The role of keratins in response to osmotic shock...severity of epidermolysis bullosa simplex. Cells with severe mutations were more sensitive to osmotic stress and took longer to

IntroductionIntermediate filaments remain the least-understood componentof the cytoskeleton of eukaryotic cells. The 65 or more (Hesseet al., 2001) members of this large multigene family areexpressed in a tissue-specific manner that suggests they havean important role in tissue cell differentiation and function.This has been difficult to test experimentally, owing to therobust nature of these filament proteins, with their long half-life and their relatively slow turnover when compared to actinand tubulin.

The microscopic morphology of intermediate filamentssuggests a role for these networks in physically reinforcingcells, a hypothesis that was greatly strengthened by findingmutations in several classes of intermediate filament genes thatlead to cell fragility and pathological consequences (forreviews, see Corden and McLean, 1996; Quinlan et al., 1999).The first identified of these disorders, and the best studied,is epidermolysis bullosa simplex (EBS). In this disorder,mutations in keratins K5 or K14, expressed in the basal cellsof stratified epithelia, lead to fragile epidermal basal cells thatlyse upon mild physical trauma to produce intraepithelialblisters (Bonifas et al., 1991; Coulombe et al., 1991; Lane etal., 1992). The clinical severity of the EBS phenotype variesfrom case to case and can be largely correlated with theposition of the mutation in the keratin gene (Letai et al., 1993).Mutation ‘hotspots’ at either end of the central α-helical roddomains are associated with the most severe type of EBS (theDowling-Meara form), whereas several cluster sites elsewhere

in the keratin protein are associated with milder (Weber-Cockayne) forms of the disorder affecting predominantlyhands and feet (for a review, see Lane, 1994). EBS is howeveronly one of many epithelial fragility disorders now known tobe caused by mutations in keratin genes (Irvine and McLean,1999).

We recently characterized a set of keratinocyte cell lines,derived from EBS patients, as potential model systems inwhich to analyze and try to reverse the impact of defectivekeratin intermediate filaments on cell function (S.M.M., I. M.Leigh, E.B.L. et al., manuscript in preparation). These cells arerobust in culture and amenable to various types of stress assaysand have already been tested for cell spreading and responseto heat shock (Morley et al., 1995). In such assays the mutantkeratin lines were slower to spread out after re-plating than thecontrol cells with wild-type keratins. The mutant keratinfilament networks showed a reduced stability, with a tendencyto break up into small aggregates upon thermal stress. Thisassay was, however, unable to distinguish between cellscarrying mutations with different degrees of clinical severity.

In this paper we describe the effects of a different typeof stress, hypo-osmotic shock, on keratinocytes expressingEBS-associated keratin mutations. Hypo-osmotic shock is acommon physiological stress that body tissues encounter, andit leads to transient cell swelling. The extreme cell-shapechanges induced by this shock led us to investigate thepossibility that the status of the cytoskeleton, and specificallythe keratin filaments, is important for cell survival. Volume-

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The intermediate filament cytoskeleton is thought to conferphysical resilience on tissue cells, on the basis ofextrapolations from the phenotype of cell fragility thatresults from mutations in skin keratins. There is a need forfunctional cell assays in which the impact of stress onintermediate filaments can be induced and analyzed. Usingosmotic shock, we have induced cytoskeleton changes thatsuggest protective functions for actin and intermediatefilament systems. Induction of the resulting stress responsehas been monitored in keratinocyte cells lines carrying K5or K14 mutations, which are associated with varyingseverity of epidermolysis bullosa simplex. Cells with severemutations were more sensitive to osmotic stress and tooklonger to recover from it. Their stress-activated response

pathways were induced faster, as seen by early activationof JNK, ATF-2 and c-Jun. We demonstrate that the speedof a cell’s response to hypotonic stress, by activation of theSAPK/JNK pathway, is correlated with the clinical severityof the mutation carried. The response to hypo-osmoticshock constitutes a discriminating stress assay todistinguish between the effects of different keratinmutations and is a potentially valuable tool in developingtherapeutic strategies for keratin-based skin fragilitydisorders.

Key words: Epidermolysis bullosa simplex, Keratins, Keratinocytecell lines, Osmotic shock, Stress response

Summary

Keratin mutations of epidermolysis bullosa simplexalter the kinetics of stress response to osmotic shockMariella D’Alessandro, David Russell, Susan M. Morley, Anthony M. Davies* and E. Birgitte Lane ‡

Cancer Research UK Cell Structure Research Group, University of Dundee School of Life Sciences, MSI/WTB Complex, Dow Street, Dundee DD15EH, UK*Present address: Tayside Institute of Child Health, Ninewells Hospital and Medical School, Dundee DD1 9SY, UK‡Author for correspondence (e-mail: [email protected])

Accepted 23 August 2002Journal of Cell Science 115, 4341-4351 © 2002 The Company of Biologists Ltddoi:10.1242/jcs.00120

Research Article

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regulatory responses to osmotic shock have been reported tobe sensitive to pharmacological disruption of the cytoskeletonin a number of cell models (Cornet et al., 1994; Foskett andSpring, 1985).

