735

Original Paper

Cell Physiol Biochem 2007;20:735-750 Accepted: May 31, 2007Cellular PhysiologyCellular PhysiologyCellular PhysiologyCellular PhysiologyCellular Physiologyand Biochemistrand Biochemistrand Biochemistrand Biochemistrand Biochemistryyyyy

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The Na+/H+ Exchanger, NHE1, DifferentiallyRegulates Mitogen-Activated Protein KinaseSubfamilies after Osmotic Shrinkage in EhrlichLettre Ascites CellsStine Falsig Pedersen, Barbara Vasek Darborg, Maria Rasmussen,Jesper Nylandsted1 and Else Kay Hoffmann

Dept. of Molecular Biology, University of Copenhagen, 1Apoptosis Department, Danish Cancer Society,Copenhagen

Stine F. PedersenDept. of Molecular BiologyUniversity of Copenhagen, 13, UniversitetsparkenDK-2100 Copenhagen (Denmark)Tel. +45 35321546, Fax +45 35321567, E-Mail sfpedersen@aki.ku.dk

Key WordsERK • P38 MAPK • JNK • MEK1/2 • Cell volumeregulation • Osmotic stress

AbstractOsmotic stress modulates mitogen activated proteinkinase (MAPK) activities, leading to altered genetranscription and cell death/survival balance, however,the mechanisms involved are incompletely elucidated.Here, we show, using a combination of biochemicaland molecular biology approaches, that three MAPKsexhibit unique interrelationships with the Na+/H+

exchanger, NHE1, after osmotic cell shrinkage:Extracellular Signal Regulated Kinase (ERK1/2) isinhibited in an NHE1-dependent, pHi-independentmanner, c-Jun N-terminal kinase (JNK1/2) isstimulated, in part through NHE1-mediated intracellularalkalinization, and p38 MAPK is activated in an NHE1-independent manner, and contributes to NHE1activation and ERK inhibition. Shrinkage-inducedERK1/2 inhibition was attenuated in Ehrlich LettreAscites cells by NHE1 inhibitors (EIPA, cariporide) orremoval of extracellular Na+, and mimicked by human(h) NHE1 expression in cells lacking endogenousNHE1 activity. The effect of NHE1 on ERK1/2 was pHi-

independent and upstream of MEK1/2. Shrinkage-activation of JNK1/2 was attenuated by EIPA,augmented by hNHE1 expression, and abolished inthe presence of HCO3

-. Basal JNK activity wasaugmented at alkaline pHi. Shrinkage-activation of p38MAPK was NHE1-independent, and p38 MAPKinhibition (SB203580) attenuated NHE1 activation andERK1/2 inhibition. Long-term shrinkage elicitedcaspase-3 activation and a loss of cell viability, whichwas augmented by ERK1/2 or JNK1/2 inhibition, andattenuated by p38 MAPK inhibition.

Introduction

When exposed to hyperosmotic stress, animal cellsrapidly lose water and shrink. This induces a cell type-specific constellation of regulatory and/or adaptiveresponses, including activation of volume-regulatoryosmolyte transport proteins, cytoskeletal reorganization,and changes in the protein kinase and –phosphataseactivities [see 1-3]. Persistent osmotic shrinkage isassociated with induction of programmed cell death

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(PCD) pathways and caspase/cathepsin dependent celldeath, especially in cells incapable of volume regulation[4, 5]. A number of important physiological andpathophysiological conditions are associated with tissuedamage due to shrinkage-induced cell death [1], yet themechanisms leading from osmotic shrinkage to cell deathare still incompletely understood.

A rapid and central response to osmotic shrinkageis activation of the ubiquitous plasma membrane Na+/H+

exchanger, NHE1, a major regulator of pHi and cellvolume in essentially all cell types studied [3, 6].Activation of NHE1, resulting in net uptake of ions andosmotically obliged water, plays a major role in theprocess of Regulatory Volume Increase (RVI) by whichcell volume is partially or fully restored after osmoticshrinkage [3]. NHE1 interacts directly with multiplebinding partners. Indeed, it has been suggested that NHE1acts as a scaffold for cellular signalling events, and thatsome physiological effects of NHE1 may involve suchinteractions, rather than simply reflecting NHE1-mediated ion fluxes [6, 7]. Inhibition of NHE1 has beenfound to accelerate or induce PCD in a number of celltypes after various stimuli, including osmotic shrinkage,however, the mechanisms involved are incompletelyelucidated [4, 6, 8].

Another well-established consequence of osmoticcell shrinkage is altered activity of at least three mitogenactivated protein kinase (MAPK) subfamilies,extracellular signal regulated kinase (ERK)1/2, p38MAPK, and c-Jun N-terminal kinase (JNK) [3, 9].Notably, MAPK activation is observed at clinicallyrelevant levels of osmotic shrinkage, such as thoseoccurring e.g. after mannitol treatment following braininjury or ischemia [10]. MAPK activity profoundlyinfluences cell viability and proliferation, generally,although not ubiquitously, such that p38 MAPK inhibitscell cycle progression and promotes programmed celldeath (PCD), while the converse is true for ERK [11].The effects of JNK are more variable, apparently at leastin part reflecting differential effects of JNK1 and JNK2[12; see 9]. Changes in MAPK activity have been shownto play important roles in adaptive responses to osmoticshrinkage, such as changes in the expression of genesinvolved in the control of volume-regulatory iontransport, and a few studies have also shown MAPKs tomodulate shrinkage-induced PCD [5, 9, 13].

Thus, MAPK activation by hypertonic cell shrinkagehas substantial physiological and pathophysiologicalimportance. Nonetheless, although progress has beenmade in recent years [9,14,15], the mechanisms

regulating MAPK activity after osmotic stress in highereukaryotes are poorly understood. MAPK activation iscontrolled by a three-component protein phosphorylationcascade in which each MAPK subfamily is activated bydual Tyr/Thr phosphorylation by specific MAPK kinases(MAPKKs), which are again activated by MAPKKkinases (MAPKKKs) [16]. Although significant crosstalkexists, ERK1/2, p38 MAPK, and JNK, are generallyactivated by the MAPK/ERK kinases (a.k.a. MEKs orMKKs) MEK1/2, MKK3/6, and MKK4/7, respectively[16]. At least 20 different MAPKKKs have beenidentified, each generally regulating multiple MAPKKs,and themselves regulated by a plethora of mechanisms,including small GTP-binding (G-) proteins, Ste20-relatedprotein kinases, scaffolding proteins and endogenousinhibitors [15, 17, 18]. Crosstalk directly at the MAPKlevel has also been demonstrated, such as the apparentlydirect p38 MAPK-mediated inhibition of ERK1/2 [19].

