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WATER AND ELECTROLYTE HOMEOSTASIS
Departments of 1Pediatrics and 2Neuroscience, University of California San Diego and 3The Rady Children's Hospital, San Diego, California; and Departments of 4Medicine, 5Pediatrics, and 6Human Genetics, Mount Sinai School of Medicine, New York, New York
Submitted 8 December 2006 ; accepted in final form 5 February 2007
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ABSTRACT |
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 TOP ABSTRACT EXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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intracellular pH regulation; cell proliferation; AntibodyArray; protein interaction; Srchomology 2 domain-containing protein tyrosine phosphatase
(38), heat shock protein (HSP)70 (59), and carbonic anhydrase II (39). An important characteristic of NHE1 is that it can be activated by a wide variety of stimuli including growth factors, hormones, cytokines, and mechanical stresses. This activation takes place through distinct cell surface receptors including receptor tyrosine kinase, G protein-coupled receptors, integrin receptors, and intracellular signaling networks (15, 44, 52, 73). Thus cells become alkalinized and allow the progression of certain biological processes. Modulation of NHE1 activity occurs at the COOH terminus by at least three mechanisms: 1) phosphorylation, such as by ERK-regulated p90 ribosomal S6 kinase (62) in response to growth factor signals; 2) the binding of regulatory proteins with conformational changes, such as the association of Ca2+/calmodulin (CaM) to the high-affinity CaM domain of NHE1, which relieves an autoinhibitory intramolecular interaction, thereby enhancing NHE1 activity (71, 72); and 3) affinity alteration of the transmembrane internal H+ transporter site (35).
Recent data have added another layer of complexity regarding NHE1 functions. For example, Denker and colleagues (18) uncovered a novel structural role for NHE1 in remodeling cortical cytoskeleton and cell shape via its association with ERM proteins, a function that is independent of H+ translocation. We showed recently (75, 76) that, in NHE1-null mutant mice, the expression of other membrane transporters and channels is altered, including an increase in the acid extruder NHE3 and Na+ channel and a decrease in the acid loader anion exchanger isoform 3. In addition, using cDNA microarrays, we demonstrated (81) that NHE1 deficiency has an impact on gene expression, such as downregulation of monocarboxylic acid transporter 13 and 14-3-3 genes and upregulation of secreted phosphoprotein 1/osteopontin (SP1/OPN) gene. The discovery of these changes in gene expression or their products may provide a molecular basis for the phenotype observed in NHE1-null mutant mice, namely, locomotor ataxia and tonic-clonic seizures (16).
The above observations indicate that NHE1 is not simply an acid-base transporter as originally thought. Indeed, it may play its role in relaying extracellular cues to a wide array of intracellular signaling pathways, not only via H+ flux but also through cytoskeletal anchoring and scaffolding. This prompted us to identify NHE1 interactive partners in signaling cascades and to investigate their potential physiological relevance.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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Membrane fractionation. Animal use was approved by the Institutional Animal Care and Use Committee of the University of California San Diego. Crude membranes were prepared from brain samples according to a method described by Grassl and Aronson (27). Briefly, the brains were quickly removed and placed in ice-cold lysis buffer (200 mM mannitol, 80 mM HEPES, 41 mM KOH, 1 µM pepstatin A, 1 µM leupeptin, 230 µM phenylmethylsulfonyl fluoride and 1 mM ethylenediamine tetrahydrochloride; pH 7.5) (Sigma, St. Louis, MO). The tissues were homogenized by 10–20 strokes at 2,000 rpm with a Teflon-glass homogenizer (Thomas Scientific, Swedesboro, NJ). The homogenate was then centrifuged at 1,000 g and 4°C for 10 min to remove cellular debris. The supernatant was recentrifuged at 100,000 g and 4°C in a Beckman L8-70M ultracentrifuge (Beckman Instruments, Palo Alto, CA) for 1 h. The resulting pellet as membrane fraction was resuspended in 500–1,000 µl of lysis buffer and stored at –80°C until use.