We observed that exposure to hypo-osmotic stress, whichcauses a rapid cell swelling and loss of membrane surfaceelaborations, is characterized by rapid changes in theconfiguration of actin and tubulin, and in the case of severelymutant cells, in intermediate filaments as well. Monitoring theearly stress response to hypo-osmotic shock by tracking itseffect on JNK activation revealed a faster response in EBSkeratinocyte cell lines, with a direct correlation between theseverity of the keratin mutation carried in these cell lines andthe speed of JNK activation. These observations have abearing on our understanding of the function of integratedcytoskeleton systems, as well as practical applications in genetherapy.

Materials and MethodsCell cultureFive keratinocyte cell lines, expressing EBS-associated keratinmutations of differing severity, were compared (Table 1). KEB-1,KEB-2 [both K5 E475G (Lane et al., 1992)] and KEB-7 [K14 R125P(S.M.M., unpublished)] represent severe (Dowling-Meara) diseasephenotypes, whereas KEB-3 and KEB-4 [both K14 V270M (Rugg etal., 1993)] derive from a milder Weber-Cockayne form of EBS. KEB-1, KEB-2 and KEB-3 cells were immortalized with a temperature-sensitive SV-40 T antigen (tsA 58), HPV16 (E6^E7) was used toimmortalize KEB-7 and KEB-4 and also the control (wild-type)keratinocyte line, NEB-1 (S.M.M., unpublished). All the experimentsdescribed in this paper were carried out on mutant and control celllines between 10-15 passages after immortalization.

The cell lines were cultured in DMEM with 25% Ham’s F12medium, 10% FCS, plus additional growth factors hydrocortisone (0.4µg/ml), cholera toxin (10–10 M), transferrin (5 µg/ml), lyothyronine(2×10–11 M), adenine (1.9×10–4 M) and insulin (5 µg/ml). All celllines were fibroblast feeder cell independent and were cultured at36.5°C in 5% CO2. They were grown in plastic tissue culture gradedishes and in 96 microwell plates for osmotic shock and on 13 mmglass coverslips for light microscopy.

Osmotic shock Cells were cultured for 48 hours to reach 80% confluence and thensubjected to hypo-osmotic shock by immersion in 150 mM urea for5 minutes at 37°C. Cells were then returned to normal tissue culturemedium for varying periods of recovery, before further analysis.Keratinocytes are quite resistant to hypo-osmotic effects of water,diluted DMEM or PBS, so urea was added (Kucerova and Strbak,

2001). This small permeant molecule diffuses rapidly into the cells,causing water to enter and increase the rate of cell swelling (Stein,1990). Cell survival was not affected by this procedure. To assess theeffect of cell confluence on sensitivity to osmotic shock, differentconcentrations of NEB-1 cells were grown in three 96-well plates andtested at 40%, 80% and 100% confluence. The recovery of themetabolic activity of the cell was assessed at 1 hour, 3 hours and 24hours after osmotic shock, using the CellTiter 96® AQueous OneSolution Cell Proliferation Assay (Promega).

The same assay was used to measure the recovery of metabolicactivity of the cell lines KEB-4, KEB-7 and NEB-1 after osmoticshock. The cells were grown in 96-well plates, subjected to hypo-osmotic shock and then measured before and immediately afterosmotic shock and at 30 minutes, 3 hours, 4 hours, 24 hours, 48 hours,5 days, 6 days, 7 days and 8 days after osmotic shock. Absorbancewas measured at λ=490 nm and compared with readings fromuntreated control cells.

Microscopy For scanning electron microscopy, cells were grown on 6 mmcoverslips and subjected to hypo-osmotic shock as described above.Both test and control cells were fixed with 4% paraformaldehyde for10 minutes, washed in PBS and dehydrated with absolute alcohol andthen acetone. They were critical point dried in a Polaron E3000 seriesII unit, sputter-coated (Polaron ES100 series II coating unit) withgold-palladium and viewed using a Joel JSM 35 scanning electronmicroscope operated at 15kV.