Importantly, the relation between NHE1 andMAPKs after osmotic cell shrinkage is incompletely char-acterized. MAPKs have been implicated in shrinkage-induced NHE1 activation in some cell types [20, 21],yet not in others [22]. Interestingly, recent studies haveassigned a role for NHE1 upstream of activation ofERK1/2 after exposure to angiotensin II or 5-HT [23],as well as after cardiomyocyte stretch [24]. Moreover,NHE1 was found to be upstream of p38 MAPK activa-tion following heart failure [25] and chemical ischemia[26], and upstream of both ERK1/2 and p38 MAPK in aphenylephrine model of cardiac hypertrophy [27; for areview, see 28]. However, the possible role of NHE1upstream of MAPKs after osmotic shrinkage has so farbeen unknown. Therefore, we employed a systematicapproach to delineate the relationship between the os-motic activation of NHE1 and the activity of the threemajor MAPK subfamilies. We measured in the same celltype the osmotic responsiveness of each MAPK, and theirpotential regulation by NHE1 as well as their potentialimpact on the hyperosmotic activation of NHE1, togetherwith the impact of NHE1 and the MAPKs on cell viabil-ity after osmotic shrinkage. We show here that ERK1/2is inhibited by shrinkage in an NHE1 dependent, pHi-independent manner, and JNK1/2 is stimulated by shrink-age at least in part through NHE1-mediated intracellularalkalinization. p38 MAPK is activated by shrinkage inNHE1-independent manner, and instead the volume-de-pendent activation of this kinase contributes to bothstimulation of NHE1 and inhibition of ERK. The loss ofcell viability induced by long-term osmotic shrinkage isaugmented by inhibition of ERK1/2 or JNK1/2, and at-

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tenuated by inhibition of p38 MAPK. Part of these re-sults have previously been presented in abstract form[29].

Materials and Methods

Reagents and solutionsUnless otherwise stated, reagents were analytical grade and

obtained from Sigma (St. Louis, MO, USA) or Mallinckrodt BakerB.V. (Deventer, NL). PD98059, SB203580, and SP600125 werefrom Calbiochem (Bad Soden, Germany) and were dissolved at18 mM, 10 mM, and 10 mM, respectively, in desiccated DMSO.4 - i s o p r o p y l - 3 - e t h y l s u l p h o n y l b e n z o y l - g u a n i d i n emethanesulphonate (Cariporide, HOE642) was a kind gift fromDr. Juergen Puenter, Sanofi-Aventis, and was dissolved at 5 mMin ddH2O. 5´-(N-ethyl-N-isopropyl)amiloride (EIPA) was fromMolecular Probes (Leiden, the Netherlands), and was dissolvedat 2.5 mM in ddH2O. 2´,7´-bis-(2-carboxyethyl)-5,6-carboxyfluorescein, tetraacetoxymethylester (BCECF-AM), alsofrom Molecular Probes, was dissolved at 1.2 mM in desiccatedDMSO. Latrunculin B was from Calbiochem (Darmstadt,Germany), and was dissolved at 10 mM in DMSO. All thesereagents were stored at –20°C. Stock solutions ofparaformaldehyde (20% w/v in ddH2O) were prepared freshregularly and stored at 4°C. Antibodies against p38 MAPK,phospho-38 MAPK (detecting p38 MAPK phosphorylated atThr180 and Tyr182), ERK1/2, phospho-ERK1/2 (detecting ERK1/2phosphorylated at Thr202 and Tyr204), phospho-JNK (detectingJNK1 and -2 phosphorylated at Thr183 and Tyr185) phospho-MEK1/2 (detecting MEK1/2 phosphorylated at Ser217/221), andphospho-MKK3/6 (detecting MKK3/6 phosphorylated at Ser189/

207) were from Cell Signaling (Beverly, MA), as was the antibodyagainst caspase-3 (detects full length (35 kDa) and large fragment(17/19 kDa) resulting from cleavage at Asp175).

Cell culture and saline solutionsEhrlich Lettre Ascites (ELA) cells, an adherent sub-line of

Ehrlich Ascites Tumor (EAT) cells, were obtained from AmericanType Culture Collection (ATCC, Manassas, VA, USA) andmaintained in RPMI-1640 (Sigma, St. Louis, MO, USA)supplemented with 10% foetal calf serum and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA), in a humifiedincubator at 37°C and 5% CO2. It may be noted that consistentwith their origin as a mammary tumor, ELA cells exhibit aconsiderable basal level of ERK activity. AP1 cells (a CHO cellderivative with no endogenous NHE activity, [30] and S5 cells(AP1 cells stably expressing the human (h)NHE1, a kind gift fromProf. P.M. Cala, University of California, Davis) were maintainedin α-modified Eagle’s medium with 1% L-glutamine and, for S5cells also 400 µg/ml G418-sulphate (Invitrogen). Cells werepassaged every 3-4 days and only passages 5 to 30 were used forexperiments.

The standard saline solution used for ELA cells had anosmolarity of 310 mOsm, and contained (in mM) 143 NaCl,5 KCl, 1 MgSO4, 1 Na2HPO4, 1 CaCl2, 3.3 MOPS, 3.3. TES, and5 HEPES, pH 7.4 (or, where indicated, pH 6.8 or 8.0). The standard

hypertonic (600 mOsm) saline was prepared by doubling theconcentrations of all components except MOPS, TES and HEPEScompared to the standard medium. In HCO3

- media, 25 mM ofNaCl was replaced by 25 mM NaHCO3. In KCl medium, KClwas substituted for NaCl in equimolar amounts. The standardsaline solution used for AP1 and S5 cells had an osmolarity of310 mOsm, and contained (in mM) 130 NaCl, 3 KCl, 1 MgCl2,0.5 CaCl2, 20 HEPES, 10 glucose, pH 7.4. The standard hypertonic(600 mOsm) saline solution for AP1 and S5 cells was preparedby increasing the concentration of NaCl to 275 mM. Unlessotherwise indicated, experiments were carried out at 37°C. It maybe noted that a severe hypertonic challenge (600 mOsm salinecompared to 300 mOsm under isotonic conditions) was chosenas the standard stimulus, since substantial changes in extra- orintracellular osmolarity occur in many mammalian cells both undersome physiological conditions (e.g. in the kidney medulla,gastrointestinal system, chondrocytes, and in connection withepithelial secretion or programmed cell death), and underpathophysiological/clinical conditions (e.g. cerebral and cardiacischemia, diabetes mellitus, dehydration, small volume hypertonicresuscitation); for a review, see [3].

RNA isolation and PCR analysisTotal RNA isolation was performed using standard

procedures. RNA was reverse transcribed using Superscript IIreverse transferase (Invitrogen, Carlsbad, CA) and randomprimers, and amplified using NHE-isoform specific primers. ThePCR protocol was: 95°C 2 min, 30 x (95°C 30 s, 40°C 30 s, 72°C2 min), 72°C 10 min. Samples were analyzed on ethidiumbromide-containing 1.8% agarose gels. Primer pairs used were:NHE1 fw 5’-ctg tgg tca tta tgg cc-3’, NHE1 rv 5’ tgg gtt cat aggcca gt-3’; NHE2 fw 5’-cag caa gct gtc agt ga-3’, NHE2 rv 5’-tcggga ggt tga agt gg-3’; NHE3 fw 5’-ttc gac cac atc ctc tc-3’, NHE3rv: 5’-tcc tgg tcc tgt ttc tc-3’; NHE4 fw 5’-act aat cgc ctt cac ta-3’, NHE4 rv 5’-gat gga gaa ggg gaa ag-3’. When RNA wasreplaced by ddH2O in the reverse transcription reaction, none ofthese primers produced visible bands in the PCR reactions.

