Cell Contact-dependent Regulation of Epithelial-Myofibroblast Transition via the Rho-Rho...

Molecular Biology of the CellVol. 18, 1083–1097, March 2007

Cell Contact–dependent Regulation ofEpithelial–Myofibroblast Transition via the Rho-RhoKinase-Phospho-Myosin PathwayLingzhi Fan,*†‡ Attila Sebe,*†‡§ Zalan Peterfi,*† Andras Masszi,*†

Ana C.P. Thirone,*† Ori D. Rotstein,*† Hiroyasu Nakano,�Christopher A. McCulloch,¶ Katalin Szaszi,*† Istvan Mucsi,#and Andras Kapus*†

*St. Michael’s Hospital Research Institute, Toronto, ON, Canada M5B 1W8; †Department of Surgery,University of Toronto, ON, Canada M5G 1L5; §Nephrology Research Center, Semmelweis University,Budapest, Hungary H-1089; �Department of Immunology, Juntendo University School of Medicine, Tokyo,Japan 113-8421; ¶CIHR Group in Matrix Dynamics, University of Toronto, Toronto, ON, Canada M5S 3E2;and #First Department of Internal Medicine, Semmelweis University, Budapest, Hungary H-1083

Submitted July 17, 2006; Revised December 15, 2006; Accepted January 3, 2007Monitoring Editor: Asma Nusrat

Epithelial-mesenchymal-myofibroblast transition (EMT), a key feature in organ fibrosis, is regulated by the state of intercellularcontacts. Our recent studies have shown that an initial injury of cell–cell junctions is a prerequisite for transforming growthfactor-�1 (TGF-�1)-induced transdifferentiation of kidney tubular cells into �-smooth muscle actin (SMA)–expressing myo-fibroblasts. Here we analyzed the underlying contact-dependent mechanisms. Ca2� removal–induced disruption of intercel-lular junctions provoked Rho/Rho kinase (ROK)-mediated myosin light chain (MLC) phosphorylation and Rho/ROK-depen-dent SMA promoter activation. Importantly, myosin-based contractility itself played a causal role, because the myosin ATPaseinhibitor blebbistatin or a nonphosphorylatable, dominant negative MLC (DN-MLC) abolished the contact disruption-triggered SMA promoter activation, eliminated the synergy between contact injury and TGF-�1, and suppressed SMAexpression. To explore the responsible mechanisms, we investigated the localization of the main SMA-inducing transcriptionfactors, serum response factor (SRF), and its coactivator myocardin-related transcription factor (MRTF). Contact injury en-hanced nuclear accumulation of SRF and MRTF. These processes were inhibited by DN-Rho or DN-MLC. TGF-�1 stronglyfacilitated nuclear accumulation of MRTF in cells with reduced contacts but not in intact epithelia. DN-myocardin abrogatedthe Ca2�-removal– � TGF-�1–induced promoter activation. These studies define a new mechanism whereby cell contactsregulate epithelial-myofibroblast transition via Rho-ROK-phospho-MLC–dependent nuclear accumulation of MRTF.

INTRODUCTION

Epithelial-mesenchymal transition (EMT) is a key process intissue development, carcinogenesis, and organ fibrosis (Leeet al., 2006). Recently EMT has emerged as a central mecha-nism underlying tubulointerstitial fibrosis (TIF), a progres-sive pathology common to a variety of chronic kidney dis-eases (Strutz et al., 1995; Liu, 2004). In a transgenic mousemodel of TIF, nearly 40% of fibroblasts have been shown tooriginate from the tubular epithelium that underwent EMT(Iwano et al., 2002). During this process tubular cells losetheir polygonal shape and epithelial markers (e.g., E-cad-herin), acquire fibroblast-specific proteins (e.g., FSP1), in-creasingly synthesize extracellular matrix (e.g., fibronectin),and ultimately differentiate into �-smooth muscle actin(SMA)-positive myofibroblasts (for a review see Kalluri andNeilson, 2003). Myofibroblasts represent a highly contractile

cell type that is thought to be critical for wound contrac-tion, tissue repair, and the pathogenesis of fibrocontrac-tive diseases (Gabbiani, 2003; Chaponnier and Gabbiani,2004; Desmouliere et al., 2005).

Several laboratories including our own have establishedtubular cell models to study EMT and the development ofmyofibroblasts (Fan et al., 1999; Yang and Liu, 2001; Massziet al., 2003). Both in vivo and in vitro, transforming growthfactor-�1 (TGF-�1) is the main inducer of EMT and fibro-genesis (Bottinger and Bitzer, 2002). However, our previousstudies revealed that in intact, confluent monolayers of tu-bular (LLC-PK1) cells, TGF-�1 alone is insufficient to induceSMA synthesis and thus myofibroblast formation. The ad-ditional prerequisite is a partial loss or injury of intercellularcontacts, which can be modeled by subconfluence, mechanicalwounding or disassembly of adherens junctions (AJ) via Ca2�

removal (Masszi et al., 2004). These studies have defined atwo-hit (TGF-�1 and contact injury) model, in which intercel-lular junctions are not only targets but also active regulators ofEMT. Indeed TGF-�1 and contact disassembly exert strongsynergy in the stimulation of the SMA promoter.

While searching for mechanisms responsible for the cellcontact–dependent regulation of SMA expression, we havepreviously found that �-catenin contributes to this phenom-

This article was published online ahead of print in MBC in Press(http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E06–07–0602)on January 10, 2007.‡ These authors contributed equally to this work.

Address correspondence to: Andras Kapus ([email protected]).

© 2007 by The American Society for Cell Biology 1083

enon. �-catenin, when liberated from the AJs upon contactinjury and rescued from proteolysis in a TGF-�1–dependentmanner, exerts a potentiating effect on the activation of theSMA promoter. However, the SMA promoter does not har-bor a �-catenin–responsive cis-element, and overexpressionof �-catenin alone does not activate SMA expression, indi-cating that the effect is indirect and other contact-dependentfactors must also be involved. These considerations promptedus to investigate the possible relationship between contactinjury and the main direct regulators of the SMA promoter.

In muscle cells and fibroblasts, the expression of smoothmuscle–specific genes, including SMA, is primarily con-trolled by serum response factor (SRF; Hill et al., 1995; Macket al., 2000) and its recently discovered coactivators, myocar-din and the myocardin-related transcription factors (MRTFs)also called MAL or MKL (Wang et al., 2001, 2002; Cen et al.,2003; Du et al., 2003; Selvaraj and Prywes, 2003). RhoGTPase–mediated actin cytoskeleton reorganization hasbeen long recognized as a key activator of SRF (Sotiropouloset al., 1999; Mack et al., 2001), but until recently the under-lying mechanisms remained undefined. Novel studies sug-gest that SRF is activated by the Rho-dependent nucleartranslocation of MRTF (Miralles et al., 2003; Du et al., 2004).According to the current view, in quiescent cells MRTF isassociated with monomeric (G) actin and this complex can-not enter the nucleus. Stimulus-induced Rho activationcauses enhanced incorporation of G-actin into actin fila-ments, which then leads to dissociation of MRTF followedby its nuclear translocation (Sotiropoulos et al., 1999;Miralles et al., 2003). Two Rho effector pathways have beenimplicated in the mediation of this effect: increased actinpolymerization via formin proteins (Copeland and Treis-man, 2002) and reduced F-actin severing by the LIM kinase-cofilin pathway (Geneste et al., 2002). Rho might also regu-late the localization of SRF; however, this aspect iscontroversial and the underlying mechanisms are notknown (Camoretti-Mercado et al., 2000; Liu et al., 2003a; Cenet al., 2004).