For light microscopy, cells were grown on 13 mm glass coverslipsand processed for immunostaining after 2 minutes and 4 minutes ofosmotic shock, and at 10 minutes and 4 hours recovery in normalmedium, after 5 minutes osmotic shock. Cells were fixed in 3%paraformaldehyde for 10 minutes at 4°C, permeabilized in 100%methanol-acetone (1:1) for 10 minutes at –20°C and treated withblocking buffer (5.5% normal goat serum in PBS) for 1 hour at roomtemperature. The cells were stained with mouse monoclonalantibodies LL001 [to K14 (Purkis et al., 1990)], α-tubulin(Amersham, dilution 1:200) and phospho-SAPK/JNK (dilution 1:200;New England Biolabs), and with rabbit polyclonal antibodies BL18[to K5, dilution 1:200 (Purkis et al., 1990)] and SAPK/JNK (dilution1:200; New England Biolabs). SAPK/JNK antibody detects the totalSAPK/JNK levels and is phosphorylation independent, whereas thephospho-SAPK/JNK antibody (Thr183/Tyr185) detects, after stress,the doubly phosphorylated threonine 183 and tyrosine 185 of JNK1(p46) and JNK2 (p54) (Kyriakis et al., 1994; Rochat-Steiner et al.,2000; Weiss et al., 2000; Shukla et al., 2001).

Rhodamine-conjugated phalloidin (Molecular Probes, Leiden,Netherlands; dilution 1:150) was used to detect actin. Mousemonoclonal antibodies were detected with FITC-conjugated sheepanti-mouse serum (Sigma Aldrich, UK, F3008) at 1:50 dilution; rabbitantibodies were detected using an Alexa-fluorescein-conjugated goatanti-rabbit serum (Molecular Probes, Leiden, Netherlands; dilution1:1000). Coverslips were mounted onto glass slides using CitiFluor(Sigma Aldrich, UK), air-dried and visualized with a Zeiss Axioplanepifluorescence microscope. Confocal images were collected using aBioRad MRC 600 confocal laser-scanning microscope.

ImmunoblottingCell lysates were extracted after 0 minutes, 2.5 minutes, 5 minutes,10 minutes, 30 minutes, 1 hour and 2 hours of recovery from osmoticshock. Cells were kept on ice and washed twice with ice-coldPBS. Lysis buffer, containing 100 mM Tris (pH 7.4), 50 mM β-glycerolphosphate, 0.5 mM Na3VO4, 1 mM ethyleneglycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetra-acetic acid, 1% Triton, 1 µMmicrocystin and one complete (EDTA-free) protease inhibitor cocktailtablet (Roche) per 50 ml lysis buffer, was added and left on ice for 5

Journal of Cell Science 115 (22)

Table 1. Cell lines used in this studyCell line NEB-1 KEB-1 KEB-2 KEB-3 KEB-4 KEB-7

Phenotype Normal DM DM WC WC DMsevere severe mild mild severe

Mutation – K5 K5 K14 K14 K14– E475G E475G V270M V270M R125P

Stress 1 3 3 2 2 4 response*

DM, Dowling-Meara form; WC, Weber-Cockayne form of EBS (see text).*Ranked order of strength and speed of stress response activation, 1 is the

lowest.

4343The role of keratins in response to osmotic shock

minutes. Cells were then harvested and centrifuged at 13,000 g for 10minutes at 4°C. The protein concentration of the supernatant lysatewas determined by Bradford assay (BioRad protein assay kit) andstandardized using bovine serum albumin.

For sodium dodecylsulphate (SDS) polyacrylamide gelelectrophoresis, 10 µg of each of the samples was mixed with SDSsample buffer (4% SDS, 20% glycerol, 20 mM Tris-HCl (pH 6.8), 20mM dithiothreitol and 1% Bromophenol Blue), boiled for 5 minutesand separated on SDS polyacrylamide gels. For immunoblotting,protein transfer was carried out at 300 mA for 60 minutes. Non-specific binding to the transfer membrane was blocked by incubationin Tris-buffered saline (pH 7.6), containing 0.5% TWEEN-20 and 5%BSA (blocking buffer), for at least 3 hours, with gentle agitation. Blotswere then incubated overnight at 4°C with JNK/SAPK antibody orphospho-JNK/SAPK or phospho-ATF-2 antibodies (dilution 1:1000;New England Biolabs) in 10 ml blocking buffer. The membranes werewashed with TBS with 0.5% TWEEN-20, and bound primaryantibodies were detected using a secondary antibody of swine anti-rabbit Ig antiserum for JNK/SAPK and phospho-ATF-2, and asecondary antibody of rabbit anti-mouse Ig antiserum for phospho-JNK/SAPK. The secondary antibodies were conjugated to horseradishperoxidase (diluted 1:2000 in 10 ml blocking buffer) and visualizedby electrochemiluminescence.

ResultsThe effect of osmotic shock on the cytoskeletonFollowing immersion in 150 mM urea, cells swell rapidly aswater is drawn into them (Fig. 1A,B). We found urea to be afast and effective way to induce osmotic shock that was nomore damaging than distilled water or diluted medium. Byscanning electron microscopy, it can be seen that this iscorrelated with a loss of surface detail of the apical plasma

membrane, as microvilli and filopodia are lost and thestretching plasma membrane becomes smooth (Fig. 1C,D).