Estimation of intracellular pH and cell volume changesNHE1 activity was measured as EIPA-sensitive intracellu-

lar alkalinisation, a standard procedure for assessing NHE1 ac-tivity. ELA cells were grown to confluence on HCl- and ethanol-cleaned glass coverslips and loaded with BCECF-AM (1.2 µM)in standard medium as previously described [27, 31]. The cellswere placed at a 50° angle relative to the excitation light, in thestirred, thermostatted cuvette of a PTI Ratiomaster spectropho-tometer. BCECF emission was detected at 525 nm after excita-tion at 445 nm and 495 nm. Fluorescence of unloaded cells wasdetermined for each experimental solution and subtracted priorto calculation of the 445 nm/495 nm ratio. Calibration was per-formed by 7-point nigericin/high K+ calibration, essentially as in[32]. The rate of change in pHi after hypertonic challenge wascalculated as the slope of the initial, linear part of the curve (0 to2-3 min after hypertonic challenge). This procedure was foundjustified because the initial pHi was very similar in all experi-ments.

For estimation of cell volume changes, large angle light scat-tering was determined simultaneously to pHi in the BCECF-loaded

NHE1 Regulates MAPKs After Osmotic Cell Shrinkage Cell Physiol Biochem 2007;20:735-750

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cells, by exciting the cells at 589 nm and measuring emission at595 nm. In the range of osmolarities used, an inverse linear cor-relation between large angle light scattering and relative cell vol-ume was confirmed by rapid exposure of the cells to saline solu-tions of 5 different osmolarities (not shown). To directly reflectrelative cell volume, light scattering values are presented as theinverse of the intensity, normalized to the value obtained underisotonic conditions, i.e. I0/I.

SDS-PAGE and Western blottingCells grown to confluence in 10cm Petri dishes were washed

with ice-cold phosphate-buffered saline (PBS, 136.89 mM NaCl,2.68 mM KCl, 8.10 mM Na2HPO4, 1.7 mM KH2HPO4), lysed in200 µl boiling lysis buffer (1% SDS, 10 mM Tris HCl, pH 7.5),and transferred to Eppendorf tubes followed by homogenizationthrough 27 G and 18 G syringes, and centrifugation (16.000 g) toclear debris. A 5 µl aliquot was removed for protein determina-tion using (Bio Rad). For each condition, identical amounts ofprotein were diluted 1:1 in sample buffer (10% glycerol, 141 mMTris base, 106 mM Tris HCl, 69 mM LDS, 0.51 mM EDTA,0.0075% Phenol Red), boiled for 5 min, resolved by SDS-PAGEon 10% NuPage bis-tris gels, and electrotransferred to nitrocellu-lose membranes. Membranes were stained with Ponceau Red toconfirm equal loading, and were discarded if loading differenceswere noted. The membranes were blocked for 1h at room tem-perature in blocking buffer (150 mM NaCl, 13 mM Tris, 5% non-fat dry milk), and incubated with primary antibody diluted inblocking buffer, overnight at 4°C. The membranes were washedextensively in TBS-T (150 mM NaCl, 13 mM Tris, 0.02% TritonX-100), and incubated with alkaline phosphatase-conjugated sec-ondary antibody (1:600), followed by wash and detection by BCIP/NBT. Membranes were scanned and band intensity was estimatedusing UN-SCAN-IT software. Within the maximum 1h time framemonitored, there were no detectable changes in the total proteinlevel of any of the MAPKs studied, and identical results wereobtained whether data were normalized to total cellular proteinlevel or to the total level of the relevant MAPK.

Immunocytochemistry and confocal laser scanningmicroscopyImmunocytochemistry and confocal laser scanning

microscopy (CLSM) of intact ELA cells was carried out essentiallyas previously described [31]. Cells were grown on round glasscoverslips, stimulated as indicated, fixed in 2% paraformaldehyde(15 min room temperature, 30 min on ice), washed in TBS (inmM: 150 NaCl, 10 Tris-HCl, 1 MgCl2, 1 EGTA, pH 7.3),permeabilized for 10min (0.2% Triton X-100 in TBS), blockedfor 30 min (5% BSA in TBS), incubated with primary antibodiesagainst ERK, phospho-ERK, p38 MAPK, or phospho-p38 MAPK(1:100 in TBS, overnight, 4°C), washed in TBS, and incubatedwith FITC-conjugated goat anti-mouse antibody (1:600 in TBS,1 h), washed in TBS, and mounted with N-propyl-galleate 2% w/v in PBS/glycerine. MAPK localization was visualized using a40 X/1.25 NA plan apochromat objective and the 488 nm laserline of a Leica DM IRB/E microscope with a Leica TSC NTconfocal laser scanning unit (Leica Lasertechnik GmbH,Heidelberg, Germany). Optical slice thickness was 1 µm, andpinhole size was 1 airy disc. Images shown are frame averaged

and presented in RGB pseudocolor. Essentially no labeling wasdetectable in the absence of primary antibody (not shown).

siRNA mediated knockdown of ezrinKnockdown of ezrin was carried out and verified as

previously described [31], and cells were used for experiments72h after transfection with siRNA. The desalted, annealed, 21-bpsiRNA duplexes with 2-nt overhangs were obtained fromDharmacon RNA Technologies (Lafayette, CO, USA), and thetarget sequence was 5’-caa gaa ggc acc uga cuu u-3’,corresponding to position 872-890 in mouse ezrin. A GC-matchedscrambled 21 bp oligomer was used as a control.

MTT assay for cell viabilityCell viability was evaluated from the fraction of functional

mitochondria using 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT). Cells were seeded in 96 well platesat 16.000 cells per well 24h prior to experiments. At the day ofthe experiment, the cells were incubated for 1h in 200 µl isotonicmedium in the absence or presence of inhibitors as indicated,followed by exposure to 200 µl of the relevant experimentalmedium with or without inhibitors as indicated. The hypertonicmedium was prepared by addition of NaCl to the RPMI mediumfrom a sterile 3M stock solution to a final concentration of 150mM. This resulted in a total osmolarity of approximately 600mOsm compared to 300 mOsm in the isotonic RPMI medium. Atthe end of the stimulation period (3, 6, 12, or 24 h), 100 µl of themedium was removed, and 25 µl MTT was added. After 2 h, thecells were lysed in10% SDS + 0.01 M HCl, and incubatedovernight at room temperature, protected from light andevaporation. Absorption was measured at 570 nm using a FluostarOptima plate reader (Ramcon A/S, Denmark). Viability wascalculated as the absorbance (always mean of 6 identically treatedwells) relative to that under isotonic control conditions, aftersubtraction of the background signal measured from wellscontaining the relevant media but no cells.

Statistical analysisSignificance was evaluated using Student’s t-test, with

p < 0.05 taken to indicate a statistically significant difference.Data shown are mean ± standard error of the mean (S.E.M.) orare representative of at least 3 independent experiments.