Importantly, contact injury also leads to characteristicchanges in the cytoskeleton. Previous work by us and othershas shown that disassembly of epithelial junctions leads torobust myosin light chain (MLC) phosphorylation (Frixione etal., 2001; Ivanov et al., 2004; Di Ciano-Oliveira et al., 2005), aprocess mediated by the downstream Rho effector, Rho kinase(ROK) (Szaszi et al., 2005). Epithelial wounding–induced MLCphosphorylation and acto-myosin ring formation is believed tobe critical for wound closure (Darenfed and Mandato, 2005).This scenario then raises a number of intriguing questionsabout the potential connection between cell contacts and theregulation of SMA expression. Specifically we sought to deter-mine whether contact disassembly impacts on the localizationof MRTF and/or SRF in epithelial cells, and whether such aneffect might be mediated by the Rho-ROK pathway. We alsoasked whether myosin phosphorylation per se is required forthe contact-dependent regulation of the SMA promoter. Ourresults indicate that contact injury–induced Rho-ROK–medi-ated MLC phosphorylation regulates MRTF distribution,which in turn plays a central role in epithelial-myofibroblasttransformation.

MATERIALS AND METHODS

Antibodies and ReagentsAnti-SRF, anti-Myc (9E-10), and fluorescein isothiocyanate (FITC)-conjugatedanti-Myc were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-�-SMA, anti-�-actin, and anti-FLAG antibodies were purchased from Sigma (St.Louis, MO), anti-monophospho-MLC from Cell Signaling Technology (Danvers,

MA), and anti-histones from Chemicon (Temecula, CA). The polyclonal anti-alpha-BSAC antibody raised against the mouse MKL1 protein was describedpreviously (Sasazuki et al., 2002). FITC- and Cy3-labeled, as well as peroxi-dase-conjugated anti-mouse, anti-rabbit, and anti-goat secondary antibodieswere obtained from Jackson ImmunoResearch Laboratories (West Grove, PA).DAPI used for nuclear staining was purchased from Invitrogen (Burlington,ON, Canada). Y-27632 and blebbistatin was from Calbiochem (La Jolla, CA),rhodamine-labeled phalloidin from Cytoskeleton (Denver, CO), and humanrecombinant TGF-�1 from Sigma.

Cell Culture and TreatmentsLLC-PK1 (CL4) proximal tubular cells were cultured in DMEM (Invitrogen)and Chinese hamster ovary (CHO) cells in �-minimal essential medium(�-MEM, Invitrogen), supplemented with 10% FBS (Invitrogen) and 1% pen-icillin/streptomycin at 37°C under humidified atmosphere of air/CO2 (19:1).Cells were grown on 6- or 12-well plates, on glass coverslips, or on 10-cmdishes to either 100% confluence or subconfluence as indicated in the legendof the corresponding figures, and then subjected to various treatments. Foracute Ca2� removal, cells were preincubated in an isotonic NaCl-based me-dium (140 mM NaCl, 3 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 5 mM glucose,20 mM HEPES, pH 7.4) for 10 min, and then the medium was replaced withthe same basic solution lacking CaCl2 and supplemented with 1 mM EGTA.For chronic Ca2� deprivation, the cells were washed four times with phos-phate-buffered saline (PBS, Invitrogen), and once with serum- and Ca2�-freeDMEM followed by incubation in the latter solution. Control samples wereincubated with serum-free DMEM containing Ca2�. Where applied, TGF-�1(10 ng/ml or vehicle for controls) was added to the cells for times specified atthe individual experiments. For inhibitor studies, cells were preincubatedwith 10 �M Y-27632 or 50–100 �M blebbistatin for various times as describedin the figure legends. Wounding of confluent monolayers grown on glasscoverslips was achieved by scraping a 1–3-mm gap using a rubber policeman.Cells were fixed 6 h after wounding.

PlasmidsThe PA3-Luc vector containing a 765-bp fragment of the rat SMA promoter(pSMA-Luc) was a kind gift from Dr. R. A. Nemenoff (Department of Medi-cine, University of Colorado), and was used as in our previous studies(Masszi et al., 2003). In certain experiments we used pGL3-SMA-Luc plasmid(provided by Dr. S. H. Phan, University of Michigan Medical School, AnnArbor; Hu et al., 2003), which harbors the same promoter region inserted intopGL3 luciferase vector. As internal control for transfection efficiency, thymi-dine kinase–driven Renilla luciferase vector (pRL-TK, Promega, Madison,WI) was used. Plasmids (pcDNA3.1) encoding for the C-terminally His- andMyc-tagged wild-type (WT) myosin regulatory light chain-2 (WT-MLC) andits dominant negative version in which T18 and S19 were replaced withalanine (DN-MLC), were kind gifts from Dr. H. Hosoya (Department ofBiological Sciences, Hiroshima University; Iwasaki et al., 2001; Di Ciano-Oliveira et al., 2005). FLAG-tagged MRTF-A, MRTF-B, and the dominantnegative truncation mutant (�C585) of myocardin were kindly provided byDr. E. N. Olson (Department of Molecular Biology, University of Texas), andwere described previously (Wang et al., 2001). Vectors encoding for Myc-tagged constitutive active RhoA (Q63L, CA-Rho), dominant negative RhoA(T19N, DN-Rho), and GFP-p190RhoGAP were described and used in ourprevious studies (Masszi et al., 2003). The SBE4-Luc reporter plasmid, whichcontains four tandem repeats of the SMAD-binding element, was a kind giftof Dr. A. B. Roberts (National Institutes of Health, Bethesda; Felici et al., 2003).

Transient Transfection and Luciferase Promoter ActivityAssaysIf not otherwise stated, cells were grown on six-well plates and transfected at a100% confluence using 2.5 �l FuGene6 (Roche, Laval, QC, Canada) reagent/1 �gDNA. For promoter activity measurements, cells were cotransfected with 0.5 �gpSMA-Luc (or pGL3-SMA-Luc), 0.05 �g pRL-TK, and 2 �g of either empty vector(pcDNA3.1) or the specific construct to be tested. After a 24-h incubation period,cells were washed and placed in a serum-free medium, either containing orlacking Ca2�. TGF-�1 (10 ng/ml) or its vehicle was added to the cells after 4 h,and the incubation was continued for an additional 16 h. Cells were then lysedin 500 �l passive lysis buffer (Promega), and the samples were subjected to a cycleof freezing/thawing, and then clarified by centrifugation (12,000 rpm, 5 min at4°C). Firefly and Renilla luciferase activities were measured by the Dual-Lucif-erase Reporter Assay Kit (Promega) using a Berthold Lumat LB 9507 luminom-eter (Bad Wildbad, Germany) according to the manufacturer’s instructions. Re-sults were normalized by dividing the Firefly luciferase activity with the Renillaluciferase activity of the same sample. For each condition duplicate or triplicatemeasurements were performed, and experiments were repeated at least threetimes. For immunofluorescence analysis typically 1–2 �g plasmid DNA wastransfected per coverslip.

Rho Activity AssayRho activation was assessed by an affinity pulldown assay as in our previousstudies (Di Ciano-Oliveira et al., 2003). Briefly, after the indicated treatment,

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cells grown in 10-cm dishes were lysed in 800 �l of ice-cold Rho lysis buffer(100 mM NaCl, 50 mM Tris-Base, pH 7.6, 20 mM NaF, 10 mM MgCl2, and 1%Triton X-100) supplemented with 0.5% deoxycholic acid, 0.1% SDS, 20 �l/mlprotease inhibitor cocktail, 1 mM Na3VO4, and 1 mM phenylmethylsulfonylfluoride. Lysates were clarified by centrifugation at 12,000 rpm for 1 min at4°C. Glutathione-Sepharose beads (10–15 �g/sample) covered with GST-Rho-binding domain (RBD) fusion protein were then added to the superna-tants and incubated at 4°C for 45 min. The GST-RBD beads were washed threetimes with Rho lysis buffer, and the captured proteins were diluted with 25 �lof Laemmli buffer, and subjected to electrophoresis on 15% SDS-polyacryl-amide gels followed by Western blotting using an anti-Rho antibody.