Conjugation of antibody with horseradish peroxidase. Horseradish peroxidase (HRP) was conjugated with mouse monoclonal anti-NHE1 antibody (Chemicon, Temecula, CA) with the EZ-Link Plus Activated Peroxidase kit (Pierce, Rockford, IL) according to manufacturer's instructions. Briefly, 1 mg of lyophilized EZ-Link Plus Activated Peroxidase was reconstituted with 100 µl of water and added to 500 µl of NHE1 antibody (100 µg) in BupH phosphate-buffered saline. Ten microliters of reductant solution (5 M NaCNBH3 in 1 M NaOH) was immediately added, and the mixture was incubated for 1 h at room temperature with slow shaking. The reaction was stopped by adding 20 µl of quench buffer (3 M ethanolamine, pH 9.0) for 15 min at room temperature with slow shaking. The HRP-conjugated NHE1 antibody was aliquotted and stored at –20°C until use.
Screen of NHE1 interactive proteins. NHE1 interactive proteins were screened with Signal Transduction AntibodyArray (Hypromatrix, Worcester, MA). Briefly, AntibodyArray was incubated in blocking solution [5% BSA in TBST (Tris-buffered saline-Tween; 150 mM NaCl, 25 mM Tris, 0.05% Tween 20, pH 7.5)] for 1 h at room temperature with slow shaking. Brain membrane proteins (0.5 µg/µl in blocking solution) were then incubated with AntibodyArray for 2 h at room temperature with slow shaking. AntibodyArray was washed with TBST three times for 15 min each. HRP-conjugated NHE1 antibody (2.3 µg/ml) was applied to AntibodyArray in TBST for 2 h at room temperature. AntibodyArray was washed again with TBST three times for 15 min each. The protein signals were detected with an ECL chemiluminescence system (Amersham Biosciences, Piscataway, NJ).
Coimmunoprecipitation and Immunoblotting of NHE1 Interactive Candidates
Coimmunoprecipitation of NHE1 and candidate interactive partners was performed as described previously (47). The membrane proteins from tissues or cultured cells were solubilized in immunoprecipitation (IP) buffer (20 mM HEPES/Tris, pH 7.4, 1% Triton X-100, 150 mM NaCl, and protease inhibitors). Two milligrams of membrane protein (500–800 µl) was incubated overnight at 4°C with 5 µl of rabbit anti-NHE1 polyclonal antibody (a gift from Dr. Shigeo Wakabayashi, National Cardiovascular Center Research Institute, Osaka, Japan) plus 120 µl of protein A-Sepharose CL-4B beads (Amersham Biosciences). The immunoprecipitated complex was washed five times with IP buffer and eluted with lithium dodecyl sulphate sample buffer. Proteins were resolved by 10% precast NuPAGE Bis-Tris gels (Invitrogen, Carlsbad, CA) and then electrotransferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA) and probed with mouse monoclonal anti-NHE1 antibody (Chemicon), anti-Src homology domain 2-containing protein tyrosine phosphatase (SHP-2) antibody (BD Biosciences, Pharmingen, San Diego, CA), anti-HSP70 antibody (Hypromatrix, Worcester, MA), or anti-Bin antibody (Stressgen Bioreagents, Victoria, BC, Canada). After incubation with the proper HRP-conjugated secondary antibody, proteins were visualized with the ECL chemiluminescence system (Amersham Biosciences). Three individual experiments using three different samples were performed for each condition.
In Vitro Binding Assay
Plasmid construction. Wild-type SHP-2 was cloned into pcDNA6-V5-HisA vector (Invitrogen). Mouse NHE1 COOH terminus (amino acid residues 504–821) was cloned into Flag-tagged pCMV-3Tag-6 vector (Promega, Madison, WI). The plasmids were sequenced after the cloning to confirm insertion and sequence.
In vitro transcription and translation. SHP-2 and NHE1 COOH terminus were in vitro transcribed and translated with the TNT coupled reticulocyte lysate system (Promega). Briefly, 50 µl of in vitro transcription/translation mixture {25 µl of TNT rabbit reticulocyte lysate, 2 µl of reaction buffer, 1 µl of TNT RNA polymerase, 1 µl of amino acid mixture minus methionine 1 mM, 2 µl of [35S]methionine, 1 µl of RNasin ribonuclease inhibitor, 1 µg of either SHP-2 or NHE1 COOH terminus (amino acid residues 504–821) plasmid, and nuclease-free water to final volume of 50 µl} was incubated at 30°C for 120 min. In vitro-translated 35S-labeled SHP-2 and NHE1 COOH terminus were resolved by SDS-PAGE and examined by autoradiography.
