that Nhe1 insufficiency reduced the MMP activity, cysteinyl cathepsin expression, IgE-induced apoptosis, and NF-κB activation in macrophages and blocked IgE-induced adhesion molecule expression in endothelial cells. A near-infrared fluorescent probe (LS662) together with fluorescence reflectance imaging of intact aortas showed reduced acidity in AAA lesions from Nhe-1-insufficient mice. This study revealed extracellular acidity at regions rich in macrophages, IgE expression, and cell apoptosis in human and mouse AAA lesions and established a direct role of Nhe1 in AAA pathogenesis.

TMIC-19. H+ EXTRUSION PROTEIN NA/H EXCHANGER IN METABOLIC POLARIZATION OF GLIOMA-ASSOCIATED MICROGLIA/MACROPHAGES AND TUMOR IMMUNITY

Abstract Immunosuppressive tumor microenvironment (TME) in glioblastoma (GBM) attributes to poor efficacy of chemotherapy and/or immunotherapy. We recently reported that SLC9A1 gene expression (Na/H exchanger 1, NHE1) in GBM tumor correlates with increased macrophage tumor infiltration and worsened patient survival. Temozolomide (TMZ) monotherapy, the standard care therapy for GBM, stimulates NHE1 protein expression in mouse syngeneic glioma models. We speculate that in response to TMZ therapy, NHE1 promotes immunosuppressive TME development via deregulation of glucose metabolism and anti-tumor function of immune cells. CD57BL/6 mice bearing GL26 glioma tumor were treated with NHE1 inhibitor HOE-642 (H, 0.3 mg/kg/day), TMZ (T, 2.5 mg/kg/day), and/or anti-PD-1 (10 mg/kg/day) monotherapy or in combination. The T+H+anti-PD-1 combination therapy increased glucose uptake (2-NBDG) and mitochondrial mass (mitotracker fluorescent intensity) of tumor infiltrating CD8+ T-cells by ~2-fold compared to the monotherapies. This suggests that NHE1 protein is involved in metabolic transformation of T-cells and affects their responses to chemo and/or immunotherapy. Furthermore, we detected that specific deletion of Nhe1 in Cx3Cr1CreErT2r+/-;Nhe1f/f mice (Nhe1 KO) alters activation of glioma-associated microglia/macrophages (GAM) and T-cells in response to TMZ therapy. TMZ monotherapy stimulated macrophage (CD11b+CD45hi) infiltration by ~2.5-fold in Cx3Cr1CreErT2-/-;Nhe1f/f (Nhe1 Con) mice bearing SB28-GFP glioma tumor with abundant proinflammatory CD16/32+ GAMs. In contrast, TMZ treated Nhe1 KO mice displayed ~3-fold reduction in tumor infiltrating GAMs with nearly abolished Ym-1+ tolerogenic macrophages, ~3-fold reduction of tumor-promoting CD4+CD25+FoxP3+ regulatory T-cells (Treg) and increased CD8+/Treg ratio by ~2-fold than the Nhe1 Con mice. Importantly, TMZ followed by anti-PD-1 therapy prolonged survival of Nhe1 KO mice but not Nhe1 Con mice. Taken together, our study suggests that NHE1 is involved in transformation of GAMs and PD-1 check point activation of T-cells. Blocking NHE1 function in combination with TMZ and anti-PD-1 may restore glucose metabolism of immune cells necessary for their anti-tumor function.