Western BlottingAfter treatments cells were scraped into Triton lysis buffer (30 mM HEPES,pH 7.4, 100 mM NaCl, 1 mM EGTA, 20 mM NaF, 1% Triton X-100, 1 mMNa3VO4, 1 mM phenylmethylsulphonyl fluoride, 20 �l/ml protease inhibitorycocktail (BD Biosciences, Mississauga, Ontario, Canada), the protein concen-tration was determined by the Bradford method (Bio-Rad Laboratories, Her-cules, CA), and the samples were mixed in a 1:1 ratio with 2� Laemmli bufferand boiled for 5 min. For pMLC blots, the cells were lysed in ice-cold acetonecontaining 10% trichloroacetic acid and 10 mM dithiothreitol, followed bycentrifugation for 10 min at 12,500 rpm at 4°C. The resulting pellet waswashed with pure acetone, allowed to air dry, and dissolved in 60 �l ofLaemmli sample buffer. Equal amounts of protein were separated on 10%SDS-polyacrylamide gel, and transferred to nitrocellulose membranes. Blotswere blocked with Tris-buffered saline (TBS), containing 0.1% Tween 20 and5% albumin for an hour. Membranes were incubated for an additional hour(or overnight for pMLC) with the primary antibody (in TBS-Tween plus 0.5%albumin), extensively washed, and incubated with the corresponding perox-idase-conjugated secondary antibody. After final washes immunoreactivebands were visualized with the enhanced chemiluminescence reaction.

Nuclear ExtractionNuclear extracts were prepared from confluent layers of LLC-PK1 cells grownon 10-cm dishes, using NE-PER Nuclear Extraction Kit from Pierce Biotech-nology (Rockford, IL) according to the manufacturer’s recommendation. Thenuclear extracts were collected, their protein concentration was determined,and samples of equal protein content were analyzed by Western blotting.Anti-histone antibody was used to check for equal loading of nuclear pro-teins.

Quantification of Cellular F-Actin ContentF-actin was measured by the rhodamine phalloidin extraction method, essen-tially as described (Pedersen and Hoffmann, 2002). This technique allowsreliable determination of a few percent change in F-actin. Briefly, confluentLLC-PK1 cells grown on six-well plates were serum-deprived, treated withvarious inhibitors, and then fixed in Tris-buffered saline containing 2% para-formaldehyde. After repeated washes the cells were permeabilized with 0.1%saponin buffer and incubated in 250 �l of a 0.33 �M rhodamine phalloidinsolution for an hour. The cells were then thoroughly washed, and the boundphalloidin was extracted by incubating the cells for 30 min with 2 ml of puremethanol per well. Rhodamine phalloidin fluorescence in the samples wasdetermined by a Photon Technology cuvette fluorimeter (Lawrenceville, NJ)using 537 nm for excitation and 576 nm for emission.

Immunofluorescence MicroscopyCells grown on coverslips were fixed with 4% paraformaldehyde for 30 min,washed with PBS, and incubated with 100 mmol/l glycine in PBS for 10 min.Cells were then permeabilized in PBS containing 0.1% Triton X-100, blockedfor an hour with 3% albumin, and incubated with the primary antibody orantibodies (in case of costaining) for 1 h. After extensive washes, fluorescentlylabeled secondary antibodies were added for another hour. The coverslipswere washed and then mounted on slides using Fluorescence MountingMedium (DAKO, Carpinteria, CA). When directly labeled, FITC-conjugatedmouse anti-Myc antibody was used together with another mouse primaryantibody, and the cells were initially processed for staining with the unlabeledprimary and corresponding secondary antibodies, blocked again with mouseserum (1:100), and then incubated with the directly labeled primary antibodyfor an hour. Samples were analyzed by an Olympus IX81 microscope (60� or100� objectives, Melville, NY) coupled to an Evolution QEi Monochromecamera controlled by the QED InVivo Imaging software (Media Cybernetics,Silver Spring, MD). Images were processed by the ImagePro Plus 3DS 5.1software (Media Cybernetics). Bars on the microscopic images correspond to20 �m.

Quantification of Nuclear/Cytoplasmic Distribution ofProteinsStaining was quantified using the ImagePro Plus software: fluorescence in-tensities were determined at three random nuclear and cytoplasmic pointsalong a line, or in three equal rectangular areas within the nucleus or the

cytoplasm. An average of three determinations per cell was used, and thenuclear/cytoplasmic ratio was calculated. Ratios measured along lines orwithin rectangular areas were identical. Nuclei were independently visual-ized by DAPI staining. MRTF distribution was categorized as cytosolic ornuclear when the nucleus was clearly demarcated either by exclusion oraccumulation of the label. Otherwise the distribution was regarded as even(or pancellular). To make these categories exact, distribution data were veri-fied using the nuclear/cytoplasmic ratios as �0.75 (cytosolic), 0.75–1.25(even), and �1.25 (nuclear). In the vast majority of cells within the nuclearcategory the ratio was �2.

Statistical AnalysisData are presented as blots or images from at least three similar experimentsor as the means � SE for the number of experiments (n) indicated. Statisticalsignificance was determined by Student’s t test or one-way ANOVA using theGraphPad InStat software (San Diego, CA).

RESULTS

Contact Disassembly Induces Rho/ROK-dependentMyosin Phosphorylation and SMA Promoter ActivationTo assess whether the disassembly of intracellular contactsaffects Rho signaling in LLC-PK1 cells, we tested the effect ofCa2� removal, a classic maneuver that dismantles Ca2�-dependent intercellular junctions. Figure 1A shows that re-placement of the normal medium with a Ca2�-free solutioncaused rapid and robust (approx. threefold) Rho activation,as detected by an affinity pulldown assay that precipitatesactive (GTP-bound) Rho from the cell lysates. Concomitantwith this response, the cells exhibited a large increase intheir staining for the monophosphorylated myosin lightchain (pMLC; Figure 1B, a and b), which occurred predom-inantly at the cell periphery. This observation together withour earlier finding that Ca2� removal caused sizable rise inperipheral diphospho-MLC staining as well (Di Ciano-Oliveira et al., 2005) indicates that contact disruption en-hances contractility via both mono- and diphosphorylationof MLC. Importantly, MLC phosphorylation is a sustainedresponse, because under Ca2�-free conditions peripheralpMLC levels remained high in �60% of the cells for days(Figure 1, Bc and C), i.e., through the time course of ourtransfection and promoter studies (see below). To testwhether Rho activity was required for increased monophos-phorylation of MLC, 2 d before Ca2� removal, cells weretransfected with a Myc epitope–tagged dominant negative(T19N) Rho construct. Double staining for Myc and pMLCrevealed that DN-Rho prevented the contact injury-trig-gered increase in pMLC (Figure 1B, e and e�, and C). More-over, the Rho kinase inhibitor Y-27632 also abolished theenhanced MLC phosphorylation (Figure 1Bd), suggestingthat Rho-mediated ROK activation is indispensable for thisprocess.

Next we addressed whether the Rho/ROK pathwaymight contribute to the Ca2� removal–induced stimulationof the SMA promoter. Cells were transfected with the SMA-Luc reporter plasmid along with empty pcDNA vector orDN-Rho, and subsequently the culture medium was ex-changed for serum-free DMEM, either containing or lackingCa2�. Ca2� deprivation caused a 6–10-fold increase in theactivity of the SMA promoter (Figure 1D). Importantly, DNRho, although exerting no significant effect on the basalpromoter activity, entirely prevented the Ca2� depletion–induced stimulation (Figure 1D). Similar results were ob-tained when Y-27632 was used to inhibit ROK: this treat-ment also abolished the contact-dependent activation ofthe SMA promoter (Figure 1E). Taken together these dataimply that the disruption of intercellular contacts leads toRho activation and ROK-dependent myosin phosphoryla-tion. Moreover, the Rho/ROK pathway is a key mediator

Cell Contacts and Myosin Regulate SMA Expression

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of the contact injury–provoked activation of the SMApromoter.