In vitro binding assay. Seven microliters of 35S-labeled SHP-2 was mixed with 70 µl of Flag-tagged NHE1 COOH terminus plus 10 µg of anti-Flag monoclonal antibody (Stratagene, La Jolla, CA) in the binding buffer (20 mM HEPES/Tris, pH 7.4, 150 mM NaCl, and 1 mM EDTA) and incubated at 4°C overnight with shaking. Subsequently, 100 µl of 50% protein A-Sepharose CL-4B beads (Amersham Biosciences) was added and incubated at 4°C for 2 h. SHP-2-NHE1 mixture without anti-Flag antibody was also examined to monitor nonspecific binding. The supernatant was collected separately as nonbinding controls for immunoprecipitation. The beads were rinsed with the binding buffer five times for 5 min each. The bound samples were resolved by SDS-PAGE and visualized by autoradiography.
Construction of BaF3:PTPN11 Stable Cell Lines
The wild-type human PTPN11 cDNA sequence (gene encoding SHP-2) (20) was cloned into pcDNA6-V5-HisA (Invitrogen). Constructs were then transfected into the interleukin (IL)-3-dependent murine myeloid progenitor cell line BaF3 to produce stable cell lines with constitutive PTPN11 expression. For each transfection, 2 x 107 BaF3 cells were washed once in additive-free RPMI 1640 (Invitrogen) and resuspended in 400 µl of additive-free RPMI 1640 in a 4-mm gap electrocuvette. Twenty micrograms of DNA was added, and the cells were incubated for 10 min. Cells were then electroporated at 72 
, 220 V, and 2,800 µF in a BTX Electro Cell Manipulator 600 (Harvard Apparatus, Holliston, MA) and recovered at room temperature for 10 min. Cells were plated into 10 ml of RPMI 1640 with 10% fetal bovine serum (FBS) and 100 ng/l recombinant murine IL-3 (rmIL-3, BD Pharmingen) and grown for 24 h. Transfected cells were plated at 5 x 104 cells/ml in 1.3% methylcellulose containing 100 ng/l rmIL-3 and 10 µg/ml blasticidin (Invitrogen) as a selective agent. Multiple clonal lines were isolated for each construct, expanded, and screened for equivalent levels of SHP-2 proteins. The cells were maintained in RPMI 1640 medium (Mediatech, Herndon, VA) supplemented with 100 ng/l IL-3 (R&D Systems, Minneapolis, MN), 1:100 dilution of -mercaptoethanol (Sigma), 10% FBS, 50 µg/ml penicillin, 50 µg/ml streptomycin, and 10 µg/ml blasticidin (Invitrogen).
Expression of human SHP-2 transgene as well as endogenous mouse SHP-2 and mouse NHE1 in transgenic and control BaF3 cells were tested by reverse transcription-polymerase chain reaction (RT-PCR) as previously described (76). Briefly, total RNA was extracted with TRIzol reagent (GIBCO BRL, Grand Island, NY) according to the manufacturer's instructions. Two micrograms of total RNA was reverse transcribed to first-strand cDNA in a 20-µl reaction volume. Negative control reactions excluding reverse transcriptase were performed to demonstrate the lack of contamination by genomic DNA in the RNA samples. PCR reactions were conducted in a 50-µl mixture containing 1 µl of cDNA from RT. The specific primers were mouse SHP-2, 5'-GATGGTTTCACCCCAACATC-3' and 5'-GACGTGGGTCACTTTGGACT-3'; mouse NHE1, 5'-TGCCAGCTATGACTCTGTGG-3' and 5'-CTGCACAAAGACGGTGAAAA-3'; and human SHP-2, 5'-AGAGCCACCCTGGAGATTTT-3' and 5'-ATCCGCCAAAAGTCATTCAC-3'. The reaction was started at 94°C for 2 min and amplified for 35 cycles of 30 s at 94°C, 30 s at 58°C, and 1 min at 72°C, finally extended at 72°C for 10 min. The PCR products were separated on 2% agarose gel, visualized with ethidium bromide staining, and captured and analyzed by the Chemi Doc system (Bio-Rad, Hercules, CA). -Actin was used as an internal control to confirm equal loading of the samples. Three individual cell samples were tested by separate RT-PCR.