Within the cell, these changes correlate with pronouncedreorganization of cytoskeleton proteins (Fig. 2). Byimmunofluorescence microscopy, it was observed that themicrotubules became fragmented within 2 minutes of theinitiation of stress (Fig. 2B). This was rapidly reversible,and microtubules were clearly repolymerizing after 4minutes of urea exposure (Fig. 2C). Ten minutes afterreturn to normal medium, the microtubules wereindistinguishable from control cells (Fig. 2D). This fastrecovery probably explains why microtubule disturbanceswere not reported in earlier studies (Cornet et al., 1994; Langet al., 1998).

Actin reorganization was also evident. Concomitant withthe loss of surface detail, there was a depletion of the actinassociated with the apical cell membrane (Fig. 2G). F-actinappeared to be relocated to sites further into the cell’s interior,and large actin filament bundles were often apparent deep inthe cell. After 2 minutes of exposure to urea, residualfilopodia were still evident at the free cell margins (Fig. 2G).At this time, the regulatory volume decrease (RVD) wouldhave been initiated, by the activation of ion pumps in themembrane, and the cells were beginning to shrink again.Actin became increasingly reassociated with the cell surfacewith time (Fig. 2H,I), and cells had all regained their normalmorphology by 4 hours after osmotic shock (Fig. 2J). All theabove changes were seen equally in wild-type or mutantkeratin cells.

No signs of keratin fragmentation or disassembly weredetected in wild-type cells during the shock and recoveryperiods (data not shown), although the profound cell-shapechanges triggered by osmotic shock must displace filamentsystems to some extent in all cells. By contrast, the KEB-7 cellline (severe phenotype EBS mutation) showed a degree ofkeratin filament fragmentation following osmotic shock (Fig.2K-O) that could be monitored by immunofluorescence. Thesechanges developed more slowly than the changes in actin andmicrotubules. Tiny keratin aggregates started to form at the cellperiphery and were clearly seen 10 minutes after osmoticshock, as shown by staining for keratin 14 (Fig. 2N). After 4hours, the aggregates appeared bigger (Fig. 2O). Theaggregates only ever represented a small proportion of the totalkeratin in the cell, and the majority of keratin intermediatefilaments appeared intact throughout the time course ofobservation.

Keratin aggregate formation depends on mutationseverityIn EBS patients, the position of the mutation in the keratinprotein is generally correlated with the severity of the diseasephenotype. When cell lines expressing different keratinmutations were subjected to osmotic stress, differences wereseen. Fig. 3 shows this comparison, illustrating the integrity ofthe keratin filament network 4 hours after stress. Clearaggregates were seen in cells with a severe mutation (Fig. 3F),whereas less severe mutations were associated with somefilament fragmentation, but no aggregates were present (Fig.3B,C). Mild mutations (Fig. 3D,E) showed little deviation fromcontrols (Fig. 3A).

Fig. 1. Hypo-osmotic shock causes rapid cell swelling (B) and lossof membrane surface elaborations (D). Cells before (A,C) and after(B,D) 5 minutes exposure to hypo-osmotic medium. (A,B) Phasecontrast microscopy (Bar, 10 µm) and (C,D) scanning electronmicroscopy (Bar, 1 µm).

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Fig. 2.Changes in the cytoskeleton following exposure to hypotonic medium. Tubulin disruption recovers more rapidly than actin and keratinintermediate filament systems. KEB-7 cells, an EBS-derived cell line expressing the K14 R125P mutation, were fluorescently stained for tubulin(A-E, anti-α tubulin), actin (F-J, phalloidin) and intermediate filaments (K-O, antibody LL001 to K14). Cells were exposed to 150 mM urea for 5minutes then transferred back to normal tissue culture medium for recovery. Cell were fixed and stained before hypo-osmotic shock (A,F,K), after 2minutes (B,G,L) and 4 minutes (C,H,M) exposure to urea, and at 10 minutes (D,I,N) and 4 hours (E,J,O) recovery after osmotic shock. Bar, 10 µm.


Sensitivity to hypo-osmotic shock varies with cultureconfluenceAlthough the short-term effects of osmotic shock werereproducible, initial experiments indicated that longer-termsurvival of the cells was quite variable and dependent on thetime elapsed since last trypsinization and re-plating of thecells and on the degree of confluence of the culture at thetime the osmotic shock was applied. Comparison of cellsurvival after osmotic shock between cells plated at differentdensities revealed that cells that were 80% confluent uponhypo-osmotic shock were more sensitive to this stress thancells that were either 40% confluent or 100% confluent(Fig. 4).

