epithelial-to-mesenchymal transition of peritoneal...

INTRODUCTIONPeritoneal dialysis (PD) is an alternative to hemodialysis for thetreatment of end-stage renal disease. During this process, theperitoneal membrane (PM) acts as a permeability barrier acrosswhich ultrafiltration and diffusion take place (Aroeira et al., 2007;Krediet et al., 1999). The PM consists of a single layer of mesothelialcells (MCs) covering a submesothelial region composed ofconnective tissue and small numbers of fibroblasts, mast cells,macrophages and blood vessels. Continuous exposure tohyperosmotic, hyperglycemic and acidic dialysis solutions, as wellas episodes of peritonitis and hemoperitoneum, may cause acuteand chronic inflammation and injury to the PM, whichprogressively undergoes fibrosis, angiogenesis and hyalinizingvasculopathy (Aroeira et al., 2007). PM injury is thus a seriousconcern during PD because it can lead to the loss of dialyticfunction.

Of the cytokines and growth factors produced during peritonealinflammation, transforming growth factor (TGF)-β1 is consideredthe master molecule in the genesis of peritoneal fibrosis, sinceincreased levels of TGF-β1 in peritoneal dialysate correlate with

worse PD outcomes (Lin et al., 1997). Moreover, combinedtreatment with interleukin (IL)-1β and TGF-β1 induces biochemicaland morphological changes in omental MCs that are reminiscentof those that occur during epithelial-to-mesenchymal transition(EMT) (Yañez-Mo et al., 2003). EMT is a complex step-wisephenomenon that occurs during embryonic development andtumor progression, and that has more recently been described inchronic inflammatory and fibrogenic diseases (Thiery and Sleeman,2006). EMT is characterized by the disruption of intercellularjunctions, replacement of apical-basolateral polarity with front-to-back polarity, and acquisition of migratory and invasive phenotypes.Cells that have undergone EMT also acquire the capacity toproduce extracellular matrix components and a wide spectrum ofinflammatory, fibrogenic and angiogenic factors.

The establishment and progression of EMT is controlled bymultiple molecular mechanisms that appear to be cell-type specific.One key regulator is the transcription factor snail homolog 1(hereafter referred to as Snail1), a potent transcriptional repressorof E-cadherin (Cano et al., 2000; Barrallo-Gimeno and Nieto, 2005).Snail1 expression integrates a complex network of intracellularsignals, including integrin-linked kinase (ILK), phosphatidylinositol3-kinase (PI3-K), the mitogen-activated protein kinases (MAPKs),glycogen synthase kinase (GSK)-3β, and the transcription factorNF-κB (De Craene et al., 2005). NF-κB has recently been shownto play a major role in EMT induction in a Ras-transformed cancercell model (Huber et al., 2004). In resting cells, cytoplasmic NF-κB is complexed to inhibitor of kappaB (IκB), which upon activationis phosphorylated and subsequently degraded, allowing the releaseand nuclear translocation of NF-κB to activate target genes. Various

RESEARCH ARTICLE

Disease Models & Mechanisms 1

Disease Models & Mechanisms 1, 000-000 (2008) doi:10.1242/dmm.001321

Epithelial-to-mesenchymal transition of peritonealmesothelial cells is regulated by an ERK/NF-κB/Snail1pathwayRaffaele Strippoli1, Ignacio Benedicto2, Maria Luisa Pérez Lozano2, Ana Cerezo1, Manuel López-Cabrera2,3,‡ and Miguel A. del Pozo1,*,‡

SUMMARY

Epithelial-to-mesenchymal transition (EMT) occurs in fibrotic diseases affecting the kidney, liver and lung, and in the peritoneum of patients undergoingperitoneal dialysis. EMT in the peritoneum is linked to peritoneal membrane dysfunction, and its establishment limits the effectiveness of peritonealdialysis. The molecular regulation of EMT in the peritoneum is thus of interest from basic and clinical perspectives. Treatment of primary humanmesothelial cells (MCs) with effluent from patients undergoing peritoneal dialysis induced a genuine EMT, characterized by downregulated E-cadherinand cytokeratin expression, cell scattering, and spindle-like morphology. This EMT was replicated by co-stimulation with transforming growth factor(TGF)-β1 and interleukin (IL)-1β. Retroviral overexpression of a mutant inhibitor of kappaB (IκB) demonstrated that NF-κB activation is required forE-cadherin and cytokeratin downregulation during EMT. Pre-treatment with the MAP kinase kinase (MEK)-1/2 inhibitor U0126 showed that cytokine-triggered NF-κB nuclear translocation and transcriptional activity are mediated by activation of extracellular regulated kinase (ERK). Cytokine-mediatedinduction of mRNA expression of the transcription factor Snail1, a repressor of E-cadherin expression and a potent inducer of EMT, was preventedby blockade of ERK or NF-κB. Finally, blockade of ERK/NF-κB signaling in ex vivo MCs that were cultured from peritoneal dialysis effluents revertedcells to an epithelioid morphology, upregulated E-cadherin and cytokeratin expression, and downregulated Snail1 expression. Modulation of theERK/NF-κB/Snail1 pathway may provide a means of counteracting the progressive structural and functional deterioration of the peritoneal membraneduring peritoneal dialysis.

1Integrin Signaling Laboratory, Department of Vascular Biology and Inflammation,Centro Nacional de Investigaciones Cardiovasculares (CNIC), Melchor FernándezAlmagro 3, 28029 Madrid, Spain2Unidad de Biología Molecular, Hospital Universitario de la Princesa, 28006 Madrid,Spain3Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Cantoblanco, 28049Madrid, Spain*Author for correspondence (e-mail: [email protected])‡These authors contributed equally to this work

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http://dmm.biologists.org/lookup/doi/10.1242/dmm.001321Access the most recent version at First posted online on 28 October 2008 as 10.1242/dmm.001321

http://dmm.biologists.org/lookup/doi/10.1242/dmm.001321

kinases, including MAPK, have been implicated in the regulationof IκB (Perkins, 2007; Neumann and Neumann, 2007; Rangaswamiet al., 2004).

Although the molecular regulation of EMT has been extensivelystudied in other cell systems, mostly in tumor cells, the signalingpathways underlying this process in MCs have not been reported.Here, we demonstrate that the extracellular regulated kinase(ERK)/NF-κB/Snail1 signaling pathway is a key regulator of EMTin MCs. Given that EMT by MCs is central to the onset of peritonealfibrosis and angiogenesis, and that there is no effective treatmentfor the progressive loss of peritoneal dialytic capacity in PDpatients, these results provide possible routes for therapeuticintervention.