Tyrosine Phosphatase SHP-2 Activity Assay
SHP-2 activity was measured according to a method described by Fragale et al. (20). Briefly, the BaF3 cells were washed twice with ice-cold PBS and collected in ice-cold lysis buffer containing 25 mM HEPES pH 7.4, 150 mM NaCl, 2 mM EDTA, 0.5% Triton X-100, and 1:100 protease inhibitor cocktail (Sigma). Three hundred micrograms of proteins were immunoprecipitated with mouse anti-SHP-2 monoclonal antibody (BD Biosciences, Pharmingen) for 2 h at 4°C. Fifty microliters of protein A-Sepharose CL-4B beads (Amersham Biosciences) were then added and incubated for another 2 h at 4°C. Immunocomplexes were washed twice in lysis buffer and once in protein tyrosine phosphatase (PTP) buffer (20 mM HEPES pH 7.5, 1 mM EDTA, 5% glycerol, 1 mM DTT), resuspended in 50 µl of PTP buffer supplemented with 250 µM Src phosphopeptide (TSTEPQ-pY-QPGENL; Upstate Biotech, Waltham, MA), and incubated for 30 min at 37°C. The supernatants were transferred to 96-well plates with 100 µl of Malachite Green solution (Upstate Biotech) and incubated for 15 min at room temperature. Their 630-nm absorbances were measured as an index of phosphatase activity. Ratios of 630-nm absorbance between SHP-2 transgenic and control BaF3 cells were calculated and used as relative activity of transgenic cell line compared with the vector-transfected control. The 630-nm absorbance value of no-antibody sham immunoprecipitate was used as background for each assay. Three samples were assayed for each condition.
Intracellular pH Measurement with BCECF
Cells were placed on coverslips attached to the bottom of a thermostatically controlled holding chamber (Warner Instrument, Hamden, CT). Cells were incubated with 2 µM membrane-permeant ester 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-AM (Molecular Probes, Eugene, OR) for 20 min. The chamber was then secured on the stage of the microscope and connected to a perfusion system (ALA Scientific Instrument, Westbury, NY) that supplied different solutions as needed.
pHi was determined in individual cells with fluorescence microscopy and digital image processing. A 175-W xenon lamp and an ultrahigh-speed wavelength switcher (Lambda DG-4, Sutter Instrument, Novato, CA) provided alternate 440/490-nm fluorescence excitation. The emission from BCECF-loaded cells was detected at a wavelength of 535 nm with a F-Fluar x40/1.3 numerical aperture oil-immersion objective (Zeiss Axiovert 200M microscope, Zeiss, Yena, Germany) and an attached 12-bit charge-coupled device camera. A computer controlled the light source, wavelength switcher, microscope, and camera (Universal Imaging). Data were recorded and analyzed with MetaFluor imaging-processing software (Universal Imaging, Downington, PA). The 440- to 490-nm ratio was converted to pH values by constructing a calibration curve by the nigericin/high-K+ method (10, 50, 69). Briefly, cells were treated with a series of seven nigericin (5 µM)/high-K+ (150 mM) solutions of varying pH values. Since K+/H+ ionophore nigericin clamps the pHi to the same value as extracellular pH, the results give the relationship between the 440- to 490-nm fluorescence ratio and pHi. An NH4Cl (20 mM) prepulse technique was used to acidify the cell in the study of proton extrusion. HOE-642 (10 µM) was used to block NHE1 activity. The intrinsic intracellular buffering power was obtained by decreasing the external concentration of the weak base NH3 and measuring the corresponding pHi in a stepwise manner as previously described (6). The rate of proton flux was computed as the product of the rate of pHi recovery and the intrinsic buffering capacity.
Cell Proliferation Assay
The [3H]thymidine incorporation method was used to measure cell proliferation. BaF3 cells were seeded in 12-well plates at a density of 106 cells/ml. An aliquot of the cell mixture was saved for protein concentration determination. The cells were pretreated with either NHE1-specific blocker HOE-642 (10 µM in 0.1% DMSO; a gift from Dr. Hans-J. Lang, Aventis Pharmaceuticals, Frankfurt, Germany) or sham control (0.1% DMSO) for 3 h. [3H]thymidine (PerkinElmer Life and Analytical Sciences, Boston, MA) was then added at 1 µCi/ml per well and incubated for an additional period of 6 h. The cells were washed three times with ice-cold PBS. [3H]thymidine incorporation was measured by dissolving cell material in 0.1 M NaOH, and radioactivity was read in a liquid scintillation counter (Pharmacia LKB, Gaithersburg, MD). The cell density and treatment period were chosen within the linear range of the assay (data not shown). Data points represent the average of three experiments, and each experiment was carried out with three replicates.