Results

Osmotic shrinkage of ELA cells elicits NHE1activation and partial RVIWhen ELA cells were osmotically shrunken by

exposure to hypertonic saline (600 mOsm compared to310 mOsm under isotonic conditions), an intracellularalkalinization of about 0.06 pH units/min was observedafter a lag time of 1-2 min (Fig. 1A-B). Upon hypertonicexposure, the cells rapidly shrunk, followed by a gradualregulatory volume increase (RVI), which was incompletewithin the time frame monitored (Fig. 1 C-D). In the

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60 min, ERK1 phosphorylation had essentially recoveredto the isotonic level. Notably, EIPA (5 µM) fully blockedthe shrinkage-induced inhibition of ERK1/2phosphorylation, even elevating ERK phosphorylationlevels above those seen in isotonic controls. Visualizationof total and phosphorylated ERK1/2 in intact ELA cellsconfirmed this pattern, and demonstrated thatphosphorylated ERK1/2 was located predominantly inthe nucleus under isotonic conditions, and that cellshrinkage was associated with a loss of nuclear phospho-ERK1/2 labeling (n=4, data not shown). To establishwhether these effects of EIPA reflected NHE1 inhibition,the experiment was repeated in the presence of thestructurally different NHE1 inhibitor cariporide (Fig. 2B)and in the absence of extracellular Na+, i.e. reversing thegradient for Na+/H+ exchange via NHE1 (Fig. 2C). Asseen, similar to EIPA, both treatments strongly attenuatedthe shrinkage-induced ERK1 dephosphorylation.

To unequivocally establish the role of NHE1, theeffect of hypertonic exposure on ERK activity wasevaluated in wild type AP1 cells, which are devoid ofNHE activity [30], compared to AP1 cells stablyexpressing human (h) NHE1 (Fig. 3). In AP1 cells,

Fig. 1. Osmotic cell shrinkage elicits activation of NHE1 andpartial RVI in ELA cells: A-D: Intracellular pH and cell volumewere monitored simultaneously in ELA cells using BCECF andlarge angle light scattering. The arrow indicates the time ofexposure to hypertonic saline (600 mOsm compared to 300 mOsmunder isotonic conditions). Panel A and C show pHi and relativecell volume (shown as the inverse of the relative light scatteringsignal, I0/I; see Experimental Procedures) respectively, over timein the absence (open symbols) or presence (black symbols) ofEIPA (5 µM) to inhibit NHE1. The blue lines indic summarizedin panel B and D, respectively, as the initial change in pHi pr minafter hypertonic challenge (calculated as the slope of the initiallinear part (0 to 2-3 min) of the pHi over time curve) (B) or theinitial change in I0/I (D). Data are representative (A,C) or mean± S.E.M. (B,D) of 3 experiments. E: Total RNA from ELA cellswas isolated and reverse transcribed (random primers), andamplified using NHE-isoform specific primers (95°C 2 min, 30 x(95°C 30 s, 40°C 30 s, 72°C 2 min), 72°C 10 min). Control rea-ctions containing no primers were included for each sample. Theprimer pairs used were: NHE1 fw 5’-ctgt ggt cat tat ggc c-3’,NHE1 rv 5’ tgg gtt cat agg cca gt-3’; NHE2 fw 5’-cag caa gctg tcagtg a-3’, NHE2 rv 5’-tcg gag gtt gaa gtg g-3’; NHE3 fw 5’-ttc gaccac atc ctc tc-3’, NHE3 rv: 5’-tcc tgg tcc tgt ttc tc-3’; NHE4 fw5’-act aat cgc ctt cac ta-3’, NHE4 rv 5’-gat gga gaa ggg gaa ag-3’.PCR products were separated on 1.8% agarose gels using Kb+DNA markers. The gel shown is representative of 3 independentexperiments. *) indicates that the value is significantly differentfrom isotonic control.

NHE1 Regulates MAPKs After Osmotic Cell Shrinkage

presence of the NHE1 inhibitor 5‘-(N-ethyl-N-isopropyl)amiloride (EIPA, 5 µM), both the intracellularalkalinization and the RVI were completely blocked. Thissuggests that in contrast to the related suspension cellline, Ehrlich Ascites Tumor (EAT) cells, in whichNKCC1 contributes more than NHE1 to RVI [33], NHE1is the main ion transport protein mediating acute RVI inELA cells. PCR analysis using isoform-specific primersindicated that ELA cells express NHE1, while NHE2,NHE3, and NHE4 could not be detected (Fig. 1E). Thus,in ELA cells, NHE1 appears to be activated by hypertoniccell shrinkage, resulting in intracellular alkalinization andat least a partial RVI.

Osmotic shrinkage inhibits ERK1/2 in a mannerdependent on NHE1 activityWe proceeded to test the hypothesis that NHE1

modulates MAPK activity after osmotic cell shrinkage.ERK activity was strongly attenuated after hypertonicexposure (600 mOsm, increased extracellular ionicstrength) (Fig. 2A). The shrinkage-induced inactivationof ERK1 was rapid and transient: maximal inhibitionwas seen 5 min after hypertonic exposure, and at time

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osmotic shrinkage induced a minor decrease in ERK1/2activity, followed by a substantial increase. Notably, theexpression of hNHE1 strongly attenuated shrinkage-induced ERK1 phosphorylation (normalized to the totalERK1 level quantified from parallel blots), and also

Pedersen/Darborg/Rasmussen/Nylandsted/Hoffmann

Fig. 3. Effect of osmotic shrinkage and NHE1 expression onERK1/2 activity in AP1 cells:AP1 (devoid of endogenous NHEactivity) and S5 cells (AP1s stably expressing human NHE1) wereexposed to high salt hypertonic saline (600 mOsm) for the timeindicated, and proteins separated by SDS-PAGE as described inExperimental Procedures. Total ERK1/2 levels and ERK1/2activity were assessed by Western blotting and quantified bydensitometric scanning. Only ERK1 was quantified, however, anapparently similar pattern was seen for ERK2. Data are mean± S.E.M. of 3 independent experiments. *) indicates that the valueis significantly different from isotonic control. #) indicates thatthe value is significantly different from the correspondinghypertonic control.

tended to decrease basal ERK phosphorylation. Thus,taken together, these data strongly indicate that NHE1plays a major role in shrinkage-induced ERK1/2inhibition, and may also attenuate basal ERK1/2 activity.

Fig. 2. Effect of osmotic shrinkage and NHE1 activation onERK1/2 activity in ELA cells: ELA cells were exposed to highsalt hypertonic saline (600 mOsm) for the time indicated, in theabsence or presence of inhibitors or Na+ as indicated. SDS-PAGEwas carried out as described in Experimental Procedures usingequal amounts of protein in each well. ERK1/2 activity wasassessed by Western blotting using phospho-ERK1/2 antibodies,and quantified by densitometric scanning. Only ERK1phosphorylation levels were quantified, however, an apparentlysimilar pattern was seen for ERK2. A: ERK1/2 phosphorylationin the absence (open bars) or presence (black bars) of 5 µM EIPA.B. ERK1/2 phosphorylation in the absence (open bars) or presence(black bars) of 5 µM cariporide. C. ERK1/2 phosphorylation inthe presence (open bars) or absence (black bars) of extracellularNa+ (replaced with NMDG+). Data are mean ± S.E.M. of 7 (A), 4(B), and 4 (C) independent experiments. *) indicates that the valueis significantly different from isotonic control. #) indicates thatthe value is significantly different from the correspondinghypertonic control.