RESULTSTreatment of primary MCs with either peritonitis effluent or TGF-β1 plus IL-1β induces morphological and biochemical alterationsconsistent with EMTEffluent-derived MCs from PD patients show phenotypic changesreminiscent of EMT – these changes correlate with PD andepisodes of peritonitis or hemoperitoneum (Yañez-Mo et al., 2003).To test whether these changes could be produced in vitro, weexposed omentum-derived MCs from healthy donors (seesupplementary material Fig. S1 and Methods for isolationprocedures and purity) to peritoneal effluent from patientsexperiencing acute peritonitis during PD. Treatment withperitonitis effluent for 72 hours induced a loss of intercellularjunctions, cell scattering, and adoption of a spindled fibroblasticphenotype (Fig. 1A), all of which are characteristic of EMT.Confocal immunofluorescence analysis showed that the treatmentdownregulated the expression of cytokeratin, an epithelial markerthat is highly expressed in untreated MCs (Lopez Cabrera et al.,2006) (Fig. 1B). Expression of E-cadherin, another epithelial marker,was markedly reduced after 24 hours (Fig. 1C). Peritonitis effluentalso upregulated proteins, such as fibronectin and N-cadherin,whose expression is associated with EMT (Fig. 1C; Fig. 5B). Weconfirmed these results in a non-dialysis setting by co-stimulatingMCs with TGF-β1 plus IL-1β. Combined treatment was usedthroughout this study because, although individual stimulation witheither cytokine induces changes associated with MC EMT, such asdecreased E-cadherin and increased β1 and α2 integrin expression(Yañez Mo et al., 2003) (supplementary material Fig. S2A-B), anadditive effect was obtained with the combination of bothcytokines. Similarly to treatment with peritonitis effluent, TGF-β1plus IL-1β induced a spindled phenotype (Fig. 2A) and a loss ofepithelial, and acquisition of mesenchymal markers (Fig. 2B,C).These experiments indicate that both peritonitis effluent and TGF-β1 plus IL-1β induce a genuine EMT in primary MCs.

TGF-β1- and IL-1β-induced downregulation of E-cadherin andcytokeratin in MCs is mediated by NF-κBThere is increasing evidence that NF-κB activation has a role inthe initial stages of EMT during tumor progression (Huber et al.,2004). We found that TGF-β1 and IL-1β, both in combination orseparately, potently induced NF-κB nuclear translocation (Fig. 3A;supplementary material Fig. S2); use of peritonitis effluent gave thesame result (Fig. 3B). To test whether this activation pathwaymediates the observed EMT, we infected primary MCs with a pRV-

IRES-CopGreen bicistronic retroviral vector encoding an IκBαsuper-repressor, a non-degradable mutant form (S32A and S36A)of the repressor IκBα. The IκBα super-repressor blocked NF-κBnuclear translocation induced by TGF-β1 and IL-1β, whereasinfection with empty pRV-IRES-CopGreen retroviral vector hadno effect (Fig. 3C).

Infection with the IκBα super-repressor vector also interferedwith biochemical changes associated with EMT. In cells stimulatedwith TGF-β1 plus IL-1β for 24 hours, expression of the IκBα super-repressor partially blocked the downregulation of E-cadherinprotein expression, whereas this was unaffected in cells infectedwith the pRV-IRES-CopGreen control vector (Fig. 4A). This effectwas also evident at the mRNA level (data not shown), although themagnitude of the effect of the IκBα super-repressor was hamperedby the limited efficiency of viral infection in these primary MCs(10-40%; see Methods). This experiment was therefore repeated inthe human MC line MeT-5A, which, similar to primary MCs,undergoes EMT upon treatment with TGF-β1 and IL-1β.Fluorescence-activated cell sorter (FACS)-purified cultures ofretrovirally infected MeT-5A cells were stimulated, and quantitativereverse transcriptase (RT)-PCR demonstrated that expression of

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ERK/NF-κB/Snail1 in peritoneal EMTRESEARCH ARTICLE

Fig. 1. Omentum-derived MCs undergo EMT upon exposure to peritonitiseffluent. (A) Photomicrographs of confluent monolayers of human primaryomental MCs, non-treated (NT) or treated for 72 hours with peritoneal effluent(diluted 1:1 with culture medium) from a PD patient suffering acute peritonitis.(B) Confocal immunofluorescence (red) of non-treated and treated MCs stainedwith monoclonal antibodies against pan-cytokeratin (72-hour stimulation).Nuclei were stained with Hoechst 33342 (blue). (C) Western blots showing theexpression of E-cadherin and fibronectin in total cell lysates of MCs treated asindicated for 1, 24 or 72 hours. Expression of α-tubulin was detected as a loadingcontrol. Data are representative of three independent experiments.

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the IκBα super-repressor completely blocked cytokine-induced E-cadherin downregulation (Fig. 4B). Expression of the IκBα super-repressor in primary MCs also limited cytokine-induceddownregulation of cytokeratin expression, as shown by confocalimmunofluorescence analysis after 56 hours of treatment (Fig. 4C).The NF-κB activation pathway thus controls both E-cadherin andcytokeratin downregulation during EMT in primary MCs.

Downregulation of E-cadherin and cytokeratin during EMT in MCsis controlled by the ERK pathwayWe analyzed upstream signaling events that might account for NF-κB activation in our experimental system. Recent reports in variouscellular models have documented a role for MAPKs, particularlyERKs, in the establishment of EMT (Zavadil and Bottinger, 2005).

Treatment of MCs with TGF-β1 plus IL-1β induced a high degreeof ERK phosphorylation (Fig. 5A); peritonitis effluent produced asimilar effect (Fig. 5B).

To examine the role of ERK activation in EMT, we pretreated MCswith U0126, a pharmacological inhibitor of MAPK kinase (MEK)-1/2 – the upstream activator of ERK. U0126 markedly suppressedcytokine-mediated downregulation of E-cadherin and cytokeratinprotein expression (Fig. 5C,D). U0126 also inhibited cytokine-


ERK/NF-κB/Snail1 in peritoneal EMT RESEARCH ARTICLE

Fig. 2. Omentum-derived MCs undergo EMT upon TGF-β1 and IL-1βstimulation. (A) Photomicrographs of confluent monolayers of human primaryomental MCs, non-treated (NT) or treated with TGF-β1 (0.5 ng/ml) incombination with IL-1β (2 ng/ml) for 24 hours (T/I). (B) Confocalimmunofluorescence (red) of non-treated and treated MCs stained withmonoclonal antibodies against E-cadherin (24-hour stimulation) and with pan-cytokeratin (56-hour stimulation). Nuclei were stained with Hoechst 33342(blue). (C) Western blots showing the expression of E-cadherin, N-cadherin andfibronectin in total cell lysates of MCs treated as indicated with TGF-β1 and IL-1βfor 24 or 48 hours. Expression of α-tubulin was detected as a loading control.Data are representative of more than ten independent experiments.