Designing a Stapled Peptide Inhibitor of CHP‐NHE1 Protein‐Protein Interactions

The Sodium Hydrogen Exchanger (NHE1) is a membrane transporter that exchanges an intracellular proton for an extracellular sodium ion. NHE1 plays a critical role interacting with, directing, and regulating proteins that drive cell motility. We have shown that NHE1 plays a pivotal role in pH homeostasis and migration of lung tumor cells. The Calcineurin homologous B protein isoforms 1 and 2 (CHP1/2) interact with NHE1 at the same 40aa domain (CHP binding domain (CBD)) both impacting NHE1 function. CHP1 is ubiquitously expressed and its interaction with NHE promotes basal activity. While CHP2 expression is limited to the gut in non‐diseased tissue, it is highly expressed in lung and other tumor cells. When CHP2 or NHE1 expression is diminished, tumor cell growth is nearly abrogated. This supports that CHP2‐NHE1 interactions have potential as an anticancer target. Models of both CHP proteins bound to NHE1 show each bind in the CBD of NHE but with subtle structural differences. To analyze the potential differences between CHP‐NHE binding, recombinant proteins were generated and used to determine the thermodynamics of CHP/NHE interaction through CD thermal melts. Recombinant CHP2 bound to NHE1 exhibited a lower free energy of binding (−1.08 kJ mol−1) compared to CHP1 (−0.48 kJ mol−1), a ΔΔG of 0.60 kJ mol−1. Supporting this are in‐vitro ligand binding assays where we show a 2.23 fold greater affinity of CHP2‐NHE than CHP1‐NHE1. In order to create an effective peptide inhibitor, Large‐scale Atomic/Molecular Massively Parallel Simulator (LAMMPS) computations were carried out using know crystal structures of each CHP isoform bound to NHE1. From these computations CHP1 was determined to have a two‐state binding interaction, whereas CHP2 had a one‐state binding interaction with NHE1. Unique initial binding contacts of NHE1 were determined for each CHP isoform. Stapled peptides mimicking the CHP‐NHE1 binding region were synthesized via a sulfenylation reaction with tryptophan amino acids. Peptides were then screened using a competitive fluorescent plate binding assay in which equal amounts of tagged CHP1/2 in the presence of NHE1. These peptide inhibitors will allow for increased CHP1‐NHE1 activity in targeted (cancer) cells while simultaneously reducing CHP2‐NHE1 activity.Support or Funding InformationSupported in part by NSF Che 1664565This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Diverse residues of intracellular loop 5 of the Na+/H+ exchanger modulate proton sensing, expression, activity and targeting

The mammalian Na+/H+ exchanger isoform 1 (NHE1) is an integral membrane protein that regulates intracellular pH (pHi) by removing a single intracellular proton in exchange for one extracellular sodium ion. It is involved in cardiac hypertrophy and ischemia reperfusion damage to the heart and elevation of its activity is a trigger for breast cancer metastasis. NHE1 has an extensive 500 amino acid N-terminal membrane domain that mediates transport and consists of 12 transmembrane segments connected by intracellular and extracellular loops. Intracellular loops are hypothesized to modulate the sensitivity to pHi. In this study, we characterized the structure and function of intracellular loop 5 (IL5), specifically amino acids 431–443. Mutation of eleven residues to alanine caused partial or nearly complete inhibition of transport; notably, mutation of residues L432, T433, I436, N437, R440 and K443 demonstrated these residues had critical roles in NHE1 function independent of effects on targeting or expression. The nuclear magnetic resonance (NMR) solution spectra of the IL5 peptide in a membrane mimetic sodium dodecyl sulfate solution revealed that IL5 has a stable three-dimensional structure with substantial alpha helical character. NMR chemical shifts indicated that K438 was in close proximity with W434. Overall, our results show that IL5 is a critical, intracellular loop with a propensity to form an alpha helix, and many residues of this intracellular loop are critical to proton sensing and ion transport.