Cell recovery after hypo-osmotic shockFollowing osmotic shock, there was a similar cell loss rate inall the cell lines tested, and an 80-85% drop of metabolicactivity was detected in all the cultures within 24 hours. Aclear difference was observed in the cell lines’ long-termrecovery from osmotic shock, depending on the state of theirkeratins. The cell lines expressing EBS mutations in K5 orK14 took longer for their metabolic activity to build up topre-osmotic shock levels compared with cells expressingwild-type keratins. In the case of the wild-type control cellline NEB-1, for example, metabolic activity of the culture hadreached its pre-shock level by 48 hours, whereas it took 6days for the cell line carrying a mild EBS mutation (KEB-4)to recuperate. The cell line with the most severe mutation(KEB-7, K14 R125P mutation) had still not recovered by 8days (Fig. 5).

Different levels of JNK and phospho-JNK in mutantkeratin cell lines In view of the variable cell recovery, it was decided to restrictthe investigation to immediate or short-term effects of osmoticshock on the cells, to seek a more consistent, cell-number-

Fig. 3. Different effect of osmotic shock on the keratin cytoskeleton in the five EBS-derived cell lines studied. All cell lines were subjected tohypo-osmotic shock and fluorescently stained for K14 (antibody LL001) after 4 hours recovery. (A) NEB-1 (wildtype); (B) KEB-1, (C) KEB-2,(D) KEB-3, (E) KEB-4, (F) KEB-7. Some filament fragmentation was seen in KEB-1 and KEB-2 (arrows in B and C) and clear peripheralaggregates were seen in KEB-7 (arrows in H). Bar, 10 µm.

Fig. 4. The effect of cell confluence on sensitivity to osmotic shock.NEB-1 (wild-type keratin) cells were grown to different degrees ofconfluence (40%, 80% and 100%), before subjecting them to hypo-osmotic shock; metabolic activity was measured at 1, 3 and 24 hoursof recovery (CellTiter 96, Promega). At all time points, cultures at80% confluence showed the greatest sensitivity to osmotic shock.

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independent readout. We therefore examined the speed ofcells’ response to this shock, as reflected by induction of thestress-activated protein kinases (SAPKs) pathway, which isknown to be triggered by hypo-osmotic stress (Haussinger,1996b). On assessing JNK/SAPK activation, byimmunoblotting with antibodies to JNK and phosphorylated(activated) JNK, we confirmed the induction of this pathwayand noted in addition some differences in the response ofcells expressing mutant keratins. Firstly, the total amount ofJNK was higher in cell lines with ‘severe’ mutations (KEB-1, KEB-2 and KEB-7) than either in the control (NEB-1) orin the ‘mild’ mutant lines (KEB-3 and KEB-4) (Fig. 6).Densitometric measurement of JNK levels showed that thisprotein was about seven times more concentrated in KEB-7than in NEB-1 (data not shown).

JNK activation also varied between the cell lines, as shownby immunoblot analysis with a phospho-JNK/SAPKantibody (New England Biolabs). A weak signal wasdetected in the control cell line NEB-1 (wild-type keratin)after 2.5 minutes of recovery from osmotic shock. Thissignal peaked at around 10 minutes into the recovery periodand then gradually reduced over the next three hours (Fig.6). Two phospho-JNK isoforms, identified by the NewEngland Biolabs antibody as phospho-JNK1 and -JNK2,were detectable before osmotic shock in the severe mutant celllines (Fig. 6). These bands had both increased greatly inintensity by 2.5 minutes recovery after osmotic shock, and a

strong signal was still present 1 hour after osmotic shock.Among all the EBS cell lines tested, KEB-7 (K14 R125P, theone carrying the most severe mutation) consistently showed the


Fig. 6. Induction of JNKphosphorylation followinghypo-osmotic shock. JNK1(p46) and JNK2 (p54) weredetected by immunoblottingwith rabbit antisera to nativeJNK or with mouse antiseraagainst phospho-JNK pJNK1and pJNK2 (New EnglandBiolabs), as indicated andvisualized bychemiluminescence (seeMaterials and Methods). Celllines varied in the time courseof JNK1 and JNK2phosphorylation. Severelymutated cell lines have higherlevels of JNK1 and JNK2 thanmilder mutants or controls, andphosphorylation of JNK isinduced faster. B, before OS; 0,start of recovery time after 5minutes osmotic shock.

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Fig. 5. Cell viability after hypo-osmotic shock (OS). Cells wereassayed for metabolic activity (CellTiter 96, Promega) at differenttime intervals of recovery after 5 minutes exposure to 150 mM urea.Metabolic activity is presented as a percentage of the activity in theuntreated control cell population. Wild-type cell cultures returned tostarting levels of metabolic activity after 2 days; KEB-4 reached thislevel in 6 days, and KEB-7 cultures showed no recovery in 8 days.


highest levels of phospho-JNK, before, during and afterosmotic shock. KEB-1 and KEB-2 (K5 E475G) were the nexthighest. KEB-3 and KEB-4 (K14 V270M) gave the weakestsignal, with the two phospho-isoforms present after 2.5minutes recovery from osmotic shock, and greatly increased by5 minutes after shock (Fig. 6). All cells reached a peak ofactivation after 10 minutes recovery from osmotic shock. Thisgradation in response to osmotic shock between the cell linesis directly correlated with the gradation in clinical severity ofthe EBS seen in patients with these mutations (see Table 1).