Fig. 3. NF-κB nuclear translocation is inhibited by IκBα super-repressorexpression. (A) Confocal immunofluorescence of NF-κB expression andlocalization. Human primary peritoneal MCs were co-stimulated with TGF-β1and IL-1β for the indicated times; NT, non-treated controls. Fixed andpermeabilized cells were stained with a polyclonal antibody against p65 NF-κB.The histogram shows mean fluorescence intensities of nuclear NF-κB stainingquantified using the software LAS-AS from Leica. Bars represent s.e.m. A total of50 cells were analyzed per condition; AU, arbitrary units. (B) Human primaryperitoneal MCs stimulated with peritonitis effluent or control medium wereanalyzed as in (A). ***P<0.0001 compared with medium-treated cells. (C) Primaryomental MCs were infected with either a Cop Green-tagged retrovirus encodingan IκBα super-repressor (IκB, lower panels) or with empty Cop Green-taggedvirus (empty vector, upper panels). Cells were then co-stimulated for 30 minuteswith TGF-β1 and IL-1β. Fixed and permeabilized cells were stained for p65 NF-κB(yellow in overlay image). Nuclei were stained with Hoechst 33342 (blue inoverlay). Green fluorescence shows infected cells. The spectral confocaltechnology allowed discrimination between yellow and green emittedfluorescence. Overlay images show overlapping of Hoechst 33342, anti-NF-κBand Cop Green staining. Data shown are representative of three independentexperiments.

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induced downregulation of E-cadherin mRNA expression at all timesanalyzed (Fig. 5E). Similar results were obtained with the MEK-1/2inhibitor PD98059 (20 μM; not shown). These results strongly suggestthat the ERK activation pathway mediates the TGF-β1- and IL-1β-induced downregulation of E-cadherin and cytokeratin.

ERK regulates NF-κB nuclear localization and transcriptionalactivity during cytokine-induced EMTWe investigated whether the role of ERK signaling in EMT mightbe mediated through the induction of NF-κB activity. Pretreatmentof MCs with U0126 reduced the intensity and persistence of NF-

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Fig. 4. Downregulation of epithelial markers in cytokine-stimulated MCsrequires NF-κB signaling activity. (A) Western blots showing the expression ofE-cadherin and IκB in total MC lysates. Primary omental MCs were infected witheither Cop Green-tagged retrovirus encoding an IκBα super-repressor (IκB) orwith empty Cop Green-tagged virus and stimulated for 24 hours with TGF-β1and IL-1β (T/I) as indicated; NT, non-treated cells. Expression of α-tubulin wasdetected as a loading control. The histogram shows E-cadherin/α-tubulin bandintensity ratios from a representative experiment. (B) Effect of NF-κB inhibitionon E-cadherin mRNA expression in MCs. MeT-5A MCs were infected as abovewith retrovirus encoding an IκBα super-repressor or empty control plasmid(CopG). After infection, MeT-5A cultures were sorted for green fluorescence toobtain near pure cultures of infected cells. Cultures were left untreated (graybars) or co-stimulated for 24 hours with TGF-β1 and IL-1β (black bars) andquantitative RT-PCR was performed on total RNA. Histone H3 mRNA levels wereused for normalization. Bars represent the means ± s.e.m. from six independentexperiments. (C) Confocal immunofluorescence analysis of cytokeratinexpression. Cells infected and stimulated as in (A) were fixed, permeabilized, andstained with a monoclonal antibody against pan-cytokeratin. Green, Cop Greenfluorescence.

Fig. 5. ERK controls E-cadherin and cytokeratin downregulation during EMTof cytokine-stimulated MCs. (A) Western blot showing expression ofphosphorylated (active) ERK (pERK, top) in MC total lysates. Primary omentalMCs were left untreated (NT) or co-stimulated with TGF-β1 and IL-1β (T/I) for thetimes indicated. As a loading control, samples were probed for total ERKexpression (bottom). (B) Primary omental MCs incubated for 24 hours withdialysis fluid (D), non-peritonitis effluent (E) or peritonitis effluent (P) wereanalyzed as in (A). (C) Western blot showing expression of E-cadherin andphosphorylated (active) ERK in MC total lysates. Omental MCs were pretreatedwith DMSO or U0126 (20 μM), and treated for 24 hours with TGF-β1 and IL-1β asindicated. Total ERK expression was detected as a loading control. (D) Confocalimmunofluorescence analysis of cytokeratin expression. Omental MCs werepretreated with DMSO or U0126 (20 μM) and then co-stimulated with TGF-β1and IL-1β for 56 hours. Cells were fixed, permeabilized, and stained with amonoclonal antibody against pan-cytokeratin. (E) Effect of ERK inhibition on E-cadherin mRNA expression in MCs. Cells were pretreated with DMSO (gray bars)or U0126 (black bars) and co-stimulated for the times indicated with TGF-β1 andIL-1β (T/I); NT, non-stimulated controls. Quantitative RT-PCR was performed ontotal RNA. Histone H3 mRNA levels were used for normalization. Bars representthe means ± s.e.m. of duplicate determinations from three independentexperiments.

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κB nuclear staining upon combined cytokine treatment (Fig. 6A).To test the effect of ERK on NF-κB transcriptional activity, wetransfected MCs with a luciferase reporter construct containingmultiple NF-κB binding sites (KBF-luc). Pretreatment of these cellswith U0126 markedly reduced cytokine-induced luciferase activity(Fig. 6B). To directly examine the role of ERK signaling in NF-κB-mediated gene expression, we analyzed the effect of U0126 on theexpression of cyclooxygenase-2 (COX-2), a gene whose expressionis regulated by NF-κB and is induced during EMT. U0126pretreatment potently inhibited COX-2 mRNA expression inducedby TGF-β1 plus IL-1β (Fig. 6C), supporting a role for ERK in theregulation of NFκB-mediated gene expression during EMT.

The ERK/NF-κB activation pathway regulates Snail1 expressionNext, we analyzed signaling events downstream of NF-κB thatmight account for EMT induction. Snail1 is the best-knownmember of a family of zinc-finger transcriptional regulatorsinvolved in EMT induction. Snail1 directly blocks transcriptionfrom the E-cadherin promoter and appears to be involved incytokeratin gene repression (Cano et al., 2000; Guaita et al., 2002).Quantitative RT-PCR showed that stimulation of primary MCs withTGF-β1 and IL-1β induced an increase in Snail1 mRNA expression,which was blocked by pretreatment with U0126 (Fig. 7A). This wasaccompanied by increased Snail1 expression and nuclearlocalization (Fig. 7B, top). Similar to its effect on Snail1 mRNAexpression, pretreatment with U0126 markedly decreased Snail1protein expression in cytokine-stimulated cells (Fig. 7B, bottom).