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741NHE1 Regulates MAPKs After Osmotic Cell Shrinkage

Fig. 4. Effect of osmotic shrinkage and NHE1 activity on p38MAPK activity in ELA cells: ELA cells were exposed to high salthypertonic saline (600 mOsm) in the absence or presence of EIPA,and p38 MAPK phosphorylation levels were assessed by Westernblotting as described in the legend to Fig. 2, except that the primaryantibody recognized p38 MAPK phosphorylated on Thr180 andTyr182. Data are mean ± S.E.M. of 5 independent sets ofexperiments in both panels. *) indicates that the value issignificantly different from isotonic control. It may be noted thatno values in the presence of EIPA are significantly different fromthe corresponding values in the absence of EIPA.

Osmotic shrinkage activates p38 MAPK in a mannerindependent of NHE1 activityPrevious reports have pointed to a role for NHE1

upstream of p38 MAPK activation after other stimulithan cell shrinkage [25-27]. As seen, p38 MAPK wasactivated within 5min of osmotic shrinkage of ELA cells(Fig. 4). Activation was transient, peaking at time 30 minafter hypertonic exposure. The p38 MAPK activationwas unaffected in the presence of EIPA (5 µM), arguingagainst a role for NHE1. Visualization of total andphosphorylated p38 MAPK in intact ELA cells confirmedthis pattern, and demonstrated that the shrinkage-inducedincrease in p38 MAPK phosphorylation occurredpredominantly in the nucleus (n=4, data not shown).

Conversely, it was noted that p38 MAPK may playa role in the shrinkage-induced activation of NHE1 inELA cells. Thus, shrinkage-induced intracellularalkalinization in ELA cells was significantly reduced by35% by pretreatment with SB203580 to inhibit p38MAPK (n=5, p < 0.05), whereas it was unaffected bypretreatment with 10 µM PD98059 to inhibit ERK1/2(n=5) or with 10 µM SP600125 to inhibit JNK (n=6)(not shown).

Osmotic shrinkage activates JNK in a mannerpartially dependent on NHE1 activityFigure 5 shows the effect of osmotic shrinkage on

JNK activity. Both JNK1 (p46) and JNK2 (p54) werequantified, as markedly different effects of the twoisoforms on cell death and proliferation have beenreported [12]. The pattern of regulation by shrinkage was

comparable for both isoforms, although the JNK1phosphorylation level was lower under isotonicconditions, and increased more after osmotic shrinkage,than that of JNK2. In accordance with previous findingsin other cell types [5, 22], the shrinkage-induced increasein JNK phosphorylation in ELA cells was slightly slowerthan that of p38 MAPK (Fig. 5A-B, compare Fig. 3),being detectable at time 15 min after hypertonic exposureand peaking at time 30 min. In the presence of EIPA,shrinkage-induced JNK phosphorylation wassignificantly, yet only partially, reduced. Substantiatingthese findings, osmotic cell shrinkage elicited JNKphosphorylation in AP1 cells, and notably, in these cells,JNK phosphorylation was significantly increased by thestable expression of hNHE1 (Fig. 5C-D). Taken together,these findings strongly indicate that JNK activation byosmotic shrinkage is partially dependent on NHE1activity.

Mechanisms involved in the NHE1-dependent,shrinkage-induced ERK inhibitionNext, we addressed the mechanisms by which

osmotic shrinkage, and specifically NHE1 activation,modulated MAPK phosphorylation. Since shrinkage-induced NHE1 activation in nominally HCO3

- -free salineis associated with intracellular alkalinization (Fig. 1),which is attenuated or abolished in the presence of HCO3

-

due to the parallel activity of the Cl-/HCO3- exchanger,

AE [34], we first evaluated the effect of hypertonicexposure in double ionic strength saline in which 25 mMNaCl was replaced by 25 mM NaHCO3. ERK inhibition

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after hypertonic exposure was unaffected by HCO3-

addition indicating that the NHE1-mediatedalkalinization in shrunken cells is not the cause of theNHE1-dependent ERK inhibition (Fig. 6A). To verifythis, the cells were incubated for 1 h in isotonic saline ofpH 6.8, 7.4, or 8.0, resulting in pHi values of 6.73 ± 0.077,7.01 ± 0.021, and 7.20 ± 0.059, respectively (n=3 foreach condition). This treatment was without effect onERK activity (Fig. 6B), indicating that pHi does notmodulate ERK activity in ELA cells.

To determine the signaling step at which NHE1interferes with ERK activity after shrinkage, wemonitored the effect of EIPA on MEK1/2 activation(MEK1/2 phosphorylation on Ser217/221) (Fig. 6C). Asseen, MEK1/2 phosphorylation followed the pattern ofERK1/2 activation, being rapidly attenuated by cellshrinkage in an EIPA-sensitive manner. Thus, together,

these findings indicate that ERK inhibition by shrinkageof ELA cells involves a pHi-independent effect of NHE1at or above the level of MEK1/2. Osmotic cell shrinkageis associated with F-actin reorganization and a netincrease in cellular F-actin content [2], and somephysiological effects of NHE1 have been proposed to bedependent on interactions between NHE1 and the actincytoskeleton (see [7]. We therefore tested the effect ofLatrunculin B (Latr. B), which we have previously foundto elicit a marked loss of F-actin in ELA cells [35], onERK activity. In Latr. B treated cells, ERK activity wasmore than doubled under isotonic conditions and after15min of hypertonic exposure, ERK activity was similarto that seen under isotonic control conditions (Fig. 6D).The relative decrease in ERK activity after 15 min ofhypertonic exposure was, however, only marginallyattenuated in Latr. B treated cells compared to that in

Pedersen/Darborg/Rasmussen/Nylandsted/Hoffmann

Fig. 5. Effect of osmotic shrinkage and NHE1on JNK activity in ELA and AP1 cells: A-B. ELAcells were exposed to high salt hypertonic saline(600 mOsm) in the absence or presence of EIPA(5 µM), and JNK1 (A) and JNK2 (B)phosphorylation levels were assessed by Westernblotting as described in the legend to Fig. 2, exceptthat the primary antibody recognized JNKphosphorylated on Thr183 and Tyr185. Data aremean ± S.E.M. of 5 independent experiments foreach condition. C-D: AP1 cells (no endogenousNHE) or S5 cells (stably expressing humanNHE1) were exposed to hypertonic saline (600mOsm) for the time indicated, and total and Thr183/Tyr185-phosphorylated JNK1 (C) and JNK2 (D)levels were assessed by Western blotting andquantified by densitometric scanning. Data aremean ± S.E.M. of 6-7 independent experimentsfor each time point. *) indicates that the value issignificantly different from isotonic control. #)indicates that the value is significantly differentfrom the corresponding hypertonic control