Myosin Phosphorylation Plays a Critical Role in theCa2� Removal–triggered Activation of the SMA PromoterAlthough the activation of Rho is known to participate in theregulation of SRF-dependent gene expression, the down-stream pathways mediating this effect have not been entirelyelucidated. Particularly, the potential role of myosin activityor phosphorylation has not been addressed. Given the ro-bust pMLC response upon contact disassembly, we soughtto determine whether this event contributes to the activationof the SMA promoter. Initially we applied blebbistatin, aspecific inhibitor of myosin ATPase (Straight et al., 2003).Figure 2A shows that pretreatment of the cells with bleb-bistatin did not affect the basal promoter activity but entirelyprevented its activation by Ca2� removal. Next we testedwhether the drug also impacts the TGF-�1–triggered stim-ulation and its contact-dependent potentiation. In agree-ment with our previous results, TGF-�1 added to confluentlayers caused only a modest increase in SMA promoteractivity (Figure 2A). This effect was significantly reduced butnot entirely abolished by blebbistatin. The combined treat-ment (Ca2� removal � TGF-�1) led to a robust rise in

promoter activity, and this synergism was fully eliminatedby blebbistatin. Although our previous results (Masszi et al.,2004) have implied that contact disruption does not actsimply via increasing receptor availability for TGF-�1, wefurther substantiated this point by testing the effect of Ca2�

removal on another TGF-�–induced effect, the activation ofthe Smad-binding element (SBE; Felici et al., 2003; Figure 2B).TGF-�1 exerted a similar stimulation of SBE in the presenceor absence of calcium, indicating that altered receptor acces-sibility does not play a key role in the observed effects, andthat the Ca2�-removal–induced potentiation is specific forthe SMA promoter.

In our subsequent experiments we chose an alternative andmore specific way to interfere with myosin function. We usedtransfection with a construct encoding for a Myc epitope-tagged, nonphosphorylatable myosin mutant in which thecritical target residues T18 and S19 were exchanged withphenylalanine (AA-MLC). This approach offers the advan-tage over blebbistatin in that it prevents myosin phosphor-ylation and activation without interfering with basal myosinATPase activity (Di Ciano-Oliveira et al., 2005). AA-MLCeffectively prevented the Ca2�-removal–induced rise in pe-ripheral pMLC (Figure 2C), implying that this mutant acts asa dominant negative (DN-MLC). This conclusion is sup-

Figure 1. Contact disassembly induces Rho/Rho kinase–dependent myosin phosphorylation and SMA-promoteractivation. (A) Confluent LLC-PK1 cell cultures wereserum-starved for 3 h and then preincubated with aCa2�-containing NaCl-based medium for 10 min. Subse-quently the medium was aspirated and either replacedwith the same solution (control) or with a Ca2�-freesolution containing 1 mM EGTA (no Ca) to rapidlydisrupt the intercellular contacts. Five minutes later thecells were lysed, and samples of equal protein contentwere subjected to the Rho activity assay as described inMaterials and Methods. Total Rho was determined fromthe same lysates. One representative blot of three sepa-rate experiments is shown. Densitometry (bars) was per-formed for each experiment, and Rho activation wasexpressed as fold increase compared with the control. (B)LLC-PK1 cells were grown on coverslips to confluence,and after various treatments were stained with anti-monophospho-MLC antibody: (a) No treatment; (b) cellswere exposed to acute Ca2� removal for 5 min usingEGTA as in A; (c and d) for chronic Ca2� removal, thenormal, serum-free DMEM was replaced with a nomi-nally Ca2�-free DMEM for 24 h. Thirty minutes beforeCa2� removal cells were preincubated with vehicle (c) or10 �M Y-27632 (d), which remained present throughoutthe whole experiment. To visualize cells, nuclei werestained with DAPI; and (e and e�) cells grown to conflu-ence were transfected with Myc-tagged DN-Rho for 24 h,exposed to nominally Ca2�-free conditions for an addi-tional 24 h, and then doubly stained for monophospho-MLC (red) and for the Myc epitope (green). (C) Thefrequency of peripheral phospho-MLC staining wasquantified in control and DN-Rho expressing cells after24-h incubation in nominally Ca2�-free DMEM. Notethat more than 60% of controls cells showed peripheralmyosin phosphorylation, whereas this response wasnegligible in DN-Rho expressing cells. (n 3, in eachexperiment �60 cells were counted in each cell popula-tion). (D) Confluent cells were transfected with pSMA-Luc plus pRL-TK along with either empty vector(pcDNA3.1) or with DN-Rho (see Materials and Methods).After 24 h the cells were incubated in serum-free (Cont)or serum- and Ca2�-free DMEM (no Ca) for an addi-

tional 24 h, followed by determination of luciferase activity (n 3). (E) The same conditions as in D, except cells were treated for 30 min beforeCa2� depletion with vehicle or 10 �M Y-27632 (n 3).

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ported also by our previous observation using other stimuli,including osmotic stress and depolarization. To assess theeffect of MLC phosphorylation per se on the activation of thepromoter, we cotransfected the cells with DN-MLC alongwith SMA-Luc and subjected them to various stimuli. DN-MLC nearly abolished the Ca2� deprivation–induced in-crease in promoter activity, and strongly abrogated the syn-ergistic effect induced by Ca2� removal and TGF-�1 (Figure2D). To substantiate these results, we performed two kindsof control experiments. First, to verify that the type of theexpression vector was not critical, and that the observedeffect was indeed exerted on the promoter, we repeatedthese experiments using an alternative (pGL3) plasmid har-boring the same 765-base pair promoter sequence. DN-MLCeffectively inhibited the Ca2� depletion–induced luciferaseresponse in this system as well (Figure 2E). Second, to verifythat the mutation is indeed the determining factor for theinhibitory effect, cells were transfected with the Myc-taggedWT (T18, S19) MLC as well (Figure 2F). Overexpression ofWT myosin had no effect on the basal promoter activity anddid not alter its activation by Ca2� removal. Together thesedata imply that myosin activity as well as myosin phosphor-ylation are important contributors to the contact-dependentregulation of the SMA promoter. Together these experi-

ments strongly argued for the participation of myosin activ-ity and activation in the regulation of the SMA promoter.

Because the level of actin polymerization is known toregulate CArG-dependent genes, we considered that theeffect of myosin inhibition might be, at least partially, due toan impact on F-actin. Phalloidin staining reveled that inconfluent cultures LLC-PK1 cells contained relatively fewand fine stress fibers near their ventral surface, and punctateF-actin distribution corresponding to microvilli at their api-cal surface. Blebbistatin induced substantial stress fiber dis-assembly in accord with our previous findings (Di Ciano-Oliveira et al., 2005) and a decrease in microvillar F-actinlabeling (Figure 3A, top and bottom). These observationssuggest that basal myosin activity is necessary for normalF-actin arrangement, but they do not provide quantitativeinformation about any potential change in the size of theF-actin pool. To address this issue, we compared the F-actinlevels in control and blebbistatin-treated cells using a phal-loidin extraction assay. As shown on Figure 3C, blebbistatindid not alter the total F-actin level, in sharp contrast with theeffect of the actin monomer-sequestering drug, LatrunculinB, which was applied as a positive control for the assay.Next we assessed the effect of DN-MLC on the actin skele-ton. As opposed to blebbistatin treatment, we were not able

Figure 2. Inhibition of myosin ATPase activity or my-osin phosphorylation strongly suppresses the contactdisruption–induced activation of the SMA promoterand its potentiation by TGF-�1. (A) Confluent monolay-ers were transfected with p-SMA-Luc and pRL-TK, andafter 24 h were treated with vehicle or 100 �M blebbista-tin for 2.5 h. Subsequently the cells were incubated inserum-free, Ca2�-containing or Ca2�-free DMEM, in thepresence or absence of blebbistatin. After 4 h, 10 ng/mlTGF-�1 was added to the samples where indicated.Sixteen hours later the cells were lysed, and their lucif-erase activity was determined (n 3). (B) Ca2� removaldoes not act through increasing receptor availability forTGF-�1. Cells were transfected with the TGF-�1–re-sponsive SBE reporter (p-SBE-Luc) and left untreated orchallenged with Ca2� removal, TGF-�1, or the combi-nation of these stimuli as in A. (C) DN-MLC inhibitsthe Ca2� removal–induced MLC phosphorylation. Cellsgrown on coverslips in 6-well plates were transfectedwith Myc-tagged DN-MLC for 24 h, incubated in serumand Ca2�-free DMEM for another 24 h, and then fixedand doubly stained for the Myc epitope (green) andphospho-MLC (red). Note the absence of pMLC stainingin the clusters of transfected cells. (D) The effect ofDN-MLC on the activation of the SMA promoter. Con-fluent cells were transfected with empty vector (pcDNA3) orDN-MLC, and after 24 h were subjected to Ca2� re-moval where indicated. Four hours later, 10 ng/mlTGF-�1 was added for 20 h to the indicated samples,followed by lysis and determination of luciferase activ-ity. (E) Cells were transfected with pGL3-SMA-Luc, analternative vector harboring the same 765-base pairsSMA promoter region as PA3-SMA-Luc. Other condi-tions were identical as in D. (F) Cells were transfectedwith wild-type (WT) MLC. Other conditions were iden-tical with D.