To analyze the role of NF-κB activity in Snail1 expression, weinfected primary MCs with either the retrovirus encoding the IκBαsuper-repressor or the empty vector pRV-IRES-CopGreen, andstimulated them with TGF-β1 and IL-1β. Exogenous expression ofthe IκBα super-repressor partially reduced cytokine-inducedexpression of Snail1 mRNA (Fig. 7C, left). To control for the limitedinfection efficiency in these cells, this experiment was repeated inFACS-purified cultures of retrovirally-infected MeT-5A cells. Inthese cells, expression of the IκBα super-repressor effectivelyinhibited cytokine-induced Snail1 mRNA expression (Fig. 7C,right). These results strongly suggest a causal role for the ERK/NF-κB activation pathway in Snail1 expression in MCs.

Inhibition of ERK or NF-κB restores the epithelial phenotype intransdifferentiated MCs from peritoneal effluentEMT by peritoneal MCs in PD patients is linked to PM dysfunction(Aroeira et al., 2007; Yañez Mo et al., 2003; Aroeira et al., 2005; DelPeso et al., 2008). Given the role of ERK and NF-κB signaling inthe genesis of EMT in cytokine-stimulated MCs in culture, wewondered whether this pathway might control the maintenance ofthe mesenchymal phenotype in PD effluent-derived MCs that havealready undergone EMT in vivo. To investigate this, we obtainedMCs from the effluents of 13 patients undergoing PD (Table 1).The parameters used to evaluate the different stages oftransdifferentiation of these cells were both morphological(epithelial-like or non-epithelioid) and biochemical [reduced levelsof E-cadherin and cytokeratins, increased expression of vascularendothelial growth factor (VEGF) and vimentin], as described inpublished studies (Yañez Mo et al., 2003; Aroeira et al., 2005) (datanot shown). Western blot analysis showed that, compared withcontrol omentum samples, untreated PD effluent-derived MCs had

significantly increased levels of active ERK (Fig. 8A). PD effluent-derived MCs were either treated with U0126 or infected withretrovirus encoding the IκBα super-repressor. Microscopy of non-epithelioid effluent-derived MCs showed that treatment withU0126 reverted cells to an epithelial morphology (Fig. 8B). Westernblot analysis showed that expression of E-cadherin, in bothepithelial-like and non-epithelioid effluent-derived MCs, wasrestored in both U0126-treated and IκBα super-repressor-expressing cells (Fig. 8C). Furthermore, confocalimmunofluorescence showed that U0126 treatment increased thelevels of cytokeratin in non-epithelial MCs from PD effluents (Fig.



Fig. 6. ERK controls NF-κB nuclear translocation and transcriptional activityinduced by TGF-β1 and IL-1β stimulation in MCs. (A) Confocalimmunofluorescence analysis of NF-κB expression and localization. OmentalMCs were pretreated with DMSO or U0126 (20 μM) and then co-stimulated forthe times indicated with TGF-β1 and IL-1β (T/I); NT, non-treated. Fixed andpermeabilized cells were stained with a polyclonal antibody against p65 NF-κB.The results shown are from a single experiment that was representative of threeindependently performed experiments. (B) Effect of ERK inhibition on NF-κBtranscriptional activity. MeT-5A cells were transiently transfected with the KBF-luc reporter plasmid together with Renilla luciferase. Cells were pretreated asindicated with U0126 (20 μM) and then left untreated (NT, gray bars) or co-stimulated for the times indicated with TGF-β1 and IL-1β (T/I, black bars). Barsrepresent means ± s.e.m. determinations from three independent experimentscarried out in duplicate. *P<0.05 compared with DMSO-treated cells.(C) Omental MCs were pretreated with DMSO or U0126 (20 μM) and treated asindicated. Qualitative RT-PCR was performed with specific primers for COX-2 andHistone 3.

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8D). U0126 also impaired NF-κB nuclear translocation (Fig. 8D).Finally, U0126 treatment of non-epithelioid effluent-derived MCsalso downregulated Snail1 mRNA expression and upregulated E-

cadherin mRNA expression (Fig. 8E). The reversal of EMT byblockade of ERK or NF-κB in these experiments strongly supportsa role for the ERK/NF-κB activation pathway in the maintenanceof the mesenchymal phenotype in the peritoneum of patientsundergoing PD.

DISCUSSIONThis study aimed to characterize the signaling pathways controllingthe establishment of EMT in primary MCs, stimulated with eitherperitoneal effluent from peritonitis patients or TGF-β1 and IL-1β.Our results, obtained with pharmacological inhibitors and infectionwith retroviral vectors, demonstrate that an ERK/NF-κB/Snail1signaling pathway controls cytokine-induced downregulation of E-cadherin and cytokeratin during EMT in MCs. Moreover, blockadeof this signaling pathway in transdifferentiated MCs isolated fromPD effluents reverses EMT.

There is increasing evidence that EMT, far from being limitedto development and cancer, also occurs in other pathophysiologicalsituations, including chronic inflammatory and fibrotic diseasesaffecting the kidney, liver and lung (Thiery and Sleeman, 2006;Kalluri and Neilson, 2003; Iwano et al., 2002). In peritoneal fibrosis,the presence of transdifferentiated MCs in the peritoneum andeffluent of patients undergoing PD is associated with recurrentacute and chronic inflammation, and has been linked to a declinein peritoneal function (Aroeira et al., 2007; Yañez Mo et al., 2003;Aroeira et al., 2005; Del Peso et al., 2008). Our results demonstratethat the exposure of normal MCs to peritonitis effluent frompatients undergoing PD is sufficient to drive MC EMT. Moreover,we obtained the same results by stimulating MCs with TGF-β1 andIL-1β at concentrations comparable to those found in inflamedperitoneum of PD patients (Yañez Mo et al., 2003; Lai et al., 2000).