Lactate Dehydrogenase Activity Assay
Cellular injury was evaluated by measuring lactate dehydrogenase (LDH) activity released in the medium with the CytoTox96 nonradioactive cytotoxicity assay kit (Promega) and quantitated with wavelength absorbance at 490 nm. Briefly, the BaF3 cells were treated under different conditions (21% O2 normoxia vs. 1% O2 hypoxia, with or without 10 µM NHE1 inhibitor HOE-642) in six-well plates. Three individual assays were performed, and samples were triplicated for each testing condition. At the end of each treatment, culture medium from each well was collected separately and stored at –80°C. To normalize to total amount of LDH, cultured cells in the corresponding wells were lysed with 0.1% Triton X-100 in PBS for 45 min in a 37°C, 5% CO2 incubator, the plates were then centrifuged at 250 g for 4 min, and the supernatant was harvested individually for intracellular LDH determination. To measure LDH activity, 50 µl of substrate mixture provided in the kit and 50 µl of either culture medium or cell lysate was added together into 96-well plates, incubated for 30 min at room temperature, and protected from light. The reaction was stopped by adding 50 µl of 1 M acetic acid. Absorbance (A) in samples was measured at 490 nm with the aid of the Ultramark Microplate Imaging System (Bio-Rad). LDH release (%) was determined with the ratio (A490 in medium)/(A490 in medium + A490 in cell lysate).
Statistical Analyses
All values are represented as means ± SE. Statistical significance was calculated by the two-tailed Student's t-test. Differences in means were considered significant if P < 0.05.
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RESULTS |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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Interactions between NHE1 and other molecules, such as CHP, 14-3-3, and ERM proteins, have been reported. These have been shown to regulate NHE1 function and localization (4, 44). In this study, we reasoned that NHE1 may, besides being an exchanger, act as a plasma membrane protein that affects intracellular signaling or be affected by other signaling molecules. As a first step to address this hypothesis, we decided to identify signaling molecules that might interact with NHE1 protein. We used a specific signal transduction AntibodyArray that contained 400 high-quality antibodies against well-studied signaling proteins and probed with membrane proteins from mouse brain. Among 400 signaling molecules tested, 10 proteins appeared to be highlighted by HRP-conjugated NHE1 antibody (Fig. 1), indicating possible physical association between each of these molecules and NHE1 protein. Some of these molecules are apoptosis related, such as HSP70, Fas/CD95/APO-1, cell death-inducing DFF45-like effector-B (CIDE-B), cellular apoptosis susceptibility (CAS), and death receptor-4 (DR4). Others are involved in cell growth and proliferation, such as SHP-2 and bone marrow X kinase (BMX) (Table 1).
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SHP-2 Binds Directly to NHE1 COOH Terminus
To investigate whether SHP-2 binds directly or indirectly to NHE1 protein, we performed an in vitro binding assay. In vitro-translated SHP-2 was radiolabeled with 35S and allowed to incubate with in vitro-translated NHE1 COOH terminus at 4°C for overnight. Since NHE1 COOH terminus constructs were Flag tagged, anti-Flag antibody-conjugated protein A beads were used to pull down NHE1 COOH terminus-associated protein complex. The results showed (Fig. 3) that SHP-2 was precipitated with NHE1 COOH terminus, indicating that SHP-2 interacts directly with NHE1 COOH terminus. The protein mixture of SHP-2 and NHE1 COOH terminus was also incubated with protein A beads without anti-Flag antibody as a control: there was no SHP-2 band, demonstrating the specificity of the binding.
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To study the functional implications of SHP-2-NHE1 interaction, a mouse BaF3 cell line was used to overexpress SHP-2. The BaF3 cell line is a murine bone marrow-derived pro-B cell line. BaF3 cells were transfected with either an expression plasmid encoding human SHP-2 (BaF3-2) or plasmid alone as a control (BaF3-1). The stable clonal cell line was established as described in EXPERIMENTAL PROCEDURES. As expected, the expression of human SHP-2 mRNA was detected in the BaF3-2 transfected cell line but not in the vector-transfected BaF3-1 cell line (Fig. 4A). In addition, positive baseline expression of endogenous mouse NHE1 and SHP-2 was observed in both BaF3-1 and BaF3-2 cells (Fig. 4A). To determine whether the expression of human SHP-2 in BaF3-2 resulted in increased SHP-2 activity, SHP-2 tyrosine phosphatase activity was also measured in the BaF3-1 and BaF3-2 cell lines. SHP-2 enzyme was first purified by immunoprecipitation, and the enzyme activity was then assayed by a PTP assay kit using Src tyrosine phosphopeptide as substrate. A significant increase (80%) of SHP-2 basal activity was found in BaF3-2 cells compared with BaF3-1 controls (Fig. 4B).