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to detect any obvious change in the F-actin arrangement ofDN-MLC–expressing (Myc-positive) cells compared withtheir nontransfected neighbors (Figure 3B). This finding isconsistent with the preservation of basal myosin activity inthese cells. To assess the impact on F-actin, we had togenerate a cell population, in which the majority of cellsexpressed DN-MLC. Using a selection protocol, we obtaineda population with �85% expressors. In these cells there wasa slight (�9%) decrease in the total F-actin compared withnonexpressors. Neither blebbistatin nor DN-MLC alteredtotal actin expression (Figure 3D). Taken together, these datashow that alteration in myosin activity markedly suppressedthe activation promoter without (blebbistatin) or with a small(DN-MLC) change in the total F-actin. Although these experi-ments do not rule out that myosin might impact on specificactin pools or on stimulus-induced F-actin polymerization,

they suggest that other mechanisms are likely to contribute tothe overall effect of myosin inhibition (see Discussion).

Cell Contact Disassembly Promotes NuclearAccumulation of Serum Response FactorSerum response factor (SRF) is the key cis-element drivingSMA expression, whose activity could be regulated by nu-cleocytoplasmic shuttling (Camoretti-Mercado et al., 2000),although both this possibility and the involvement of theRho pathway in this process remain controversial (Cen et al.,2004). Therefore we asked whether contact disruption affectsSRF localization. Figure 4A shows that SRF exhibited nu-clear accumulation even in resting, nonstimulated cells. In-terestingly, nuclear distribution was more pronounced insubconfluent cultures (not shown), whereas upon reachingconfluence, the cytosolic staining increased concomitant

Figure 3. The effect of myosin inhibition on F-actinorganization and content. (A) LLC-PK1 cells were eitherleft untreated or exposed to blebbistatin as in Figure 2A,and then fixed and stained with rhodamine phallodin.F-actin arrangement was visualized near the apical sur-face (top, microvilli) and the ventral surface (bottom,level of stress fibers). (B) Cells were transfected withMyc-tagged DN-MLC as in Figure 2C, and then doublystained using an anti-Myc antibody (green) and rhoda-mine phalloidin (red). No obvious differences were ob-served in the organization of F-actin between controland DN-MLC–expressing cells. (C) Quantification ofthe cellular F-actin content. Cells were treated with bleb-bistatin (blebbi) as in Figure 2A or with 10 �M latrun-culin B (LB) for 2 h (as a positive control), and their totalF-actin content was determined with the phalloidin ex-traction assay as described in Materials and Methods. Toassess the effect of DN-MLC, transiently transfectedcells were exposed to a selection procedure using G-418,until �85% transfection efficiency was achieved. Thetotal F-actin in these cells was then measured as above.(D) Total actin was assessed by Western blotting underthe same conditions as in C.

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with a drop in nuclear labeling. Nonetheless, the latter stillremained significantly higher than the extranuclear signal(Figure 4A). Ca2� depletion of the cultures caused a sizableand time-dependent increase in nuclear SRF staining. Toverify that this effect was not an optical artifact due to cellcontraction–associated cytosolic shrinkage, we performedWestern blots on nuclear extracts obtained from control andCa2�-deprived cultures. Figure 4B shows that an increase innuclear SRF upon Ca2� removal was detected also by thistechnique. Next we tested whether a Myc-tagged constitu-tively active Rho mutant was able to impact on SRF local-ization. We observed enhanced nuclear accumulation of SRFin active Rho-transfected cells (Figure 4C). Curiously how-ever, this effect was clearly visible only in cells that showeda modest Rho expression (as visualized by Myc staining),whereas it was not apparent in cells with high level (andpossibly longer lasting) expression of active Rho. This find-ing suggests that the increase in nuclear SRF accumulationmay be transient, or various Rho-dependent pathwaysmight be involved both in nuclear import and export pro-cesses. Expression of DN-Rho substantially mitigated orcompletely prevented the increase in nuclear SRF (Figure4D). Indeed in many DN-Rho–expressing, stimulated cellsthe level of nuclear SRF was lower than in nonstimulatedcontrols (not shown). Finally we tested whether inhibition ofmyosin phosphorylation might impact on SRF traffic. To thisend cells were cotransfected with DN-MLC, and after Ca2�

removal doubly stained for Myc (DN-MLC) and SRF. DN-MLC expression appeared to mitigate the increase in nuclearSRF accumulation (Figure 4E). To quantify the results, thefluorescence intensity of SRF staining was determined in thenucleus and cytosol of individual (control or DN-MLC express-ing) cells and the nucleocytoplasmic ratio was calculated undervarious conditions (Figure 4F). Under resting conditions incontrol cells, SRF exhibited a �1.4-fold nuclear accumulationover the cytosol, which upon Ca2� removal increased to �2.1-fold. The expression of DN-MLC did not have a significanteffect on the resting SRF distribution, but it reduced the Ca2�

deprivation–induced rise by 60%. These data are consistentwith some contribution of myosin phosphorylation in the con-tact-dependent traffic of SRF. However, given the fact thatthere is a substantial amount of SRF in the nucleus even underresting conditions, and that the effect of DN-MLC was onlypartial, we continued to examine the contribution of anotherRho-dependent process, namely localization of MRTF.

Localization of MRTF Isoforms and the Effect of theRho-F-Actin Pathway on MRTF Distribution in TubularCellsBecause the distribution of MRTF, and its regulation hashitherto not been characterized in epithelial cells, initiallywe sought to compare the localization of MRTF isoforms inLLC-PK1 cells and fibroblast-like CHO cells. To achieve this,and also to overcome the restricted availability of MRTF

Figure 4. Contact disassembly facilitates the nuclearaccumulation of serum response factor (SRF) in a Rhoand MLC phosphorylation–dependent manner. (A)Confluent monolayers were serum-starved for 3 h andthen bathed in Ca2�-free DMEM for the indicated times.Cells were then fixed and stained for SRF. (B) Nuclearextracts were prepared from Control or Ca2�-deprived(3 h) cells followed by Western blotting for SRF and H3histones as a nuclear marker. (C) Confluent cells weretransfected with constitutive active Myc-tagged Rho(CA-Rho) for 24 h, and then doubly stained for SRF(red) and Myc (green). Note the robust nuclear accumu-lation of the transfected cells compared with their non-transfected neighbors. (D) Cells were transfected withMyc-tagged dominant negative Rho (DN-Rho) for 24 hfollowed by incubation in nominally Ca2�-free DMEMfor another 24 h. Cells were then fixed and stained forSRF (red) and Myc (green). To facilitate the identifica-tion of the same cells on the two corresponding fluores-cent images, successfully transfected cells or clusters ofcells are circled with dashed lines. Note the substantialreduction in the nuclear SRF staining of DN-Rho–ex-pressing cells. (E) Conditions were as in D, except thecells were transfected with Myc-tagged DN-MLC. (F)The intracellular distribution of SRF was quantified bymeasuring the nucleo-cytoplasmic ratio of the fluores-cence intensity. For each cell determinations were madealong lines drawn across the nucleus (see dashed line inE). The ratios were calculated for control (pcDNA3) andDN-MLC–transfected cells, which were incubated eitherin Ca2�-containing or nominally Ca2�-free medium fora day. In each category at least 60 cells were analyzed.Ca2� removal significantly enhanced the nuclear accu-mulation of SRF (p � 1010), and this effect was signif-icantly suppressed (p � 106) by DN-MLC.