Our approach of combining TGF-β1 and IL-1β attempts toreproduce something of the complex mixture of proinflammatoryand profibrotic stimuli induced during peritoneal EMT in vivo.TGF-β1 alone has been widely reported to induce EMT, includingin MCs (Yañez Mo et al., 2003). However, combined cytokinetreatment has additive effects on cell morphology (Yañez Mo etal., 2003), E-cadherin downregulation (supplementary material Fig.S2) and increased β1 and α2 integrin expression, and enhancesmigration by transdifferentiated cells (Yañez Mo et al., 2003)(supplementary material Fig. S2). Moreover, there are severalpoints of cross-talk between signaling pathways activated by TGF-β1 and IL-1β (Lu et al., 2007), thus their combined use provides amore physiological analysis for specific biological responses.

Reproduction of the effects of peritonitis effluent on primaryMCs (loss of intercellular junctions, cell scattering, and acquisitionof a spindle-like morphology) by the cytokine combinationestablishes the experimental system used here as a valid model forthe study of peritoneal EMT. Cytokine treatment also triggers rapidE-cadherin internalization and degradation. In addition, we foundthat both peritonitis effluent and cytokine treatment induced denovo expression of fibronectin and increased expression of N-cadherin. The switch towards the expression of non-epithelialcadherins, such as N-cadherin, may be related to increased motilityand invasiveness of transdifferentiated MCs. Meanwhile, expressionof the extracellular matrix component fibronectin may be linkedto the role of MCs in the genesis of peritoneal fibrosis (Maeda etal., 2005).

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Fig. 7. ERK and NF-κB control Snail1 expression induced by TGF-β1 plus IL-1β co-stimulation in MCs. (A) Effect of ERK inhibition on Snail1 mRNAexpression in MCs. Omental MCs were pretreated with DMSO (gray bars) or20 μM U0126 (black bars) and co-stimulated as indicated with TGF-β1 and IL-1β(T/I); NT, non-treated. Quantitative RT-PCR was performed on total RNA. HistoneH3 mRNA expression was used for normalization. Bars represent means ± s.e.m.of duplicate determinations from three independent experiments. (B) Effect ofERK inhibition on Snail1 protein immunofluorescence. Omental MCs werepretreated with DMSO or U0126 (20 μM) and treated as indicated for 24 hours.LiCl (40 mM) and MG132 (10 μM) were added to cells 4 hours before the end ofthe stimulation. Cells were fixed, permeabilized, and stained with a polyclonalantibody against Snail1 before being subjected to confocal microscopy analysis.Panels show Hoechst 33342 staining of cell nuclei (nuclei) andimmunofluorescence staining of Snail1 expression. The results shown are from asingle experiment that was representative of three independently performedexperiments. (C) Effect of NF-κB inhibition on Snail1 mRNA expression in MCs.Human primary omental mesothelial cultures (omentum, left panel) or the MeT-5A MC line (right panel) were infected with a retrovirus encoding an IκBα super-repressor (IκB) or empty control plasmid (CopG). After infection, MeT-5A cultureswere sorted for green fluorescence to obtain near pure cultures of infected cells.Cultures were left untreated (NT, gray) or co-stimulated for 24 hours with TGF-β1and IL-1β (black), and quantitative RT-PCR was performed on total RNA. HistoneH3 mRNA expression was used for normalization. Bars represent means ± s.e.m.of duplicate determinations from six independent experiments.

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A crucial role for ERK, p38 and JNK MAPK signaling pathwaysin the genesis of EMT has been widely demonstrated; however, theireffects appear to be cell-type specific (Grotegut et al., 2006;Santibanez, 2006; Bhowmick et al., 2001). Our results underlinethe importance of ERK signaling in the onset of mesothelial EMTin response to stimulation with TGF-β1 plus IL-1β. We found thatinhibition of ERK signaling prevents cell scattering and theacquisition of a spindle-like phenotype, and also blocksdownregulation of E-cadherin and cytokeratin during EMT. E-cadherin appears to be regulated by ERK at the level oftranscription, although an effect on the E-cadherin endocytosis-degradation pathway cannot be excluded.

NF-κB activation plays a major role in EMT in a Ras-transformedcancer model (Huber et al., 2004). In our study, expression of aretrovirally encoded IκBα super-repressor demonstrates that NF-κB controls both E-cadherin and cytokeratin downregulation duringMC EMT. To our knowledge, the current report is the first todemonstrate involvement of NF-κB in a primary cell culture modelof EMT. Moreover, this report is the first to demonstrate a linkbetween ERK and NF-κB signaling in relation to EMT, as indicatedby impairments in both NF-κB nuclear translocation andtranscriptional activity in cells pretreated with the MEK inhibitorU0126 before cytokine stimulation. This link is further supportedby U0126-induced blockade of COX-2 expression – an NF-κB-induced gene that can be upregulated during EMT. Althoughregulation of NF-κB transcriptional activity by the ERK activationpathway has been demonstrated previously, the molecularmechanism of this effect remains unclear. The Ras-ERK pathway hasbeen shown to mediate NF-κB-induced gene expression inmacrophages through an effect on IκB kinase (IKK) α/β activationand IκBα degradation (Chen et al., 2004). ERK can phosphorylateIκB in vitro and can also bind to, and directly phosphorylate, NF-κB (Dhawan and Richmond, 2002). Our results indicate that ERKregulates NF-κB translocation in MCs, possibly via an action on IκB.There may also be a role for ERK-mediated NF-κB phosphorylation,which has been demonstrated to affect NF-κB transcriptional activity.

However, it should be emphasized that the link between ERK andNF-κB does not exclude the possibility that ERK regulates EMTindependently of NF-κB.

Snail1 is considered a key transcriptional regulator of EMTbecause of its direct inhibitory action on the E-cadherin promoter(Cano et al., 2000; Batlle et al., 2000). In addition to this, Snail1 isemerging as an overall regulator of EMT (Barrallo Gimeno andNieto, 2005). In our experimental system, combined cytokinetreatment induces a marked increase in Snail1 expression, whichinversely correlates with E-cadherin expression levels. Thedemonstration, in pharmacological and retroviral assays, thatinduction of Snail1 expression is dependent on ERK and NF-κB isconsistent with studies of Snail1 promoter activation (Barbera etal., 2004) and the recent confirmation that Snail1 expression isdownstream of NF-κB activation (Bachelder et al., 2005). Snail1activity has recently been shown to be regulated byphosphorylation, which regulates its subcellular localization(Dominguez et al., 2004). Our confocal immunofluorescenceexperiments show that cytokine-induced Snail1 expression andnuclear localization in MCs is dependent on ERK activity. Theinteraction between ERK, NF-κB and Snail1 may bemultidirectional; ERK activation can be induced by the IKKcomplex (Huber et al., 2004), and can also be regulated by Snail1(Barrallo Gimeno and Nieto, 2005). Furthermore, Snail1 alsoregulates ERK expression (Peiro et al., 2006). Thus, there may bea negative feedback mechanism in the regulation of Snail1 activity,which might be dysregulated under pathological conditions.