Palmitoylation Regulates the Functions of NHE1

The sodium hydrogen exchanger isoform 1 (NHE1) is a ubiquitously expressed transmembrane protein that regulates intercellular pH (pHi) and interacts with proteins involved in cell migration and adhesion. Within the extended cytosolic tail of NHE1, there are multiple sites of post‐translational modification including phosphorylation and lipidation. Our understanding of impact of reversible palmitoylation on membrane transporters is growing. 2‐bromopalmitate (2BP) irreversibly inhibits the enzyme, palmitoyl acyltransferase, that transfer palmitate to cysteines. We found NHE1 is reversibly palmitoylated and here, demonstrate the impact of the lipidation on NHE1 function. Lung fibroblasts expressing NHE1 (PSN) showed minimal decrease (8.5 ± 2.5% SEM) in cell viability after treatment with 50 μM 2BP after 72 h. Palmitoylation plays a critical role in cell adhesion and motility. Cells that were incubated in 15 μM 2BP for 18 hrs displayed a significant decrease cell adhesion in low serum conditions and cells treated with 2BP did not fully migrate into vacated space after 24 hrs in a wounding assay. Impact on cytoskeletal structure by 2BP treatment was also investigated. Lysophosphatidic acid (LPA) increased actin stress fiber formation 40% over control cells. This LPA‐induced stress fiber formation was inhibited more than two‐fold in the presence of 2BP. This is important as LPA‐induced stress fiber formation requires NHE1; therefore, palmitoylation is required for NHE1‐dependent actin stress fiber formation. To determine the effect of palmitoylation on NHE1 transport activity, PSN cells were treated with 2BP and the pHi was measured. Addition of increasing concentrations of 2BP inhibited NHE1 transport in cells with and without serum, but the effect was enhanced in low serum conditions. Interestingly, LPA stimulation of NHE1 activity was inhibited by 60%, but not to baseline. This indicates that in addition to cell adherence, motility, and cytoskeletal formation, palmitoylation is critical for NHE1 transport.Support or Funding InformationThis research was partially funded by the Beckman FoundationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Differential Impacts of Hypoxia on Na+/H+ Exchanger Isoform 1 and Calcineurin B Homologous Protein Isoform 2 Expression in Non‐small Cell Lung Cancer

The Na+‐ H+ exchanger isoform 1 (NHE1) is a key regulator of cell proliferation, migration and invasion in cells from a variety of solid tumors. The calcineurin B homologous proteins (CHP1 and CHP2) appear to be essential cofactors to support NHE1 function. CHP1 appears to be expressed ubiquitously in healthy tissue, while CHP2 is predominantly expressed in tumor cells. While both CHP isoforms are highly homologous and bind in nearly identical regions on NHE1, each has been identified to have a distinct impact on NHE1 function. Our research has demonstrated an elevation in CHP2 expression in tumor samples from patients with either adenocarcinoma or squamous cell carcinoma of the lung as well as in a broad range of NSCLC cell lines. To evaluate the role of CHP2 in NSCLC cells we used NCI‐H1299, a carcinoma cell line, as the parent cell line and developed two new cell lines. One of these cell lines lacked CHP2 expression (H1299 CHP2KD) and the other lacked NHE1 expression (H1299 NHE1KD). We hypothesized that CHP2 expression enhances cell survival and the tumorigenic potential of these cells. We evaluated the impact of a low serum and hypoxic microenvironment on CHP2 expression and determined CHP2 levels increased when H1299 cells are grown in 0.5% serum and 1% O2 compared to cells grown in 10% serum and 21% O2 (normal conditions) after 24 and 48 hours; however, at approximately 72 and 96 hours, CHP2 expression in the same conditions decreased while NHE1 expression increased. CHP2 may be involved in initial survival mechanisms during the time NHE1 expression is increasing to further allow for cancer progression. To evaluate the role of CHP2 and NHE1 biologically, proliferation and migration were assessed in all conditions. Proliferation and migration rates in normal conditions for H1299 CHP2KD cells were approximately 50% of H1299 cells. When these experiments were performed in low serum and hypoxic conditions, the proliferation and migration rates of the H1299 cells was reduced to about 50% of that seen in normal conditions, while the rates of proliferation and migration in H1299 CHP2KD was reduced to a rate equivalent to the H1299 NHE1KD cells. These data suggest a major role for CHP2 expression in the development and progression of non‐small cell lung cancer.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Biallelic CHP1 mutation causes human autosomal recessive ataxia by impairing NHE1 function.