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control cells (6D, insert). The plasma membrane-cytoskeleton linker protein ezrin directly links NHE1 toF-actin (see [7]. We recently found ezrin to be rapidlyactivated by osmotic shrinkage of ELA cells [31], and asezrin has been implicated in regulation of MAPK activity[36], we tested the potential involvement of ezrin in theshrinkage-induced, NHE1 dependent ERK inhibition.However, siRNA mediated knockdown of ezrin (verifiedto elicit 75-80% knockdown, see ref. [31]) had no

Fig. 6. Mechanisms of NHE1-dependent,shrinkage-induced ERK inhibition in ELAcells: ERK1/2 (panels A, B, D) or MEK1/2(panel C) phosphorylation levels were assessedby Western blotting and densitometricscanning as described in ExperimentalProcedures, after the treatments indicatedbelow. A. Cells were exposed for 15 min tohypertonic saline (600 mOsm) prepared byaddition of NaCl, mannitol, or NaCl with25 mM NaCl replaced by NaHCO3 asindicated. Isotonic HCO3

- saline similarlycontained 25 mM NaHCO3. Data are mean± S.E.M. of 7 independent experiments. B:Cells were preincubated for 1h at pHo 6.8, 7.4,and 8.0, resulting in pHi values of 6.73 ± 0.077,7.01 ± 0.021, and 7.20 ± 0.059, respectively(n=3 for each condition), as measured byfluorescence spectrophotometry in BCECF-loaded cells (see Experimental Procedures).Data shown are mean ± S.E.M. of 3independent experiments. C. Shrinkage-induced MEK1/2 phosphorylation in controlcells or after preincubation with 5 µ EIPA. Dataare mean ± S.E.M. of 2-7 independentexperiments for each time point. It may benoted that at the 30 min time point, variationin MEK1/2 phosphorylation was considerable,and no individual experiment at this time pointreflected precisely the mean. D: Effect ofF-actin disruption on ERK activity. Cells werepreincubated with 10 µ Latrunculin B for 1 hprior to stimulation as indicated. Data are mean± S.E.M. of 7 independent experiments. E:Shrinkage-induced ERK1/2 phosphorylationin control cells or after preincubation with thep38 MAPK inhibitor SB2003580 (10 µM,1 h). Data are mean ± S.E.M. of 4 independentexperiments. *) indicates that the value issignificantly different from that in the isotonic

detectable effect on shrinkage-induced ERK inhibitioncompared to parallel experiments using scrambled siRNA(n=2, not shown). Together, these findings indicate thatthe NHE1 dependent ERK inhibition was not ezrindependent.

Finally, crosstalk between p38 MAPK and ERK1/2has been proposed to inhibit ERK activity after somestimuli, including osmotic shrinkage [5, 19], hence, thispossibility was also investigated. Preincubation of ELA

NHE1 Regulates MAPKs After Osmotic Cell Shrinkage

control condition. ‡) indicates that the value is significantly different from that in the correspondingly treated isotonic condition(i.e. HCO3

- treated in A, Latr. B treated in D). #) indicates that the value is significantly different from that in the correspondinghypertonic control condition.

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Fig. 7. Mechanisms of NHE1-independent, shrinkage-induced p38 MAPK activation: The p38 MAPK or MKK3/6 phosphorylationlevels in ELA cells were assessed by Western blotting and densitometric scanning as described in Experimental Procedures, after thetreatments indicated below. A. Cells were exposed for 15 min to hypertonic saline (600 mOsm) prepared by addition of NaCl, mannitol,or NaCl with 25 mM NaCl replaced by NaHCO3 as indicated. Isotonic HCO3

- saline similarly contained 25 mM NaHCO3. Data aremean ± S.E.M. of 7 independent experiments. B: Cells were preincubated for 1h at pHo 6.8, 7.4, and 8.0 to reach the indicated pHi, asoutlined in the legend to Fig. 6. Data shown are mean ± S.E.M. of 3 independent experiments. C. Shrinkage-induced MKK3/6phosphorylation in control cells or after preincubation with 5 µM EIPA. Data are mean ± S.E.M. of 3 independent experiments foreach time point. *) indicates that the value is significantly different from the isotonic control.

cells with 10 µM of the p38 MAPK inhibitor SB203580abolished the inhibitory effect of osmotic shrinkage onERK1/2 phosphorylation and tended to increase ERK1/2 phosphorylation above that in isotonic controls (Fig.6E), strongly indicating that in addition to NHE1, p38MAPK is also necessary for the shrinkage-induced ERKinhibition.

Mechanisms involved in the shrinkage-induced p38MAPK activationSimilar to the ERK inhibition, the increase in p38

MAPK phosphorylation after a 15 min hypertonicexposure was unaffected by HCO3

- addition (Fig. 7A).Thus, consistent with its lack of NHE1-dependence,shrinkage-induced p38 MAPK activation was pHi-independent, in spite of the fact that basal p38 MAPKactivity exhibited an alkaline pHi optimum (Fig. 7B). TheMAPKK MKK3 has been assigned a major role inshrinkage-induced p38 MAPK activation [14] (althoughMKK3 has, to our knowledge, not been directly shownto be shrinkage-activated; see Discussion). MKK3/6phosphorylation at Ser189/207, indicative of activation, wasslightly increased after 15 and 30 min of osmoticshrinkage and unaffected by EIPA (5 µM), in congruencewith the lack of involvement of NHE1 in p38 MAPK

activation by shrinkage (Fig. 7C). A role for NKCC1 asa scaffold in stress-induced regulation of p38 MAPK hasbeen proposed in the nervous system [37]. However,although ELA cells exhibit robust NKCC1 expression,inhibition of NKCC1 by 10 µM bumetanide had nodetectable effect on shrinkage-induced p38 MAPKactivation (not shown, n=2). This argues against a rolefor NKCC1, although it should be noted that the drivingforce for inward ion transport by NKCC1 may benegligible in hypertonically shrunken ELA cells (as alsosupported by the complete inhibition of RVI by EIPA,Fig. 1).

Mechanisms involved in the partially NHE1-dependent, shrinkage-induced JNK activationIn marked contrast to the effects of shrinkage on

ERK and p38 MAPK activity, the shrinkage-induced JNKactivation was strongly attenuated in the presence ofHCO3

- (Fig. 8A). Moreover, basal JNK activity washighly pHi dependent with an alkaline pHi optimum (Fig.8B). Thus, these data strongly indicate that the role ofNHE1 in shrinkage-induced JNK activation reflects theNHE1-dependent intracellular alkalinization under theseconditions.

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Fig. 8. Mechanisms of partially NHE1-dependent,shrinkage-induced JNK activation: JNK1/2 phosphorylationlevels in ELA cells were assessed by Western blotting anddensitometric scanning as described in ExperimentalProcedures, after the treatments indicated below. A. Cellswere exposed for 15 min to hypertonic saline (600 mOsm)prepared by addition of NaCl, mannitol, or NaCl with 25mM NaCl replaced by NaHCO3 as indicated. Isotonic HCO3

-

saline similarly contained 25 mM NaHCO3. Data are mean± S.E.M. of 7 independent experiments. B: Cells werepreincubated for 1h at pHo 6.8, 7.4, and 8.0 to reach theindicated pHi, as outlined in the legend to Fig. 6. Data shownare mean ± S.E.M. of 3 independent experiments. *)indicates that the value is significantly different from theisotonic control.