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antibodies, we transfected cells with constructs encodingFLAG epitope–tagged MRTF-A and MRTF-B, and followedtheir localization through staining with an anti-FLAG anti-body. As expected, in CHO cells both MRTF-A and -B ex-hibited predominantly cytosolic staining (in 67 and 82% ofthe cells, respectively), although the rest of the cells showedeven (pancellular) staining (nuclear/cytosolic ratio 0.75-1.25) or nuclear accumulation (nuclear/cytosolic ratio �1.25;Figure 5A). In contrast, in LLC-PK1 cells MRTF-A wasmostly nuclear (�70%), whereas MRTF-B was mainly cyto-solic (�70%; Figure 5A). This finding indicates that there aresignificant cell type–specific differences in MRTF distribu-tion between fibroblasts and LLC-PK1 epithelial cells. Nextwe investigated whether the distribution of MRTF-B, whichunder resting conditions partitioned mostly in the cytosol,was responsive to Rho signaling and cytoskeletal changes inepithelial cells. Coexpression of constitutively active GFP-Rho resulted in a large (�8-fold) increase in nuclear MRTF-Baccumulation (Figure 5, B and D, a and a�). Overall �80% ofRho-transfected cells showed pancellular or fully nucleardistribution, whereas this fraction was only 15% in the con-trol (GFP expressing) cells. To verify that the changes inactin organization are indeed able to redistribute MRTF-B inkidney epithelial cells, we used jasplakinolide, a potent ac-tin-polymerizing agent. Similar to active Rho, this drug alsoprovoked robust nuclear accumulation of MRTF-B (Figure5Db).

Next we investigated the effect of contact disassembly onMRTF-B distribution. Ca2� removal caused a more than2.5-fold increase in nuclear localization of MRTF-B. More-over, inhibition of Rho signaling by the overexpression ofRho-GAP suppressed the contact disruption–promoted nu-clear translocation of MRTF-B (Figure 5C).

Next we studied whether forced F-actin polymerization orMRTF overexpression might result in SMA promoter acti-vation and protein expression in LLC-PK1 epithelial cells.Jasplakinolide (in the absence of any other stimulus) wasable to provoke a large increase in SMA promoter activity(Figure 6A) and induced SMA protein expression (Figure6B), indicating that drastic actin polymerization is sufficientto trigger myofibroblast transformation of normal epithelialcells. Consistent with an important role of MRTF in theregulation of SMA expression, transfection of MRTF iso-forms led to a robust increase in the SMA promoter activity(Figure 6C), which—in agreement with the localization da-ta—was stronger in case of MRTF-A than MRTF-B. Further,expression of the MRTF constructs was often accompaniedwith SMA protein expression, as verified by double immu-nostaining for SMA and the FLAG epitope (Figure 6E). Theexpression of SMA was robust considering that transienttransfection of a few percent of the cells with MRTF-A or Bresulted in SMA synthesis that was readily detectable intotal cell lysates by Western blotting (Figure 6D). Control ormock-transfected epithelial cells never expressed SMA (Fig-ure 6, D and E).

Taken together these results indicate that although MRTFisoforms may be differentially regulated in fibroblasts andcertain epithelial cells, Rho-dependent cytoskeleton remod-eling is a key factor in the control of MRTF distribution alsoin epithelial cells. Furthermore, MRTF is a potent inducer ofSMA expression in an epithelial setting as well.

Contact Integrity Regulates the NucleocytoplasmicTransfer of Endogenous MRTF in a Rho- and MLCPhosphorylation–dependent MannerTo substantiate the relevance of these findings, we followedthe localization of endogenous MRTF using a polyclonal

antibody raised against BSAC, the mouse homologue ofMKL1/MRTF-A. In resting LLC-PK1 cells endogenousMRTF showed entirely cytosolic distribution with strongnuclear exclusion (Figure 7, Aa and B). Expression of con-stitutive active Rho redistributed MRTF into the nucleus(Figure 7A, c and c�, and B), verifying the Rho responsive-ness of the endogenous protein in this epithelial setting.Importantly, Ca2� removal also caused a marked nuclear

Figure 5. The impact of the Rho-F-actin pathway and cell contactinjury on the localization of MRTF isoforms in epithelial cells. (A)CHO cells or LLC-PK1 cells were transfected with either FLAG-tagged MRTF-A or -B and 2 d later stained with an anti-FLAGantibody. FLAG-expressing CHO cells (145 and 167 cells forMRTF-A and -B, respectively) and LLC-PK1 cells (335 and 3606 forMRTF-A and -B, respectively) were counted for nuclear, even orcytosolic distribution. These categories were objectified as describedin Materials and Methods. Typical examples of distribution are shownon the right panels. (B) Confluent layers of LLC-PK1 cells werecotransfected with FLAG-MRTF-B and GFP or GFP-CA-Rho, and48 h later analyzed for intracellular distribution of FLAG staining.Note that CA-Rho induced large nuclear accumulation of MRTF-B.(C) Cells were either transfected with FLAG-MRTF-B alone or alongwith p190 RhoGAP, and after 48 h, cells were serum-deprived for3 h. Subsequently, the medium was replaced with Ca2�-free DMEMwhere indicated, and after 2.5 h incubation the cells were fixed andstained with an anti-FLAG antibody. Data are from 3 to 9 separateexperiments, and in each category 300-1800 cells were counted.Ca2� removal raised the percentage of cells with fully nucleardistribution from 10.1 � 1.8 to 26.2 � 5.1% (p � 0.005, n 9), andthis effect was entirely prevented by Rho-GAP. (D) In a and a�typical images showing the entirely nuclear accumulation ofMRTF-B (FLAG-staining, red) in a GFP-CA-Rho–expressing cell(green); (b) jasplakinolide (Jas) treatment (0.5 �M for 12 h) inducesstrong nuclear accumulation of the transfected MRTF-B.

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shift: after this treatment �16% of the cells exhibited strongnuclear accumulation, whereas the nuclear exclusion disap-peared in the majority of cells (Figure 7, Ab and B). Domi-nant negative Rho strongly mitigated the contact disrup-tion–induced nuclear transfer (Figure 7, A, d–d�, and B).Importantly, expression of DN-MLC significantly sup-pressed the translocation as well, suggesting the contribu-tion of myosin phosphorylation in the process (Figure 7, A,e–e�, and B).

These findings raised the possibility that the contact-depen-dent regulation of MRTF distribution might play an importantrole in the differential responsiveness of confluent and noncon-fluent cultures to the EMT-inducing effect of TGF-�1. To testthis assumption, we compared MRTF distribution in confluentand nonconfluent cultures exposed to TGF-�1 for varioustimes. Endogenous MRTF was entirely cytosolic in confluentcultures (Figure 8A, top panels). Treatment of intact confluentlayers with TGF-�1 (0–24 h) did not induce nuclear transloca-

tion of MRTF, and most cells showed no change in MRTFlocalization at all, whereas some exhibited a punctate, perinu-clear labeling. A radically different picture was observed insubconfluent cultures. Under resting condition, �75% of thecells located at the free edges of cellular islands showed cyto-solic MRTF staining, whereas �17% showed clear nuclear ac-cumulation and 8% had even cytosolic and nuclear distribution(Figure 8A, bottom panels and graph). The extent of the nu-clear accumulation of MRTF in subconfluent layers was ingood agreement with the values obtained in cells in which thecontacts were disassembled by Ca2� removal. In cells locatedin the intact inner regions of these multicellular islands, MRTFwas fully cytosolic. In subconfluent layers (as opposed to theconfluent ones), TGF-�1 exposure induced a dramatic changein MRTF distribution: in cells at the free edges, perinuclearMRTF condensation was apparent after 1 h treatment (notshown), whereas after 6 h, 95% of peripheral cells showedstrong nuclear accumulation of MRTF (Figure 8A). Cells inrows adjacent to the peripheral row also showed increasednuclear localization (Figure 8A), whereas in the inner areasMRTF remained cytosolic (not shown). To our surprise nuclearaccumulation of MRTF in the peripheral cells was transient:after 24 h of TGF-�1 treatment, the response significantly de-creased: only 25% of the cells showed clear nuclear MRTFlocalization, whereas even distribution or punctate, perinuclearlabeling was visible in 12% of the cells (Figure 8A).