Objective:To ascertain the genetic and functional basis of complex autosomal recessive cerebellar ataxia (ARCA) presented by 2 siblings of a consanguineous family characterized by motor neuropathy, cerebellar atrophy, spastic paraparesis, intellectual disability, and slow ocular saccades.Methods:Combined whole-genome linkage analysis, whole-exome sequencing, and focused screening for identification of potential causative genes were performed. Assessment of the functional consequences of the mutation on protein function via subcellular fractionation, size-exclusion chromatography, and fluorescence microscopy were done. A zebrafish model, using Morpholinos, was generated to study the pathogenic effect of the mutation in vivo.Results:We identified a biallelic 3-bp deletion (p.K19del) in CHP1 that cosegregates with the disease. Neither focused screening for CHP1 variants in 2 cohorts (ARCA: N = 319 and NeurOmics: N = 657) nor interrogating GeneMatcher yielded additional variants, thus revealing the scarcity of CHP1 mutations. We show that mutant CHP1 fails to integrate into functional protein complexes and is prone to aggregation, thereby leading to diminished levels of soluble CHP1 and reduced membrane targeting of NHE1, a major Na+/H+ exchanger implicated in syndromic ataxia-deafness. Chp1 deficiency in zebrafish, resembling the affected individuals, led to movement defects, cerebellar hypoplasia, and motor axon abnormalities, which were ameliorated by coinjection with wild-type, but not mutant, human CHP1 messenger RNA.Conclusions:Collectively, our results identified CHP1 as a novel ataxia-causative gene in humans, further expanding the spectrum of ARCA-associated loci, and corroborated the crucial role of NHE1 within the pathogenesis of these disorders.

Expressional and Functional Characterization of Intracellular pH Regulators and Effects of Ethanol in Human Oral Epidermoid Carcinoma Cells

Background/Aims: To functionally characterize intracellular pH (pH_i) regulating mechanisms, such as Na⁺-H⁺ exchanger (NHE) and Na⁺-HCO₃^- co-transporter (NBC), and further examine effects of ethanol on the pH_i regulating mechanism in human oral epidermoid carcinoma (OEC-M1) cells. Methods: OEC-M1 cells were a gift from Tri-Service General Hospital. Changes of pH_i were detected by microspectrofluroimetry with a pH-sensitive fluorescent dye, BCECF. Isoforms of transporters were examined by Western blot technique. Results: i) the steady-state pH_i value shifted from alkaline (7.35∼7.49) to acidic (7.0∼7.03) following acid/base impacts; ii) in HEPES-buffer system, pH_i recovery following induced-acidification was totally blocked by either removing [Na]_o⁺ or adding HOE 694 (a NHE1 specific inhibitor), which demonstrates existence of NHE1; iii) in HCO₃^-/CO₂-buffer system, the pH_i recovery following induced-acidification was entirely blocked by either removing [Na]_o⁺ or adding HOE 694 plus DIDS (a NBC specific inhibitor), which suggests existence of Na⁺- and HCO₃^–dependent acid-extruder, i.e. NBC; iv) the isoforms of the two acid extruders were NHE1, NBCn1, NBCe1 and NDCBE; v) ethanol (10-1000 mM) showed a biphasic and concentration-dependent effect on resting pH_i (i.e. increase then decrease) by changing the activity of NHE1 and NBC accordingly; vi) treatment with ethanol for 24 hr (<unterline>></unterline> 300 mM) significantly inhibited the expression of NHE1, NBCn1 and NDCBE, while up-regulated NBCe1. Conclusions: Ethanol affects pH_i in a concentration-dependent manner by changing function and expression of NHE1 and NBC isoforms in OEC-M1 cells.

NHE1 has a notable role in metastasis and drug resistance of T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) represents a spectrum of hematological malignancies that affect human health. Metastasis and chemotherapeutic drug resistance are the primary causes of mortality in patients with T-ALL. Sodium-hydrogen antiporter 1 (NHE1) is established to serve a role in metastasis and drug resistance in numerous types of cancer; however, the function of NHE1 in T-ALL remains to be elucidated. Previously, the C-C-motif chemokine ligand 25 (CCL25) was identified to be involved in metastasis and drug resistance in the MOLT4 T-ALL cell line, as was the ezrin protein. The present study investigated the role of NHE1 in the metastasis of T-ALL using a Transwell assay and scanning electron microscopy, using MOLT4 cells as a model. The association between NHE1 and ezrin was assessed using laser scanning confocal microscopy. The effect of NHE1 on resistance to the chemotherapy drug doxorubicin (DOX) was also investigated using a cell viability and cytotoxicity assay. Expression of NHE1 increased following treatment with CCL25, accompanied by morphological changes in MOLT4 cells and the co-localization of NHE1 with ezrin. In addition, wild-type MOLT4 cells exhibited an increased polarization ability compared with NHE1- or ezrin-silenced cells. NHE1- or ezrin-silenced cells exhibited higher sensitivity to DOX compared with wild-type MOLT4 cells. In conclusion, the increased expression or activity of NHE1 may potentially be a poor prognostic indicator for human T-ALL.