Discussion

Shrinkage-induced cell damage is a contributingfactor in a number of clinically important conditions [1,8]. Osmotic shrinkage elicits a number of protectivemeasures counteracting cellular damage, including theactivation and induction of volume-regulatory iontransport, cytoskeletal reorganization, and activation ofsurvival pathways [2, 3]. Regulation of MAP kinaseactivity plays an essential role in the response to osmoticshrinkage [9], yet the mechanisms regulating MAPKsunder these conditions are incompletely understood.

NHE1 is activated by hyperosmotic stress andcontributes to the volume regulation known to be crucialfor cell survival after osmotic shrinkage [39, see 3].Accordingly, the protective role of NHE1 activation afterosmotic stress [4] has been assumed to reflect its volume-regulatory capacity. Here, we demonstrate for the firsttime that NHE1 is a major regulator of MAPK activityafter osmotic cell shrinkage, adding an importantphysiologically and pathophysiologically relevant

NHE1 Regulates MAPKs After Osmotic Cell Shrinkage

Involvement of NHE1 and MAPKs in regulation ofcell viability after osmotic shrinkageFinally, it was of importance to determine the

possible relation between shrinkage-induced regulationof NHE1 and MAPK activity, and cell death under theseconditions. Osmotic shrinkage of ELA cells induced agradual loss of cell viability as measured by MTT assay,amounting to loss of about one-third of the cells at time48 h after hypertonic exposure (600 mOsm, by additionof NaCl to the growth medium) (Fig. 9A). Consistentwith the notion that shrinkage-induced cell death is aform of PCD, hypertonic exposure was associated withan increase in caspase-3 activity, the time course of whichcorrelated well with the loss of cell viability (Fig. 9B).Inhibition of NHE1 (EIPA, 5 µM) modestly, yet not quitesignificantly, exacerbated shrinkage-induced cell deathat time 48 h, as did inhibition of ERK by PD98059(10 µM, 1 h), or inhibition of JNK by SP600125(10 µM, 1h). In contrast, shrinkage-induced cell deathwas ameliorated after inhibition of p38 MAPK bySB203580 (10 µM, 1 h).

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condition to a growing body of literature implicatingNHE1 in regulation of MAPK activity after variousstimuli [23-26] A tentative working model of the relationsbetween NHE1 activity, MAPK activity, and shrinkage-induced cell death is shown in Figure 10, and thearguments leading to this model are presented below.Importantly, although not illustrated, NHE1-mediatedeffects on death/survival balance after osmotic shrinkageare of course by no means restricted to regulation ofMAPKs; most notably, the contribution of NHE1 to theRVI process plays a major role in counteractingshrinkage-induced cell death [see 7, 35].

Shrinkage-induced inhibition of ERK1/2 wasabolished by inhibition or reversal of NHE1-mediatedtransport, and mimicked by expression of hNHE1 in cellsdevoid of endogenous NHE activity. These data stronglyindicate that NHE1 negatively regulates ERK1/2 activityin osmotically shrunken cells. Interestingly, basal ERKactivity also appeared to be suppressed by NHE1,indicating that this role is not limited to shrunken cells.

The inhibitory effect of NHE1 on ERK1/2 appearsto occur upstream of MEK1/2, since the activity of thiskinase exhibited a shrinkage- and NHE1-dependencesimilar to that of ERK1/2. The mechanism by which

Pedersen/Darborg/Rasmussen/Nylandsted/Hoffmann

Fig. 9. Mechanisms of shrinkage-induced loss of viability: Cells were exposed to hypertonic conditions by increasing the osmolarityof the growth medium to 587 mOsm by addition of NaCl. A. Cell viability was evaluated from the fraction of functional mitochondriausing 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT), as described in Experimental Procedures. Cells seeded in96 well plates were incubated for 1h in the absence or presence of inhibitors as indicated, followed by exposure to iso- or hypertonicconditions with or without inhibitors. Viability was calculated as the absorbance relative to that under isotonic control conditions,after subtraction of the background signal measured from wells containing the relevant media but no cells. Loss of cell survival isshown as percent above that in cells exposed to isotonic conditions for the same time interval. Data are mean ± S.E.M. of 4-6independent experiments for each condition. Under isotonic conditions, EIPA and SB203580 had no effect on cell survival, whereasSP600125 and PD98059 treatment was associated with an ∼10% and ∼20% loss of viability, respectively (n=4-6). *) indicates that thevalue is significantly different from isotonic control. B. At the times indicated, crude membrane lysates were prepared as described inExperimental Procedures, resolved by SDS-PAGE (equal amounts of protein in each well), and caspase-3 activity was assessed as therelative intensity of the activated (cleaved) form of the protein. Data shown are mean ± S.E.M. of 3 independent experiments. *)indicates that the value is significantly different from isotonic control. C. Experiments were carried out as described for panel A,except that the cells were exposed to iso- or hypertonic conditions for 48h, in the absence or presence of EIPA (5 µM), or 10 µM eachof PD98059 to inhibit ERK1/2, SB203580 to inhibit p38 MAPK, or SP600125 to inhibit JNK, respectively. Viability was calculatedas described in A. It may be noted that under isotonic conditions, EIPA and SB203580 at the concentrations employed here werewithout effect on viability, while SP600125 and PD98059 attenuated viability by about 10 and 20%, respectively (n=4-6, not shown).Data are mean ± S.E.M. of 4-6 independent experiments for each condition. *) indicates that the value is significantly different fromthe hypertonic control value.

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Fig. 10. Working model: Interrelations between NHE1, MAPKactivity, and loss of cell viability after osmotic shrinkage of ELAcells.

NHE1 inhibits MEK1/2 phosphorylation after osmoticshrinkage remains to be fully elucidated, however, severalconclusions can be drawn from the present findings.Acidic pHi has been shown to activate ERK1/2 in severalcell types [40], however, strongly arguing against a rolefor pHi, the shrinkage-induced ERK inhibition wassimilar in the absence and presence of HCO3

-, and ERK1/2 activity in ELA cells was unaffected at acidic or alkalinepHi. A role for changes in [Na+]i is also not likely inhealthy cells, in which the NHE1-dependent Na+-influxis efficiently counteracted by the activity of theNa+,K+ATPase (for a discussion, see ref. [6]). In otherwords, it seems most likely that the effect of NHE1 onERK1/2 activity is not dependent on the ions transportedvia NHE1. This is in congruence with recent reports ofion transport-independence of other physiological effectsof NHE1 activity, at least some of which appear to involveinteractions between NHE1 and the actin-basedcytoskeleton [41, see 7]. Interestingly, F-actin disruptionstrongly increased basal and hypertonic ERK activity inELA cells, suggesting that F-actin exerts a tonic inhibitoryeffect on ERK1/2 activity in ELA cells, yet the relativedecrease upon hypertonic shrinkage was similar to thatin control cells. Shrinkage-induced ERK inhibition wasalso unaffected by knockdown of ezrin, which linksNHE1 to F-actin [42], regulates ERK activity in somecell types [36], and is activated by osmotic shrinkage inELA cells [29, 31]. Roles for the other ERM proteins,which can also bind to NHE1 in vitro, can, however, notbe excluded. Preliminary co-immunoprecipitationexperiments indicate that at least ERK2 may associatewith NHE1. However, this interaction does not appearto be shrinkage-regulated, and its potential importancefor ERK regulation remains to be assessed (SFP,unpublished, n=2). It is, however, interesting in thisregard that CD44, which is known to interact directlywith NHE1 [43], was recently shown to form a complexwith ERK1/2 in breast cancer cells [44]. In conclusion,NHE1-dependent ERK inhibition in osmoticallyshrunken ELA cells appears to be independent of iontransport by NHE1, and occurs at or upstream of theMEK1/2 level (Fig. 10). Finally, in addition to beingNHE1-dependent, the shrinkage-induced ERK1/2inhibition in ELA cells was dependent on p38 MAPKactivity, in congruence with findings in other cell typesafter osmotic and other stress stimuli [5, 19]. While thislikely in part reflects the apparent role of p38 MAPKupstream of the shrinkage-induced NHE1 activation inELA cells, a direct inhibitory interaction between p38MAPK and ERK may also contribute [19] (Fig. 10). Thus,