To determine whether JNK phosphorylation was truly areflection of the JNK/SAPK pathway activation, we looked foreffects on known downstream targets of this kinase. The ATF-2 transcription factor is a well documented substrate forphosphorylation by activated JNK (Ip and Davis, 1998; Leppaand Bohmann, 1999). Immunoblotting revealed the appearanceof phosphorylated (activated) ATF-2 (pATF-2) with the sametime course observed for JNK phosphorylation (Fig. 7). In thecontrol cell line, a weak signal was observed at 5 minutesrecovery after osmotic shock. This signal increased after 10minutes and had the strongest peak after 30 minutes (Fig. 7),in keeping with its activation downstream of JNK. In the KEB-3 and KEB-4 cell lines, a weak signal was present after 2.5minutes of recovery from osmotic shock. This then increasedprogressively, until reaching the highest intensity 30 minutesafter osmotic shock (Fig. 7). In the cell lines carrying a moresevere mutation (KEB-1, KEB-2, and KEB-7), there wasalready a signal before osmotic shock, which reached itsmaximum intensity 10-30 minutes after the shock andremained very strong at 3 hours (Fig. 7). Activation of thispathway was further confirmed by assaying phosphorylated c-Jun levels, as c-Jun is another downstream target of JNK. Threecell lines (NEB-1, KEB-4 and KEB-7) were tested, and the c-Jun phosphorylation pattern observed was identical to thatobserved for ATF-2 (data not shown).

It appears therefore that the mutated cell lines have a morerapid activation of JNK in response to osmotic shock than thecontrols and that there is a direct correlation between theseverity of the mutation and the speed of JNK activation.

Moreover, the presence of a signal, even before osmotic shock,in KEB-1, KEB-2 and KEB-7, seems to indicate that the celllines with a severe mutation have constitutively activated theJNK/SAPK pathway.

Cellular localization of JNK and phospho-JNKThe distribution of JNK, and its relocation after activation, wasmonitored by immunofluorescence on cultured cells. Beforeosmotic shock, JNK was localized at the periphery of wild-typecells, often with a distribution suggestive of focal contacts (Fig.8A). After activation (phosphorylation), JNK was foundpatchily within the nucleus, both in the control and in the KEB-7 mutant cell line (Fig. 8B,D). No phospho-JNK was detectedin control lines before stress (Fig. 8E), but it was present in thenucleus of KEB-7 both before and after osmotic stress (Fig.8G,H), again confirming its constitutive activation in thesecells. After osmotic shock, phospho-JNK was also detected inthe nucleus of the control cell line (Fig. 8F). These resultsconcur with the immunoblotting data.

DiscussionThe first stage in the development of any new therapy forkeratin-based skin fragility disorders, such as EBS, will dependon in vitro culture studies with cell lines expressing EBS-associated mutations. Useful cell model systems must be ableto distinguish between the effects of different mutations, sincethese may require different clinical strategies to treat them.Differences in response to heat shock were reported betweenkeratinocytes expressing wild-type versus EBS mutant keratins(Morley et al., 1995), but this assay was unable to distinguishbetween EBS mutations of different severity. We thereforesought to develop a more discriminating stress assay, on thebasis of hypo-osmotic shock. During initial investigations itbecame clear that the outcome of the osmotic stress wasdependent on the state of confluence of the epithelial cells, tothe extent that long-term reproducibility of the assay becamehard to establish. Thus, we concentrated on the early effects of

Fig. 7. Induction of ATF-2phosphorylation followinghypo-osmotic shock. ATF-2activation was detected byimmunoblotting with rabbitantisera to phospho-ATF-2(pATF2) (New EnglandBiolabs) and visualized bychemiluminescence. Cell linesvaried in the time course ofATF-2 phosphorylation. Inseverely mutated cell linesphosphorylation of ATF-2 wasinduced faster.

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osmotic stress and its consequent changes in c-Jun N-terminalkinase (JNK/SAPK) pathway, as a marker for cellular responseto stress in the presence of pathogenic keratin mutations.

Hypo-osmotic stress is a common physiological situationwith which tissue cells have to cope. Ischaemia and theresulting oxygen deprivation, for example, lead to energydepletion and loss of function of ion pumps in the cellmembrane, and so to a rise in the intracellular osmolarity. Cellswelling follows rapidly, with a substantial but transientdistortion of the cell architecture. Most mammalian cells havedeveloped compensatory mechanisms to respond to osmoticchanges and within minutes will have upregulated the activityof ion channels to restore the osmotic balance. This process,known as regulatory volume decrease (RVD), includes theconcerted opening of K+ and Cl– selective ion channels, leadingto an efflux of KCl, which causes the loss of cellular water(Haussinger, 1996a; Okada et al., 1994). By contrast, if cellsare exposed to hypertonic extracellular fluid, they initiallyshrink but then return to their normal volume, by a regulatorycell volume increase (RVI), achieved by the activation of ionpumps and carriers.