We argued that if myosin phosphorylation indeed plays apermissive role in nuclear translocation of MRTF, thenTGF-�1 might not be able to elicit large or sustained MLCphosphorylation in fully confluent layers. Indeed, TGF-�1exposure for 16 h had no discernable effect on phospho-MLCcontent when the cytokine was added to confluent layers(Figure 8B). In contrast substantial phospho-MLC stainingwas observed at the free edges of cells located in the periph-ery of untreated subconfluent islands (Figure 8B), i.e., at thesame locus where cells are susceptible to TGF-�1–inducedSMA expression. The phospho-MLC signal at these sitesappeared to further increase upon TGF-�1 treatment. Con-sistent with these findings, acute Ca2� removal caused ro-bust myosin phosphorylation in confluent layers (as visual-ized by Western blotting), whereas short-term treatmentwith TGF-�1 caused a much smaller response. Nonetheless,TGF-�1 was able to cause a significant and rapid albeittransient MLC phosphorylation, indicating that the conflu-ent monolayer remained responsive to the cytokine. Ca2�

removal combined with TGF-�1 gave the most robust effecton MLC phosphorylation (Figure 8C).

In addition to Ca2� removal and subconfluence, the third,and from a pathological standpoint possibly the most rele-vant, model of contact disruption is mechanical woundingof a confluent monolayer. Cells at the wound edge showedmarked MLC phosphorylation (Figure 8D). Interestingly, atthis location a number of cells exhibited nuclear accumula-tion of endogenous MRTF as well (Figure 8D).

Finally we tested whether interfering with myosin phos-phorylation indeed impacts on SMA protein expression. Asshown on Figure 8E DN-MLC reduced the number of SMA-expressing cells in sparse, TGF-�1–treated cultures. Underthese conditions approx. 22% of control cells expressedSMA, whereas this number dropped more than fourfold(approx. 4%) in DN-MLC–expressing cells (Figure 8E).

Taken together, the nuclear accumulation of endogenousMRTF is regulated in a cell contact– and contractility-depen-dent manner in tubular cells, and impaired contact integrityplays a deterministic role in the regulation of MRTF distri-bution upon TGF-�1 treatment.

Figure 6. Jasplakinolide or the overexpression of MRTF isoformsis sufficient to induce SMA protein synthesis in tubular cells. (A)Cells were transfected with the pSMA-Luc/pRL-TK system for 24 hand either left untreated for a day, or treated with jasplakinolide(Jas, 0.5 �M) for 24 h or for the last 3 h of this 24-h period. SMApromoter activity is expressed as fold increase compared with theuntreated sample. (B) Cells were left untreated or exposed to Jas for24 h, lysed, and subjected to Western blotting using an anti-SMAantibody. (C) Tubular cells were cotransfected with the pSMA-Luc/pRL-TK and either MRTF-A or -B. Twenty-four hours later SMApromoter activity was determined as in A. (D) Untransfected con-trols (none) or cells transiently transfected with MRTF-A or -B for48 h were lysed, and subjected to SDS-PAGE followed by Westernblotting using an anti-SMA antibody. Control cells do not expressSMA, whereas both MRTF-A and -B were able to induce SMAexpression. The response was stronger in the case of MRTF-A inagreement with the strong nuclear localization and greater SMApromoter–activating capacity of this construct. (E) Cells were trans-fected with MRTF-A or -B, and after 48 h, fixed and stained for SMA(red), FLAG (green) to visualize successful transfection, and thenuclear dye DAPI to visualize every cell.


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MRTF Is a Central Contributor to the CellContact–regulated and TGF-�1–modulated Activationof the SMA PromoterTo address whether in our system MRTF has a causal role inthe contact injury–dependent SMA promoter response, andits potentiation by TGF-�1, we transfected the cells with amutant FLAG-tagged myocardin (�C585), which lacks thetransactivation domain, and has been shown to act as dom-inant negative against each member of the MRTF family(Wang et al., 2001). This mutant showed spontaneous accu-mulation in the nucleus and was present in the cytosol too,as revealed by immunostaining with an anti-FLAG antibody(Figure 9A). Importantly, expression of �C585 abolished theCa2� deprivation–triggered increase in promoter activityand strongly suppressed the synergism between contactdisassembly and TGF-�1 (Figure 9B). These observationssuggest that endogenous MRTF activity is a central target ofthe cell contact– and TGF-�1–dependent regulation of theSMA promoter, and it plays an indispensable role in myo-fibroblast differentiation of kidney tubular cells.

DISCUSSION

Epithelial–mesenchymal transition is a key process in organfibrosis (Kalluri and Neilson, 2003). Injury or absence of

intercellular contacts exerts a permissive and potentiatingeffect on the transdifferentiation of epithelial cells to myofi-broblasts (Masszi et al., 2004). This phenomenon may have akey importance from a pathobiologic standpoint: althoughintact epithelia may be partially resistant to the fibrogeniceffect of TGF-�1, an initial injury may render the woundedregion susceptible for this cytokine, thereby generating fo-cally transformed areas. From these foci the process canspread to neighboring regions. Furthermore, the develop-ment of myofibroblasts in the wound has been associatedwith a change in the type of the expressed cadherins (Hinzet al., 2004). With this scenario in mind, the present studyaimed to identify contact-dependent mechanisms that canfacilitate EMT. We found that contact disassembly causesRho activation and ROK-mediated MLC phosphorylation.These events then contribute to the nuclear redistribution ofSRF and mainly its coactivator, MRTF, which in turn activatethe SMA promoter, and strongly synergize with the TGF-�1–induced SMA protein expression.

Cell Contacts, Rho Activation, and MLC PhosphorylationOur finding that the Ca2� removal–induced disruption ofcell junctions activates Rho is in good accord with the re-ported converse phenomenon, i.e., that during the Ca2�-triggered formation of intercellular junctions Rho activity is

Figure 7. Contact disassembly induces Rho- and MLCphosphorylation–dependent nuclear translocation ofendogenous MRTF in epithelial cells. (A) In a and b cellswere serum-deprived for 3 h and then placed into eitherCa2�-containing or Ca2�-free DMEM for 24 h. Cellswere then fixed and stained for endogenous MRTF us-ing a polyclonal antibody raised against BSAC, themouse MKL1 or MRTF-A protein. (c and c�) Cells weretransfected with Myc-tagged CA-Rho and 24 h laterfixed and stained for endogenous MRTF (red) and Myc(green). Note the large nuclear accumulation of MRTFin the CA-Rho-expressing cells. (d–d�) Cells were trans-fected with Myc-tagged DN-Rho and 24 h later sub-jected to Ca2� removal for 24 h, fixed, and stained forMyc (green), endogenous MRTF (red), and all nucleiwere visualized with DAPI (blue). Note the reducednuclear accumulation of MRTF in the DN-Rho–express-ing cells compared with their untransfected neighbors.(e–e�) Similar experiments were performed as in d, ex-cept the cells were transfected with DN-MLC. Note thesubstantial nuclear translocation in many nontrans-fected cells and the preservation of nuclear exclusion inthe DN-MLC–expressing cells. (B) Distribution of en-dogenous MRTF was quantified in each transfectedgroup. The number of evaluated cells was as follows:control, 283; No Ca, 438; CA-Rho, 52; DN-Rho, 52; DN-MLC, 224.