In vitro and in vivo studies have demonstrated that EMT can bereversed (Huber et al., 2004; Zeisberg et al., 2003), and cytokinessuch as bone morphogenetic protein-7 (BMP7) have been shownto play a role in this reversal in several organs and tissues, includingtransdifferentiated MCs (Zeisberg et al., 2003; Zeisberg et al., 2007;Vargha et al., 2006). Transdifferentiated cells from peritonealeffluents of patients undergoing PD express Snail1 at high levels(Yañez Mo et al., 2003). Coupled to this finding, our observationthat ERK is activated in ex vivo cultured MCs from CAPD patients



Table 1. Enumeration and staging of effluent-derived mesothelial cells employed in this study

Figure 8B 8A 8C; 8D 8C 8D 8D 8E

Patient No.

Microscopic

analysis

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phosphorylation

E-cadherin/

U0126

E-cadherin/

I B

Cytokeratin/

NF B/U0126

Cytokeratin/

U0126/WB

Snail/E-cadherin/

U0126/PCR

1 E X X X X

2 NE X X X X

3 NE X X

4 E X X

5 E X X X

6 NE X X

7 E X

8 E X

9 NE X X X

10 NE X X X X

11 NE X X

12 E X X

13 NE X X

The parameters used to evaluate the different stages of transdifferentiation of these cells were both morphological and biochemical (see Results). X refers to the use of each sample

in the experiments shown in Fig. 8.

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(Fig. 7A) suggests a role for the ERK/NF-κB/Snail1 pathway in thegenesis and maintenance of EMT in these cells. This view is stronglysupported by our finding that blockade of ERK or NF-κB signalinginduces a reverse MET (mesenchymal to epithelial transition),characterized by adoption of epithelial cell morphology andincreased protein expression of E-cadherin and cytokeratin.

Our results suggest that the ERK/NF-κB/Snail1 pathway israpidly activated during combined stimulation with TGF-β1 plus

IL-1β, and mediates the progression and stabilization of themesenchymal state in peritoneal MCs. This study is the firstextensive characterization of a signaling pathway controlling EMTin a non-tumoral primary cell culture model, and providesknowledge of basic and clinical relevance, since it could form therationale for the development of drugs able to counteract theprogressive deterioration of the PM that occurs in patientsundergoing PD.

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Fig. 8. Inhibition of ERK and NF-κB can reverse the EMT in transdifferentiated MCs from the peritoneal effluent of patients undergoing continuousambulatory peritoneal dialysis (CAPD). (A) Western blot showing expression of pERK in monolayer cultures of MCs derived from either the peritoneal effluent ofpatients undergoing CAPD or from normal omentum. Confluent monolayers of omental MCs from control donors or from CAPD patients were lysed, and lysateswere blotted with monoclonal antibodies against pERK. Total ERK expression was detected as a loading control. The histogram shows pERK/total ERK band intensityratios. *P<0.05 control donors compared with CAPD patients. (B) Photomicrographs of confluent monolayers of non-epithelioid effluent-derived MCs treated for 48hours with DMSO (NT) or U0126 (20 μM). (C) Top, western blot showing expression of E-cadherin in monolayer cultures of effluent-derived MCs or control MCs fromnormal omentum (O). Confluent monolayers of MCs were treated with DMSO or U0126. Alternatively, cells were infected with either a retrovirus encoding an IκBαsuper-repressor (IκB) or with empty virus (CopGreen). After 48 hours, cells were lysed and the lysates subjected to western blotting with monoclonal anti-E-cadherinantibody. Expression of α-tubulin was detected as a loading control. Bottom, densitometry of western blots showing E-cadherin expression upon treatment withDMSO or U0126 (patients 1-6), or infection with IκBα super-repressor (IκB) or empty virus (CopG) (patients 1, 2, 5, 6). *P<0.05 compared with DMSO-treated or empty-vector-infected cells. (D) Top, confocal immunofluorescence analysis of the effect of ERK inhibition on NF-κB and cytokeratin expression in transitional peritonealmesothelium. Confluent monolayers of effluent-derived MCs (patient 10), showing non-epithelioid morphology, were treated with DMSO or U0126 for 56 hours.Cells were stained with polyclonal anti-p65 NF-κB and monoclonal anti-cytokeratin antibodies. Nuclei were stained with Hoechst 33342 (blue). Bottom-left, thehistogram shows mean fluorescence intensities of nuclear NF-κB staining from cells treated as in the top panel (patients 1-3, 9, 10), quantified using the softwareLAS-AS from Leica. Bars represent s.e.m. A total of 250 cells were analyzed per condition; AU, arbitrary units. Bottom-center, western blots showing cytokeratin and E-cadherin expression in non-epithelioid effluent-derived MCs treated with DMSO (D) or U0126 (U0) (patients 11-13). Bottom-right, quantification of the western blotshown in the middle panel. (E) Snail1 and E-cadherin mRNA expression in effluent-derived MCs (patients 7-10) treated with DMSO (gray) or U0126 (black) for 24hours. Quantitative RT-PCR was performed on total RNA. Histone H3 mRNA expression was used for normalization. Bars represent means ± s.e.m. of duplicatedeterminations from four independent experiments using cells from four different patients. *P<0.05 compared with DMSO-treated cells.

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METHODSIsolation and culture of MCsHuman MCs were obtained by digestion of omentum samplesfrom patients who were undergoing unrelated abdominal surgery(Stylianou et al., 1990). The samples were digested with a 0.125%trypsin solution containing 0.01% EDTA. Cells were cultured inEarle’s M199 medium supplemented with 20% fetal calf serum,50 U/ml penicillin, 50 μg/ml streptomycin, and 2% Biogro-2(containing insulin, transferrin, ethanolamine, and putrescine)(Biological Industries, Beit Haemek, Israel). To induce EMT, MCswere treated with a combination of human-recombinant TGF-β1(0.5 ng/mL) and IL-1β (2 ng/mL) (R&D Systems, Minneapolis,MN) as described previously (Yañez Mo et al., 2003; Aroeira etal., 2005). Although both TGF-β1 and IL-1β separately are ableto induce EMT phenotypic changes, combined stimulationinduces a genuine EMT (Yañez Mo et al., 2003). The cytokinedoses used are in the range of those detected in peritoneal-dialysisfluids in the presence of peritonitis (Lai et al., 2000) and are similarto those used in previous studies (Yañez Mo et al., 2003; Yang etal., 1999).