The hypo-osmotic shock was carried out on 80% confluentcells, which were more sensitive to this stress than cells thatwere either 40% confluent or 100% confluent. As cells becomeconfluent, they establish junctions with all their neighboringcells, and with increasing time these desmosomes ‘mature’ anddifferentiate to a more stable state, in which for example theycannot be disrupted by removal of calcium from the medium(Wallis et al., 2000). It is possible that the greater sensitivityof cells at subconfluence is due to the transition betweencell dependence on actin-mediated attachment junctions tointermediate-filament-mediated attachment junctions, withmaturation of desmosomes still incompletely established at thetime of stress. Further studies will be needed to understand thisobservation.

Different effects of osmotic shock on actin, tubulin andintermediate filamentsOf the three cytoskeleton filament systems, the role of the actinnetwork in response to anisosmotic conditions has received themost attention. There is general agreement that hypo-osmoticshock leads to a transient actin reorganization, with which weconcur. This has been variously reported as an increase in F-actin (Czekay et al., 1994; Mountain et al., 1998; Tilly et al.,1996) or a decrease in G-actin (Theodoropoulos et al., 1992),with evidence for Rho kinase signaling involvement (Koyamaet al., 2001; Tilly et al., 1996). Our time course observationssuggest that actin is shifted from filaments subtendingmembrane structures such as microvilli to stress fiber-likebundles of filaments deeper in the cytoplasm. An intact actinfilament cytoskeleton appears to be required for activation ofat least some of the volume regulatory mechanisms. Cellswelling requires actin mobilization and is accompanied by acalcium surge, either or both of which may be involved in theactivation of membrane channels (Cornet et al., 1993; Foskettand Spring, 1985; Jessen and Hoffmann, 1992; Sudlow andBurgoyne, 1997). The resulting RVD response has been shownto be inhibited by both cytochalasin B (actin depolymerization)(Pedersen et al., 1999) and phalloidin (F-actin stabilization)(Shen et al., 1999).

Release of actin from its membrane tethering proteins (e.g.proteins in the focal adhesion or focal contact zones) couldinitiate the signal cascade that triggers the stress response,possibly by integrin-mediated detection of membranestretching (Low et al., 1997; Low and Taylor, 1998). Themovement of actin away from the plasma membrane may also


Fig. 8. Cellular localisation of JNK changes upon phosphorylation.NEB-1 wild-type cells (A,B,E,F) and KEB-7 mutant K14 cells(C,D,G,H) were subjected to hypo-osmotic shock and stained forJNK (A,B,C,D) and phospho-JNK (E,F,G,H). In wild-type cells,native JNK was localized at the cell periphery, in a pattern typical offocal adhesions (A), but, upon phosphorylation, JNK left the focaladhesions and moved to the nucleus (B). In the mutant cells, nuclearJNK was detectable even before osmotic shock (C). Phospho-JNK(pJNK) was absent in wild-type cells before osmotic shock (E) andwas only detectable after the shock (F); but it was constitutivelypresent in mutant cells even before osmotic shock (G). Bar, 10 µm.


directly help the cell to survive a sudden intracellular increasein pressure, by releasing plasma membrane to accommodatevolume expansion and so prevent the cell from rupturing.Raucher and Scheetz have demonstrated the existence of areservoir of plasma membrane with great capacity forexpansion, which is normally tethered by the cytoskeleton(Raucher and Sheetz, 1999). The physical location of suchmembrane reservoirs may actually be in the numerous smallmicrovillar structures that vanish from cell surfaces uponosmotic shock (Fig. 1). These microvilli and microridgesdecorate the free apical surface of epithelial tissues in aqueousenvironments, from internal mammalian epithelia to theepidermal surface of aquatic vertebrates (e.g. Lane andWhitear, 1982). They are readily seen by scanning electronmicroscopy, but there has been no satisfactory explanation oftheir existence to date. They may exist to protect epithelial cellsagainst hypotonic damage.

We observed that osmotic shock has a wider effect on thecytoskeleton than previously described, as even microtubulesare temporarily disrupted. Hypo-osmotic shock causes adramatic but transient tubulin depolymerization, which israpidly reversed. Tubulin has been reported to be depolymerizedby increased pressure (Engelborghs et al., 1976; Larsen et al.,2000), and the time course of tubulin disruption observed here,that is, disruption within 2 minutes and recovery by 4 minutes,is certainly compatible with a direct linkage to the transientpressure increase inside the cell. Other studies commenting onthe effect of osmotic shock on microtubules have suggested thatperturbation of microtubule kinetics either has no effect on, orinhibits, the RVD (Downey et al., 1995; Edmonds and Koenig,1990; Shen et al., 1999; Zhang et al., 1997), and thathypotonicity may stabilize microtubules and stimulate tubulinsynthesis (Haussinger et al., 1994).