Inhibition of Sodium Hydrogen Exchanger 1 Palmitoylation is Associated with Suppression of Cell Migration

The sodium hydrogen exchanger isoform 1 (NHE1) is involved in many cellular processes including regulation of intracellular pH, coordination of cell migration, control of cell volume, and anchoring protein interactions at the cell membrane. NHE1 is integral plasma membrane protein with 12 transmembrane spanning domains that exchanges an intracellular proton for an extracellular sodium ion. NHE1 is implicated in neoplastic transformation leading to tumorgenesis where the exchanger localizes to the leading edge of migrating cells initiating forward movement of cells indicating a potential role for NHE1 in cancer progression. NHE1 is regulated by many factors including multiple protein‐protein interactions and a number of phosphorylation events. Recent reports indicate that many proteins identified to play key roles in cancer are regulated by palmitoylation. S‐palmitoylation is a dynamic and reversible posttranslational modification, in which the protein undergoes cycles of palmitoylation and depalmitoylation, catalyzed by palmitoyl acyltransferases (PATs) and acyl protein thioesterases (APTs), respectively. Palmitoylation is known to regulate protein trafficking, membrane microlocalization and protein‐protein interactions. These findings prompted us to investigate the presence of this modification on NHE1 using acyl‐biotinyl exchange (ABE), an in vitro assay for post‐hoc detection of endogenous palmitoylation, which revealed that NHE1 is palmitoylated. To further evaluate the nature of NHE1 palmitoylation we treated Chinese hamster lung fibroblasts expressing human NHE1 (PSN) with the irreversible PAT inhibitor 2‐bromopalmitate (2BP). We found 2BP suppresses NHE1 palmitoylation in both a time and dose dependent manner. Additionally, PSN cell migration assessed using electric cell‐substrate impedance sensing was inhibited by 2BP in a manner consistent with the degree of inhibition of NHE1 palmitoylation. These results suggest that palmitoylation may regulate NHE1 function and its role in modulating cell migration.Support or Funding InformationNational Institutes of Health grants DA 031991 (JDF), DA13147 and 5P20‐104360 (RAV), P30‐GM103329 (UND), P20‐GM12345 (UND)

Enhanced Reactive Oxygen Species Production, Acidic Cytosolic pH and Upregulated Na+/H+ Exchanger (NHE) in Dicer Deficient CD4+ T Cells

Background: MicroRNAs (miRNAs) negatively regulate gene expression at a post-transcriptional level. Dicer, a cytoplasmic RNase III enzyme, is required for the maturation of miRNAs from precursor miRNAs. Dicer, therefore, is a critical enzyme involved in the biogenesis and processing of miRNAs. Several biological processes are controlled by miRNAs, including the regulation of T cell development and function. T cells generate reactive oxygen species (ROS) with parallel H+ extrusion accomplished by the Na+/H+-exchanger 1 (NHE1). The present study explored whether ROS production, as well as NHE1 expression and function are sensitive to the lack of Dicer (miRNAs deficient) and could be modified by individual miRNAs. Methods: CD4+ T cells were isolated from CD4 specific Dicer deficient (DicerΔ/Δ) mice and the respective control mice (Dicerfl/fl). Transcript and protein levels were quantified with RT-PCR and Western blotting, respectively. For determination of intracellular pH (pHi) cells were incubated with the pH sensitive dye bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) and Na+/H+ exchanger (NHE) activity was calculated from re-alkalinization after an ammonium pulse. Changes in cell volume were measured using the forward scatter in flow cytometry, and ROS production utilizing 2',7' -dichlorofluorescin diacetate (DCFDA) fluorescence. Transfection of miRNA-control and mimics in T cells was performed using DharmaFECT3 reagent. Results: ROS production, cytosolic H+ concentration, NHE1 transcript and protein levels, NHE activity, and cell volume were all significantly higher in CD4+ T cells from DicerΔ/Δ mice than in CD4+ T cells from Dicerfl/fl mice. Furthermore, individual miR-200b and miR-15b modify pHi and NHE activity in Dicerfl/fl and DicerΔ/Δ CD4+ T cells, respectively. Conclusions: Lack of Dicer leads to oxidative stress, cytosolic acidification, upregulated NHE1 expression and activity as well as swelling of CD4+ T cells, functions all reversed by miR-15b or miR-200b.