p38 MAPK appears to be necessary, yet not sufficient,for the shrinkage-induced ERK inhibition. It may benoted that while significant, the NHE1-dependence ofshrinkage-induced ERK inhibition was not complete inthe AP1 cells (Fig. 3), and it is possible that this reflectsdifferent relative roles of p38 MAPK in the two cell lines.

In contrast to ERK1/2, p38 MAPK was rapidly andreversibly activated after ELA cell shrinkage. This agreeswell with findings in other cell types [5, 22], and hasbeen suggested to involve the shrinkage-activated [45]small G-proteins Cdc42 and Rac [5, 14, 46], and furtherdownstream, MKK3 [14, 15] and/or MKK6 [47]. MKK3/6 phosphorylation was slow and very modest in shrunkenELA cells. This may reflect negative autoregulation, assuggested in kidney cells [15] in which a lack ofendogenous MKK3/6 activation by shrinkage wasobserved in spite of a major role of MKK3 in shrinkage-activation of p38 MAPK [15]. In contrast to the upstreamrole of NHE1 in p38 MAPK activation in various celltypes after other stimuli [25-28], shrinkage-activation ofp38 MAPK was independent of NHE1 activity.Conversely, p38 MAPK partially contributed to NHE1activation under these conditions, in agreement with ourprevious findings in the related EAT cells [21]. In

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congruence with its lack of dependence on NHE1, p38MAPK activation persisted in the presence of HCO3

-,although a weakly alkaline pHi optimum for p38 MAPKwas noted under basal conditions. Thus, p38 MAPK isactivated by osmotic shrinkage of ELA cells, in an NHE1-and pHi–independent manner, and contributes to NHE1activation under these conditions (Fig. 10).

JNK1 and JNK2 were transiently stimulated byshrinkage of ELA cells, consistent with findings in othercell types [10, 22]. JNK activation was partially inhibitedby EIPA and augmented by hNHE1 expression, indicatingthe involvement of both NHE1-dependent and –independent mechanisms. This is, to our knowledge, thefirst study demonstrating a role for NHE1 in JNKregulation. The shrinkage-induced JNK activation wasessentially abolished in HCO3-containing medium, andbasal JNK activity was potently activated at alkaline pHi,consistent with previous reports [48]. These data stronglyindicate that the NHE1 dependence of JNK activation inshrunken cells reflects the NHE1-mediated intracellularalkalinization under these conditions (Fig. 10). Sincemany cancer cells exist in an acidic, low HCO3

-, tumormicroenvironment, and exhibit increased activity of bothNHE1 [49, 50] and JNK [51], an important implicationof the present findings is that NHE1-mediatedintracellular alkalinization may play a role in theexcessive JNK activity under these conditions. Incontrast, shrinkage-activation of JNK in U937 cells wasindependent of NHE1 [48], possibly reflecting the muchsmaller shrinkage-induced alkalinization in U937 cellscompared to ELA cells.

Long term osmotic shrinkage activated caspase-3and reduced ELA cell survival, in congruence withprevious studies indicating that osmotic shrinkage elicitsPCD [4, 5, 52]. Shrinkage-induced death was slightlyexacerbated by inhibition of NHE1, consistent with ourprevious finding that NHE1 inhibition exacerbatedshrinkage-induced cell death in murine fibrosarcoma cells[4]. Inhibition of ERK1/2 and JNK1/2 likewiseexacerbated, and inhibition of p38 MAPK ameliorated,shrinkage-induced ELA cell death. While these effectsof ERK and p38 MAPK are consistent with earlier studies[5, 13], the role of JNK is more controversial [9, 13],likely reflecting cell type specific differences in the effectof shrinkage on JNK activity (see [9], or differential rolesof JNK1 and JNK2 as reported for other apoptotic stimuli[12]. Thus, it appears that the slight protective role ofNHE1 reflects the combined impact of NHE1-dependenteffects counteracting (RVI, JNK1/2 activation, and likely

intracellular alkalinization) and favoring (ERK1/2inhibition) shrinkage-induced cell death (Fig. 10).

Thus, NHE1 modulates MAPK activity inosmotically stressed ELA cells in an isoform-specificmanner. Interestingly, the Na+K+ATPase [53], NKCC1[37], and ether-à-go-go (EAG) K+ channels [54, see also28] have also been assigned roles in regulation and/orscaffolding of MAPKs have been proposed. Althoughthe present study sheds some light on the mechanisms ofNHE1-dependent regulation of MAPK activity, thepathways involved remain to be fully elucidated, at leastin the case of ERK1/2. It is interesting to note that EAGK+ channels were found to stimulate p38 MAPK activityby a mechanism apparently involving conformationalchanges associated with activation, yet independent ofion transport [54]. Future studies should address whetherNHE1 may regulate MAPK activity by similarmechanisms.

In conclusion, we identified a novel role of NHE1as a regulator of MAPK activity after osmotic shrinkage.ERK1/2 is inhibited by shrinkage in an NHE1 dependent,pHi-independent manner, JNK1/2 is stimulated byshrinkage, partly as a consequence of NHE1-mediatedintracellular alkalinization, and p38 MAPK is activatedby shrinkage in NHE1-independent manner. The loss ofcell viability induced by long-term osmotic shrinkage isaugmented by inhibition of ERK1/2 or JNK1/2, andattenuated by inhibition of p38 MAPK. Regulation ofMAPK activity may be an important physiological roleof NHE1, impacting on shrinkage-induced cell death aswell as on MAPK-dependent transcriptional effects oflong-term osmotic stress.

Abbreviations

ERK (extracellular signal regulated kinase); JNK(c-Jun N-terminal kinase); p38 MAPK (p38 mitogenactivated protein kinase).

Acknowledgements

This work was supported by the GangstedFoundation (SFP), the Augustinus Foundation (SFP), andthe Danish Natural Sciences Research Council (EKH,SFP, grant no. 21-01-0507, 21-04-0535). We gratefullyacknowledge the skilled technical assistance of BirtheJuul Hansen and Anni Bech Nielsen.

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