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To test the functional impact of SHP-2-NHE1 interaction on NHE1 activity as an acid-base exchanger, resting pHi and pH recovery from an acid load in BaF3-2 as well as control BaF3-1 cells were measured with BCECF. All experiments were done in the nominal absence of HCO3– in the bath solution. The resting pHi of control BaF3-1 cells (7.1 ± 0.1, n = 36) was remarkably lower than that of SHP-2-overexpressing BAF3-2 cells (7.3 ± 0.2, n = 42; P < 0.001). The rate of pHi recovery was also measured by NH4Cl prepulse technique (9). In control BaF3-1 cells, the withdrawal of NH4Cl acidified the cells to a pHi around 6.7 from which cells recovered in HEPES buffer at a certain rate (Fig. 5A, trace 1). In contrast, adding 10 µM HOE-642 to or omitting Na+ from HEPES buffer completely blocked this recovery (Fig. 5A, traces 2 and 3). The rate of proton flux was computed as the product of the rate of pHi recovery and the intrinsic buffering capacity (6) for both BaF3-1 and BAF3-2 cells. For example, at pH 6.7 the addition of 10 µM HOE-642 or the removal of extracellular Na+ decreased the rate of proton flux from 0.67 ± 0.18 mM/min (n = 12) to 0.05 ± 0.04 (n = 16, P < 0.05) or 0.13 ± 0.05 (n = 6, P < 0.05) mM/min, respectively. Similar results were obtained from BAF3-2 cells (Fig. 5B). For example, at pH 6.7 the addition of HOE-642 or the removal of extracellular Na+ decreased the rate of proton flux from 1.0 ± 0.27 mM/min (n = 12) to 0.03 ± 0.02 (n = 5, P < 0.05) or 0.02 ± 0.01 (n = 6, P < 0.05) mM/min, respectively. A major difference in proton flux between BaF3-1 and BAF3-2 cells was also observed (Fig. 5, C and D). For example, at pHi 6.8 the proton flux was 0.94 ± 0.16 mM/min (n = 5) for BaF3-2 cells, and this is significantly higher (almost double) than that of control BaF3-1 cells (0.52 ± 0.09 mM/min, n = 7; P < 0.05). This increase in NHE1 function could be related to either an increased protein expression in the cell membrane or an increased activity per se. Immunoblotting analysis of NHE1 protein level in the membrane showed no statistically significant difference between BaF3-2 and BaF3-1 cells (25% in average, P > 0.05; n = 3, data not shown).
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Since both NHE1 and SHP-2 proteins have been demonstrated to play a role in cell proliferation (13, 17, 18, 28, 30, 33, 42, 49, 51, 53, 54, 56, 61, 68, 74), we next examined the impact of the interaction between NHE1 and SHP-2 on cell growth. Control (BaF3-1) and human SHP-2 transfected (BaF3-2) cells were pretreated either with sham control (0.1% DMSO) or with NHE1 inhibitor HOE-642 (10 µM in 0.1% DMSO) for 3 h, followed by 1 µCi/ml [3H]thymidine for an additional 6 h. [3H]thymidine incorporation was used as an index of cell proliferation. Our results showed similar [3H]thymidine incorporation between BaF3-2 and control BaF3-1 cells in the absence of the NHE1 inhibitor (Fig. 6). However, we detected a 30–40% decrease in [3H]thymidine incorporation in the HOE-642-treated control BaF3-1 cells, suggesting that NHE1 activity is required for normal cell proliferation. Intriguingly, this HOE-642-induced suppression in [3H]thymidine incorporation was abolished in the SHP-2-overexpressing BaF3-2 cells (Fig. 6). These results indicate that there is a significant functional relationship between SHP-2 and NHE1 not only in relation to pHi regulation but also for cell growth.
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Since we cannot rule out the possibility that a reduction in proliferation in BaF3-1 cells by HOE-642 is due to an increase in cell death, we further assessed whether cell injury occurs, using medium LDH as an index. Our data revealed that NHE1 blockade did not lead to significant cell death compared with controls (without NHE1 inhibitor) in both BaF3-1 and BaF3-2 cells under normoxia (Fig. 7). However, SHP-2 overexpression led to 24% decrease (P < 0.001, compared with SHP-2 wild-type cells) in cell death caused by 1% hypoxia. Combined with NHE1 blocker HOE-642, medium LDH was almost completely normal (P > 0.05, relative to normoxia controls).