Effluent-derived MCs were isolated from 13 clinically stablePD patients using a method described previously (Lopez Cabreraet al., 2006). Cells were cultured as above, and after 10-15 dayscultures reached confluence and were split at a ratio of 1:2. Themorphological features of cells in confluent cultures werecompared and remained stable during the two to three passagesused for experiments. Confluent MC cultures from PD effluentsshow one of two major phenotypes, epithelial-like or non-epithelioid, which remain stable for two to three cell passages(Aroeira et al., 2005). Of the 13 effluent-derived MC culturesevaluated, six had the epithelial-like phenotype and seven hadthe non-epithelioid phenotype. To control for fibroblastcontamination, the purity of omentum and effluent-derived MCcultures was determined from the expression of the standardmesothelial markers, intercellular adhesion molecule (ICAM)-1and cytokeratins (Aroeira et al., 2007). MCs expressed high levelsof ICAM-1 and low levels of the fibroblast-specific marker S100,allowing MCs to be easily distinguished from peritonealfibroblasts (supplementary material Fig. S1). MC cultures werealso negative for the endothelial marker CD31 and themacrophage marker CD45 (supplementary material Fig. S1).When isolating both omentum- and peritoneal effluent-derivedMCs, we generally obtain highly purified cell populations, with<5% contaminant cells, as determined by FACS analysis (M.L.-C., unpublished results). Purified samples with >5%contaminant cells are routinely discarded.

The study was approved by the ethics committee of the HospitalUniversitario de la Princesa (Madrid, Spain). Written informedconsent was obtained from both PD patients included in this study,for the use of effluent samples, and from omentum donors priorto elective surgery.

The human MC line MeT-5A (ATCC, Rockville, MD) wascultured in Earle’s M199 medium, as above, and stimulated withthe same doses of TGF-β1 and IL-1β. MeT-5A is an untransformedMC line, which is increasingly used in peritoneal MC research; dataobtained with this cell line have shown concordance with dataobtained with primary cells (Rampino et al., 2001; Bidmon et al.,2004).

Antibodies and chemicalsThe monoclonal antibody against E-cadherin was purchased fromBD (Becton-Dickinson Laboratories, Mountain View, CA);monoclonal antibodies against tubulin and pan-cytokeratin werefrom Sigma (Saint Louis, MO); polyclonal antibodies against ERKand phospho-ERK were from Cell Signaling (Cell SignalingTechnology, Danvers, MA); polyclonal antibodies against p65 NF-κB, IκBα and Snail1 were from Santa Cruz Biotechnology (SantaCruz, CA); monoclonal antibodies against fibronectin and N-cadherin were from Zymed (Invitrogen, Carlsbad, CA); and thepolyclonal antibody against S100 was purchased from Dako(Glostrub, Denmark). The monoclonal antibodies against ICAM-1 (HU5/3) and CD31 (TP1/15) were provided by Dr SánchezMadrid (CNIC, Madrid). U0126 was from Calbiochem (EMD,Darmstad, Germany). MG132 and LiCl were from Sigma.

Western blottingMonolayers of MCs were lysed in modified RIPA buffer containing:50 mM Tris-HCl, pH 7.4; 1% NP-40; 0.1% SDS; 0.25% Na-deoxycholate; 150 mM NaCl; 1 mM EDTA; 1 mM EGTA; 1 mMPMSF; 1 μg/ml each of aprotinin, leupeptin and pepstatin; and 25mM NaF (all from Sigma). Equal amounts of protein were resolvedby SDS-PAGE. Proteins were transferred to PVDF membranes(Millipore, Bedford, VA) and probed with antibodies using standard



TRANSLATIONAL IMPACT

Clinical issuePeritoneal dialysis (PD) is a treatment for end-stage renal disease in which theblood-vessel-rich abdominal cavity lining, the peritoneum, is used essentiallyas an artificial kidney. Following the delivery of dialysis solution into theabdomen, ultrafiltration and diffusion take place across the peritonealmembrane to clean waste from the blood. However, continuous exposure todialysis solutions, as well as episodes of peritoneum infection (peritonitis) andbleeding (hemoperitoneum), may induce peritoneal cell abnormalities andeventually lead to reduced ultrafiltration activity. These alterations arereminiscent of an epithelial-mesenchymal transition (EMT), a complex step-wise transformation of cells that takes place during embryonic development,and is also seen in disease states such as tumorigenesis, chronic inflammation,and fibrosis.

Aim and resultsThis study analyzes the signaling pathways that underlie the EMT of peritonealmesothelial cells (MCs). The authors demonstrate that inflammatory cytokines,as well as peritoneal effluent from PD patients, induce EMT-like changes inprimary MCs from human peritoneum. They also show that an ERK/NF-κB/Snail1 activation pathway regulates the establishment of an EMT in thesecells. Interestingly, blockade of ERK and NF-κB activation induces the reversalof EMT in MCs collected from the peritoneal effluent of patients undergoingPD.

Implications and future directionsPD provides more advantages to the patient than hemodialysis, the traditionaltreatment for kidney failure, and is therefore increasingly used in the clinic.However, patients frequently discontinue PD owing to a loss of peritonealultrafiltration. One of the key events preceding this loss in filtration ability isEMT in the peritoneum. Studying the signaling pathways underlying EMT willfacilitate development of specific pharmacological inhibitors to counteractand/or reverse the changes that lead to peritoneal fibrosis. Additionally, thisinformation may help us to understand the other disease processes in whichEMT takes place.

doi:10.1242/dmm.001404

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procedures. PVDF-bound antibodies were detected bychemiluminescence with ECL (Amersham Life Sciences, LittleChalfont, UK).

Confocal microscopy and immunofluorescenceCells were fixed for 20 minutes in 3% formaldehyde in PBS,permeabilized in 0.2% Triton X-100/PBS for 5 minutes, and blockedwith 2% BSA for 20 minutes. For E-cadherin staining, cells were fixedand permeabilized in cold methanol for 10 minutes. For Snailstaining, cells were pretreated with LiCl and MG132 to block Snailphosphorylation, ubiquitination and subsequent degradation (Zhouet al., 2003). Secondary antibodies (conjugated to Alexa-647, -488and -541) and Hoechst 33342 were from Pierce Chemical Company(Rockford, IL). Confocal images were acquired using a Leica SP5spectral confocal microscope. The spectral technology allowsdiscrimination between yellow and green fluorescence.Quantification of NF-κB nuclear intensity was performed using theLeica LAS-AF software. Briefly, nuclei were delimited using Hoechstlabeling, and mean fluorescence intensity of NF-κB labeling wasquantified. A minimum of three different fields per condition wereacquired, with between 30 and 50 cells quantified per condition.