Few studies have been undertaken to date on the responseof intermediate filaments to anisosmotic conditions. Astrocytesdevoid of any intermediate filaments (GFAP–/–, vimentin–/–)had a less effective response to osmotic stress, as measured bytaurine efflux (Ding et al., 1998). Our results also suggesta role for keratin intermediate filaments in protecting cellsagainst osmotic stress. A keratin cytoskeleton network,compromised by dominant-negative mutations known to bepathogenic in situ, will also render cells less able to cope with,and recover from, osmotic stress.

The different responses to osmotic stress that we observe inthe three cytoskeleton filament systems also point to anotherfunctional aspect of intermediate filament evolution. Ifmicrotubules are intrinsically unable to withstand pressure, andfilamentous actin needs to be remodeled to accommodate cellswelling, this leaves the intermediate filament network as theonly one of the three cytoskeleton filament systems that can bemaintained through hypo-osmotic stress, to provide a referenceframework for the steady state of the cell. Osmotic shock is sucha universal stress in multicellular organisms, especially on theepithelial surface, that this may be one of the driving forcesbehind the evolution of the intermediate filament cytoskeleton.

Stress response activation by osmotic shockHypo-osmotic stress triggers the MAPK pathway, leading toactivation of the Jun-NH2-terminal kinase (JNK/SAPK)pathway, via MAPK kinase (MKK4) or via the extracellular

signal-regulated kinases ERK-1 and ERK-2 (Haussinger,1996b; Lang et al., 1998). JNK is known to be involved incellular responses to environmental stresses. These include UVlight (Derijard et al., 1994), γ-irradiation (Chen et al., 1996),osmotic shock (Niisato et al., 1999) and mechanical stress(Kippenberger et al., 2000). JNK activation is also induced bymitogenic signals, including growth factors, oncogenic Ras andCD40 ligation. Targets of JNK signaling pathways include thetranscription factors ATF-2 and c-Jun, which are members ofthe basic leucine zipper (bZIP) group that bind to AP-1 andAP-1-like sites (Whitmarsh and Davis, 1996) within thepromoters of many genes. JNK phosphorylates two sites withinthe activation domain of each transcription factor, leading toan increased transcriptional activity.

In this paper, we have monitored the osmotic stress responseof cell lines carrying a mutation in K5 or K14 by tracking theprogress of JNK phosphorylation. Using immunocytochemicalanalysis, we confirmed that, once activated, JNK moves towardthe nucleus, where it can signal to nuclear transcription factorsand thus activate transcription. We present evidence for a directcorrelation between the severity of the mutation carried in thecell line and the speed of JNK activation. This is the first cellculture assay with a read-out that varies with the gradedseverity of the mutations in the clinic and should therefore bea useful monitor of the effect of mutations. In addition, weobserved that the severely mutated cell lines (only) appear tobe in a permanent ‘stress’ condition, as they exhibit asignificant level of JNK activation even without osmotic shock.We speculate that this may be due to the increasedphysiological burden on the cell, incurred by the necessity ofhandling a defective keratin protein. This aspect of EBS cellphysiology may have a direct bearing on the clinical phenotypeof epidermolysis bullosa simplex.

As we come to understand the molecular basis of keratindisorders, we are faced with the challenge of devising practicaland effective therapy for these genetic diseases. Any newtherapeutic strategies will need extensive preclinicaldevelopment work, beginning with in vitro model systems, inwhich the defects can be induced and then repaired. Inconjunction with the appearance of cell culture models,response to hypo-osmotic shock is a practical surrogatemeasure of the mutation burden on keratinocytes. We expectthat this will be useful in monitoring the effect of gene therapyon EBS keratinocyte cell lines.

This work was supported by the Dystrophic Epidermolysis BullosaResearch Association, D.E.B.R.A., (LANE3 to E.B.L., supportingM.D.) and by the Cancer Research UK (SP2060/0103 to E.B.L.,supporting S.M.M.). D.R. is supported by a Medical Research Councilstudentship (G78/6810). We are grateful to John James for his helpwith the scanning electron microscopy and to Martyn Ward and PeterTaylor for their helpful discussions on the osmotic shock procedure.A special thank you to Mirjana Liovic and Mandy Gulbransen fortheir help and support.

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The role of keratins in response to osmotic shock...severity of epidermolysis bullosa simplex. Cells with severe mutations were more sensitive to osmotic stress and took longer to