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DISCUSSION |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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Intracellular acidification and cell shrinkage characterize apoptotic cell death. As one of the essential acid extruders and volume regulators, NHE1 has been linked to the cascade of programmed cell death (PCD) (36, 45, 48, 52). Our present AntibodyArray results suggested that NHE1 protein might interact directly with apoptotic-related proteins, pointing to the possible additional involvement of a scaffolding role for NHE1 in control of PCD.
In the present study, the interactions between NHE1 and SHP-2, Bin 1, or HSP70 were reciprocally confirmed by coimmunoprecipitation. HSP70 has been reported to associate directly with NHE1 by Fliegel and colleagues (59), although the regulatory mechanisms were not investigated. Recently, both HSP70 and NHE1 have been implicated in PCD and tumorigenesis (12, 43, 59). Whether they work synergistically via the same pathway(s) is not clear at present. Bin1 (also called amphiphysin II), another NHE1 interactive protein, is structurally related to amphiphysin, a breast cancer-associated autoimmune antigen, and RVS167, a negative regulator of the yeast cell cycle. By virtue of its ability to interact with and inhibit c-Myc oncoprotein function, Bin1 is thought to be a tumor suppressor that controls cell cycle transit, cell proliferation, and transformation (57, 67, 80), processes that are also affected by NHE1 (15, 44, 52, 73). In addition, both Bin1 and NHE1 have been shown to be involved in a cell survival signaling cascade, namely, mitogen-activated extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK (15, 44, 52, 67, 73).
The intriguing finding in this work is the demonstration that not only are NHE1 and SHP-2 physically associated with each other but also their interaction is functionally significant. Mammalian SHP-2 (also known as SH-PTP2, PTP1D, PTP-2C, and Syp) is a cytosolic PTP. Like NHE1, SHP-2 is ubiquitously expressed and is activated by diverse growth factors, cytokines, insulin, and interferon (42, 49, 53, 61). Recent reports have highlighted the clinical relevance of SHP-2 to some diseases, such as Noonan syndrome (20, 34, 63–66), diabetes (3, 8), and neutropenia (Kostmann syndrome) (70), which further emphasizes the importance of SHP-2 and the interactions of SHP-2 with other proteins such as NHE1. SHP-2 protein possesses two Src homology 2 (SH2) domains at the NH2 terminus and one phosphatase domain at the COOH terminus (42, 49, 53, 61). Our current in vitro binding data have shown that SHP-2 is directly associated with NHE1 COOH terminus. Normally, SHP-2 interacts with other proteins via its SH2 domains, which bind to phosphorylated tyrosine residues. Although a potential tyrosine phosphorylated sequence (RIGSDPLAY, amino acid residues 704–712 of mouse NHE1 protein) is predicted by a PredictProtein (http://cubic.bioc.columbia.edu/predictprotein), there has been no laboratory proof of this phosphorylation so far. Therefore, whether SHP-2 associates with NHE1 via this site is still an open question. Furthermore, both NHE1 and SHP-2 proteins have been proven to interact with 14-3-3 or HSP70 protein separately (38, 78, 79). Our present results are the first to demonstrate that SHP-2 directly interacts with NHE1. The functional importance of the protein complexes NHE1/SHP-2/HSP70 or NHE1/SHP-2/14-3-3 will need further investigation.
To gain insight into the physiological significance of NHE1-SHP-2 interaction, functional assays, including pHi measurement, thymidine incorporation, and cell death, were performed in SHP-2-overexpressing and control BaF3 cells. Our pHi measurements were done in HEPES buffer, thus eliminating the effects of HCO3–-dependent transporters on pHi. Under such conditions, pHi regulation is mainly governed by NHEs and possibly, depending on cell type, a proton pump. By removing extracellular Na+, we further separate the contribution of NHEs and proton pumps on pHi regulation. Finally, we used the NHE1-specific inhibitor HOE-642 to differentiate NHE1 from other NHE isoforms. The results obtained on pHi showed that SHP-2-overexpressing cells have a higher steady-state pHi and a faster, NHE1-dependent recovery rate from acid load (in HEPES buffer), indicating that SHP-2 enhances NHE activity in both basal and activated states. Furthermore, the NHE1 protein level in the membrane did not show significant difference between BaF3-1 and BaF3-2 cells in the immunoblotting study, indicating that SHP-2 overexpression modulates NHE1 activity itself rather than NHE1 protein expression.