Infection of MeT-5A cells and omentum-derived MCs withretroviral vectorsMeT-5A cells and omentum-derived MCs were infected with eithera pRV-IRES-CopGreen retroviral vector (Genetrix, Madrid, Spain)encoding a super-repressor IκBα mutant that harbored mutationsS32A and S36A, or with empty pRV-IRES-CopGreen vector ascontrol. A crucial step in NF-κB activation is phosphorylation of IκBby the high molecular weight IKK complex (Huber et al., 2004).Mutations S32A and S36A render IκBα insensitive to IKKphosphorylation. Twenty-four hours before infection, MCs wereseeded into 6-well plates (2�105 cells per well) and retrovirus-producing 293T cells were seeded at 3�106 cells per 10 cm plate.For infection, 293T cell supernatants were filtered through a 0.45μm filter (Whatman, Dassel, Germany), and 5 μg/ml polybrene(Sigma-Aldrich, St Louis, MO, USA) was added to the filtrate.Thereafter, medium was removed from the MCs and replaced with293T cell supernatants containing the retrovirus. This process wasrepeated twice at 24-hour intervals. Twenty-four hours after the finalexposure to retrovirus, infection efficiency was monitored byfluorescence microscopy (Carl Zeiss, Standort Göttingen, Germany)or FACS analysis (BD FACS Canto, Becton-Dickinson Laboratories,Mountain View, CA). FACS analysis showed that the percentage ofprimary MCs infected ranged between 10 and 40% (data not shown),indicating that infection efficiency could lead to an underestimationof the effect of the IκBα super-repressor. As a control for this, purecultures of infected MeT-5A cells were obtained by sorting using aDako MoFlo cell sorter (Glostrub, DK).

Cell transfection and luciferase assaysNF-κB transcriptional activity was measured by transienttransfection of MeT-5A cells with the KBF-luc reporter plasmidand subsequent luciferase activity assay (Castellanos et al., 1997).Briefly, 2�105 cells were transfected with 2 μg of the KBF-lucreporter plasmid together with 500 ng of the reporter plasmid pRL-null, which bears a promoter-less Renilla luciferase gene (Promega,Madison, WI). Transfections were performed by incubating cells

for 4 hours with a mixture of DNA and lipofectamine at a ratio of1:2.5 (Lipofectamine 2000; Invitrogen, Carlsbad, CA, USA) inserum-free medium. After transfection, cells were pretreatedovernight with vehicle (DMSO) or U0126 (20 μM). Cells were thenstimulated with TGF-β1 and IL-1β for the times indicated.Luciferase activity was measured with the dual-luciferase reporterassay system (Promega) according to the manufacturer’sinstructions and determined in a Sirius single tube luminometer(Berthold Detection Systems GmbH, Pforzheim, Germany). Allexperiments were carried out in duplicate.

Reverse-transcriptase PCRTotal RNA was extracted with the RNeasy kit (Qiagen GmbH,Hilden, Germany), and the cDNA was obtained from 500 ng oftotal RNA by using an Omniscript RT kit (Qiagen). QuantitativePCR was carried out in a LightCycler (Roche Diagnostics GmbH,Mannheim, Germany) using a SYBR Green kit (Roche DiagnosticsGmbH) and the following specific primer sets: 5�-TGAAG GTG -ACAGAGCCTCTG-3� and 5�-TGGGTGAATTCG GGCTTGTT-3� for E-cadherin; 5�-GCAAATACTGCAACAAGG-3� and 5�-GCACTGGTACTTCTTGACA-3� for Snail1; 5�-AAAG CC -GCTCGCAAGAGTGCG-3� and 5�-ACTTGCCTC CTGCA -AAGCAC-3� for histone H3 (used for normalization). Theannealing temperature for E-cadherin and H3 amplification was62°C, and fluorescence was measured at the end of each elongationcycle. For Snail1 amplification, the annealing temperature was 55°Cand fluorescence was measured at 88°C, after each elongation cycle.All experiments were carried out in duplicate. After amplification,the PCR products were confirmed by melting-curve analysis andgel electrophoresis. COX-2 mRNA levels were estimated by 35cycles of qualitative PCR with an annealing temperature of 63°Cand the following primers: 5�-TTCAAATGAGAT TGTG -GAAAAAT TGCT-3� and 5�-AGATCATCTCTGCCTG AGTA -TCTT-3�.

Statistical analysisStatistical significance was determined with a t-test with OriginPro7software (OriginLab Co.). P values of <0.05 were consideredsignificant.ACKNOWLEDGEMENTSThis work was supported by the MICINN (Spanish Ministry of Science andInnovation) through grants SAF2005-00493, GEN2003-20239-C06-04, and RTICC(Cancer Research Network) to M.A.d.P., EUROHORCS (European Heads Of ResearchCouncils) and the European Science Foundation (ESF) through a EURYI (EuropeanYoung Investigator) award to M.A.d.P., the EMBO Young Investigator Programme,and by the European Union 6th Framework Programme through a Marie CurieInternational Reintegration Grant (MIRG-CT-2005-016427) to M.A.d.P. This workwas also supported by MICINN grants to M.L.-C. (SAF2007-61201 and PET2006-0256). R.S. was first supported by a fellowship from the FIRC (Fondazione Italianaper la Ricerca sul Cancro) and then by a Río Hortega Contract (Instituto de SaludCarlos III). I.B. is a recipient of a CIBERehd fellowship (MICINN). Editorial assistancewas provided by Simon Bartlett. The CNIC is supported by the Spanish Ministry ofHealth and Consumer Affairs and the Pro-CNIC Foundation. Deposited in PMC forimmediate release.

COMPETING INTERESTSThe authors declare no competing financial interests.

AUTHOR CONTRIBUTIONSR.S. designed the experiments, performed biochemical assays,immunofluorescence labeling, image analysis, participated in all otherexperiments, and wrote the first draft of the manuscript. I.B. performed RT-PCR,designed experiments and contributed discussion. M.L.P.L. purified human

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ERK/NF-κB/Snail1 in peritoneal EMTRESEARCH ARTICLED

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mesothelial primary cells and participated in experiments with peritonitis effluent.A.C. participated in RT-PCR, statistical and image analysis. M.L.-C. provided generalknowledge of MC EMT, participated in design of experiments and contributeddiscussion. M.A.d.P. established the initial scientific questions, provided continuingintellectual guidance, participated in and coordinated experimental design andmanuscript writing.

SUPPLEMENTARY MATERIALSupplementary material for this article is available athttp://dmm.biologists.org/content/?/??/????/suppl/DC1

Received 29 July 2008; Accepted 12 August 2008.

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