Na+/H+ exchanger 1 has tumor suppressive activity and prognostic value in esophageal squamous cell carcinoma.

Na+/H+ exchanger 1 (NHE1) is a plasma membrane transporter that controls intracellular pH and regulates apoptosis and invasion in various cancer cells. However, the function of NHE1 in esophageal squamous cell carcinoma (ESCC) cells and the relationship between the expression of NHE1 and prognosis of ESCC remain unclear. We found that the knockdown of NHE1 in ESCC cells inhibited apoptosis and promoted cell proliferation, migration, and invasion and showed increases in Snail, β-catenin, and activation of PI3K-AKT signaling, which was consistent with the results obtained from microarrays. Microarrays results suggested that the knockdown of NHE1 suppressed Notch signaling pathway. An immunohistochemical investigation of 61 primary ESCC samples revealed that NHE1 was expressed at higher levels in well-differentiated tumors. The 5-year survival rate was poorer in the NHE1 low group (57.0%) than in the NHE1 high group (82.8%). Multivariate analyses revealed that the weak expression of NHE1 was associated with shorter postoperative survival (hazard ratio 3.570, 95% CI 1.291-11.484, p = 0.0135).We herein demonstrated that the suppression of NHE1 in ESCC may enhance malignant potential by mediating PI3K-AKT signaling and EMT via Notch signaling, and may be related to a poor prognosis in patients with ESCC.

Glioma-mediated microglial activation promotes glioma proliferation and migration: roles of Na+/H+ exchanger isoform 1.

Microglia play important roles in extracellular matrix remodeling, tumor invasion, angiogenesis, and suppression of adaptive immunity in glioma. Na(+)/H(+) exchanger isoform 1 (NHE1) regulates microglial activation and migration. However, little is known about the roles of NHE1 in intratumoral microglial activation and microglia-glioma interactions. Our study revealed up-regulation of NHE1 protein expression in both glioma cells and tumor-associated Iba1(+) microglia in glioma xenografts and glioblastoma multiforme microarrays. Moreover, we observed positive correlation of NHE1 expression with Iba1 intensity in microglia/macrophages. Glioma cells, via conditioned medium or non-contact glioma-microglia co-cultures, concurrently upregulated microglial expression of NHE1 protein and other microglial activation markers (iNOS, arginase-1, TGF-β, IL-6, IL-10 and the matrix metalloproteinases MT1-MMP and MMP9). Interestingly, glioma-stimulated microglia reciprocally enhanced glioma proliferation and migration. Most importantly, inhibition of microglial NHE1 activity via small interfering RNA (siRNA) knockdown or the potent NHE1-specific inhibitor HOE642 significantly attenuated microglial activation and abolished microglia-stimulated glioma migration and proliferation. Taken together, our findings provide the first evidence that NHE1 function plays an important role in glioma-microglia interactions, enhancing glioma proliferation and invasion by stimulating microglial release of soluble factors. NHE1 upregulation is a novel marker of the glioma-associated microglial activation phenotype. Inhibition of NHE1 represents a novel glioma therapeutic strategy by targeting tumor-induced microglial activation.