How does SHP-2, a PTP, affect the pHi regulatory activity of NHE1? Tyrosine phosphorylation has been demonstrated to modulate NHE1 activity in a number of cell types (1, 21–24, 41). Such activation of NHE1 is proposed to be mediated by Janus kinase 2 (Jak2)-dependent tyrosine phosphorylation of CaM and subsequent increased binding of CaM to NHE1. Jak2 can also recruit and activate SHP-2 (7, 29, 55). Therefore, SHP-2 may work via CaM, a known NHE1 regulator, to modify NHE1 activity. However, this possibility is very unlikely, since SHP-2, as a phosphatase, would dephosphorylate CaM, which in turn decreases the binding of CaM to NHE1 and lowers NHE1 activity. A more likely possibility is via 14-3-3 protein because 1) both SHP-2 and NHE1 are proven to physically associate with 14-3-3 (38, 79); 2) the phosphorylation status of 14-3-3 protein may interfere with its interaction with other proteins (19); and 3) the binding of 14-3-3 to NHE1 sustains NHE1 activity (38, 62). The third possibility is that SHP-2 directly associates with NHE1, leading to a conformational change and an increase in H+ sensitivity of the exchanger.
In the absence of the NHE1 blocker HOE-642, we observed no significant difference in cell proliferation between SHP-2-overexpressing and control BaF3 cells. This might indicate that overexpression of wild-type SHP-2, such as in our cells, is not sufficient to promote BaF3 cell growth, similar to previous published results in COS-7 cells (20). In the presence of an NHE1 blocker, however, BaF3-1 cells showed 30–40% reduction in cell proliferation. These data suggest that NHE1 activity is a requisite for cell growth and proliferation, a notion that is consistent with previously published results (13, 17, 18, 28, 30, 33, 51, 54, 56, 68, 74). Moreover, our cell injury data indicate that there was no significant difference in cell injury between BaF3-1 and BaF3-2 cells, with or without HOE-642, under normoxic conditions. Thus they excluded the possibility that reduced [3H]thymidine incorporation in BaF3-1 cells in the presence of HOE-642 is due to an increase in cell death rather than in growth arrest. Interestingly, in the SHP-2-overexpressing cells, this inhibition of proliferation with HOE-642 did not occur. Possible explanations for this lack of inhibition would include the following: 1) NHE1 is not the only protein affected by SHP-2 that alters cell proliferation and 2) NHE1/SHP-2 protein complex may work via a pHi-independent mechanism to regulate cell proliferation, i.e., NHE1 is not an acid-base transporter in this instance. Instead, NHE1 acts as a scaffold protein independently of its activity, associates with SHP-2, and relays a signal that is important to cell proliferation.
BaF3-2 cells were also interestingly unique when exposed to stress. Indeed, BaF3-2 cells, unlike BaF3-1 cells, were protected from hypoxic injury modestly (24% reduction relative to BaF3-1) and dramatically when NHE1 was inhibited (57% less injury compared with BaF3-1 and only 23% more injury than normoxia controls). This would suggest that 1) there is functional interaction between NHE1 and SHP-2 that is elicited during stress; 2) this interaction is highly dependent on NHE function, as HOE-642 reduces LDH release back down to almost normal; and 3) NHE1 is possibly deleterious to cell survival under hypoxic conditions.
In summary, we believe that we have made several important observations in this work: 1) potential NHE1 interactive partners are identified, such as apoptotic proteins Fas, DR4, HSP70, CAS, and CIDE-B, growth-related proteins SHP-2 and BMX, as well as amphiphysin I and II (Bin1), which play various roles such as endocytosis, actin cytoskeletal organization, and tumor suppression; 2) the associations (e.g., SHP-2 and HSP70) are widely distributed as we demonstrated in mouse brain, heart, kidney, and primary neuronal and astrocytic cultures, while the association with Bin1 is only restricted to central nervous system neurons; and 3) SHP-2 interacts with NHE1 directly to modulate NHE1 functions, especially pHi regulation, cell proliferation, and cell injury under hypoxia.
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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hypoxia vs. normoxia; #BaF3-2 vs. BaF3-1 under hypoxia; *with HOE-642 vs. without HOE-642.