Calcineurin B Homologous Protein Isoform 2 (CHP2) Expression and Activity Impact Cell Migration and Tumor Formation in Non‐small Cell Lung Cancer

The Na+‐ H+ exchanger isoform 1 (NHE1) is a key regulator of cell proliferation, migration and invasion in cells from a variety of solid tumors. The calcineurin B homologous proteins (CHP1 and CHP2) appear to be essential cofactors to support NHE1 function. CHP1 appears to be expressed ubiquitously in healthy tissue, while CHP2 is predominantly expressed in tumor cells. While both CHP isoforms are highly homologous and bind in nearly identical regions on NHE1, each has been identified to have a distinct impact on NHE1 function. Our research has demonstrated an elevation in CHP2 expression in tumor samples from patients with either adenocarcinoma or squamous cell carcinoma of the lung as well as in a broad range of NSCLC cell lines. We hypothesized that elevated CHP2 expression enhances cell survival and the tumorigenic potential of these cells. To evaluate the role of CHP2 in NSCLC cells we used NCI‐H1299, a carcinoma cell line, as the parent cell line and developed two new cell lines. One of these cell lines lacked CHP2 expression (H1299 CHP2KD) and the other lacked NHE1 expression (H1299 NHE1KD). We evaluated the impact of a low serum environment on CHP2 expression and determined that CHP2 levels increased when H1299 cells are grown in 0.5% serum compared to cells grown in 10% serum. This increased CHP2 expression drives changes that support the cells ability to survive these low serum environments. In Electric Cell‐substrate Impedance Sensing (ECIS) migration experiments in 10% serum, H1299 cells reestablished a confluent monolayer within 18 hours of wounding. In contrast, the H1299 CHP2KD cells migrated at approximately 50% of that rate and the H1299 NHE1KD cells showed extremely limited cell migration. When these experiments were performed in 0.5% serum, the migration rate of the H1299 cells was reduced to about 50% of that seen in 10% serum while the rate of migration in H1299 CHP2KD was reduced to a rate equivalent to the H1299 NHE1KD cells. In cell proliferation experiments, H1299 cells in 0.5% serum grow at a rate equivalent to H1299 CHP2KD cells in 10% serum. Finally, in a soft agar assay, a greater rate of tumor formation occurred for H1299 cells in both 10% and 0.5% serum than either the H1299 CHP2KD or H1299 NHE1KD cells. These data suggest a major role for CHP2 expression in the development and progression of non‐small cell lung cancer.

You can Never have too many kinases: The Sodium Hydrogen Exchanger Isoform 1 Regulation by Phosphorylation

Regulation of Na+-H+ exchanger isoform 1 (NHE1) activity is a dynamic, integrated system demonstrating the complexity of kinase signaling. Studies of NHE1 have described 22 confirmed and putative phosphorylation sites on the cytoplasmic domain regulated by 12 protein kinases. However the final number of sites and the impact of these sites remains unclear. By identifying the Rock phosphorylation site and its role in regulating NHE1 activity, we began to understand the functional interplay between the RhoA/Rock and Rsk/Erk pathways. This demonstrates the need to have a complete and comprehensive understanding of the relationship between each kinase involved in NHE1 modification for a true understanding of the exchanger’s role in the control of cellular biological activity. By observing how the Rock phosphorylation site impacts cellular proliferation, it was demonstrated that Rock activity is only partially responsible for NHE1’s impact on biological activity. Furthermore, data presented here shows that Rock activity is involved in both α1-adrenergic (Phenylephrine) and LPA signaling but not PDGF activation of NHE1 transport activity suggesting that growth factor signaling does not require Rock, yet the kinase is essential in the effects of GPCR pathways. Of particular interest and adding to the complexity of kinase signaling, Pyk2 activity has been found to have an inhibitory effect on NHE1. These data demonstrate an uncertainty in the exact role that different kinases play in the regulation of NHE1 function, yet succeeds in providing evidence of the intricacy of phosphorylation organization and a need for further elucidation of the interplay between phosphorylation sites. Overall, the pivotal role of NHE1 in disease establishment and progression creates a solid argument for continued efforts in understanding its regulation.