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gradually down-regulated (Noren et al., 2003). The mecha-nism whereby the destabilization of tight or adherent junc-tions stimulates Rho remains to be elucidated. It is notewor-thy that recently a Rho-specific guanine nucleotide exchangefactor, the Dbl family member GEF-H1/Lfc has been foundto localize to the tight junction, where it regulates paracel-lular permeability, a myosin-modulated function (Benais-Pont et al., 2003). Future studies should address whetherGEF-H1 is activated by contact disruption. In tubular cells,contact disassembly led to rapid and long-lasting MLCphosphorylation, which was most prominent at the cell pe-riphery. This response was mediated by the Rho/ROK path-way because it was inhibited by genetic or pharmacologicalinterference with this signaling route. The same maneuversabolished the Ca2� removal–induced activation of the SMApromoter as well, indicating that the Rho/ROK pathway hasa key role in cell contact–dependent regulation of geneexpression. In addition to the spatially restricted activationof Rho, junctional ROK and/or myosin localization or accu-mulation may also contribute to the focal MLC phosphory-lation. Indeed, a subpool of ROK was found to be associatedwith the adherens junctions (Walsh et al., 2001), and a pe-ripheral myosin ring is present in epithelial cells (Ivanov etal., 2004, 2005). Thus, each component of the Rho/ROK/MLC pathways can be junction-associated, facilitating the

preferential activation of this particular downstream Rhopathway at the contacts.

Myosin Phosphorylation and the Regulation of the SMAPromoterRho has been shown to increase the transcriptional activityof SRF on those target genes, including SMA, whose pro-moter harbors CArG boxes (Hill et al., 1995; Mack et al., 2001;Masszi et al., 2003). Elegant studies have revealed that theeffect of Rho is mediated by cytoskeletal reorganization, akey component of which is enhanced F-actin polymerization(Miralles et al., 2003). So far two downstream Rho effectorpathways have been implicated in SRF-dependent transcrip-tion: the activation of the formin protein mDia, which in-duces net F-actin polymerization (Copeland and Treisman,2002) and the activation of the Rho/ROK/LIM kinase/cofi-lin phosphorylation pathway, which stabilizes F-actin due todecreased severing (Geneste et al., 2002). The former mech-anism was predominant in fibroblasts, whereas both werecritical in neuron-like PC12 cells. Our studies point to theimportance of a third Rho effector pathway, namely ROK-dependent MLC phosphorylation. This mechanism, at leastin our epithelial cells, seems to be an important contributor,because the myosin inhibitor blebbistatin or a phosphoryla-tion–incompetent DN myosin mutant abolished the contact

Figure 8. The effect of TGF-�1 in confluent and sub-confluent layers on the intracellular distribution of en-dogenous MRTF and MLC phosphorylation. (A) Cellswere grown to 100% confluence or approx. 30% conflu-ence (subconfluent) and left untreated and fixed ortreated with 10 ng/ml TGF-�1 for the indicated timesand then fixed and stained for MRTF. The bar diagramon the right indicates the intracellular distribution ofendogenous MRTF in cells at the periphery of cellularislands, under control conditions or after treatment forthe indicated times with TGF-�1. (B) Confluent or sub-confluent layers were left untreated or exposed toTGF-�1 for 16 h and then fixed and stained for pMLC.Nuclei were visualized by DAPI. (C) The acute effect ofCa2� removal and TGF-�1 on myosin phosphorylation.Cells were treated with normal or EGTA-containingmedium in the absence or present of 10 ng/ml TGF-�1for 15 min and then processed for Western blotting withthe anti-pMLC antibody as described in Materials andMethods. Tubulin was used as loading control. (D) Awound was generated in a confluent monolayer with arubber policeman, and 6 h later the cells were fixed andstained either for MRTF or for pMLC. (E) Cells wereseeded sparsely, transfected with Myc-tagged DN-MLC, and then treated daily with 10 ng/ml TGF-�1 for3 d. Cells were then fixed and doubly stained for SMAand for the Myc epitope. Note the robust SMA expres-sion in the control cells and the absence of SMA expres-sion in the DN-MLC–expressing cells. To quantify theeffect, three separate experiments were performed, inwhich 910 randomly selected control (nontransfected)cells and 311 DN-MLC–expressing cells were assessedfor SMA expression (p � 2.0 � 105).


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disruption-provoked SMA promoter expression, eliminatedthe synergism between contact injury and TGF-�1 on thepromoter, and suppressed SMA protein expression. Periph-eral myosin activity (junctional contractility) has been pro-posed to participate in the regulation of various functionsincluding paracellular permeability (Turner, 2000), junctionremodeling (Ivanov et al., 2004, 2005; Shewan et al., 2005),cell scattering (de Rooij et al., 2005), morphogenesis (Bertet etal., 2004), and closure of epithelial wounds (Darenfed andMandato, 2005). Our data assign yet another critical role forthis process: the regulation of SRF-dependent gene expres-sion (Figure 10). This mechanism efficiently couples the me-chanical and genetic responses to wounding: Formation ofactin–myosin complexes triggers contractile wound closureand at the same time initiates genetic reprogramming, lead-ing to enhanced generation of extracellular matrix proteinsand contractile elements.

What is the mechanism whereby myosin activity regulatescontact-dependent SMA expression? Contact disassemblypromoted the nuclear translocation of SRF and its coactiva-tor MRTF, whereas interference with the Rho/ROK path-way or directly with myosin phosphorylation suppressedthe nuclear accumulation and/or retention of these factors.The effect of myosin inhibition on the accumulation or re-tention of SRF was modest, suggesting that myosin-phos-phorylation is not the major Rho-mediated pathway regu-lating this process. In addition, high Rho activity appearedto facilitate the nuclear export of SRF, pointing to a complexregulation. In any case, our results suggest that contractilityor tension per se may be critical regulators of MRTF-medi-ated gene expression. Intriguingly, active (movement-asso-

ciated) or passive (traction-transmitted) tension induced thenuclear localization of drosophila MAL (Somogyi and Rorth,2004) and has been associated with increased SMA expres-sion (Hinz et al., 2001).

The original question then translates into asking howmyosin activity impacts on MRTF localization or activity(Figure 10). Several possibilities can be evoked: 1) MRTFlocalization is regulated by the G/F actin ratio. Binding ofmonomeric actin (presumably through a yet unidentifiedprotein) to MRTF prevents its translocation to the nucleuswhereas actin polymerization removes G-actin from MRTF,thereby exposing its nuclear localization sequence (Miralleset al., 2003; Posern et al., 2004). It is conceivable that myosinactivity, which promotes actin filament bundling, can also

Figure 9. Dominant negative MRTF inhibits the contact disassem-bly–induced activation of the SMA promoter and suppresses thesynergism between contact disruption and TGF-�1. (A) Localizationof DN myocardin (DN-MyoC). Cells transfected with FLAG-taggedDN-MyoC for 24 h were serum-starved, incubated in Ca2�-contain-ing or Ca2�-free medium for 24 h, fixed, and stained using ananti-FLAG antibody. Note the predominantly nuclear localization ofthe construct irrespective of the state of the intercellular contacts. (B)Cells were cotransfected with pSMA-Luc/pRL-TK along with eitherempty vector (pcDNA3) or DN-MyoC for 24 h, and then exposed toCa2� removal, 10 ng/ml TGF-�1, or the combination of these treat-ment as in Figure 2D.

Figure 10. The synergistic effect of cell contact injury and TGF-�1in the complex regulation of the SMA promoter. Contact disassem-bly activates Rho which, in turn stimulates mDia (1; Copeland andTreisman, 2002) and ROK (2). The former process leads to increasedactin polymerization, whereas the latter activates the LIMK/cofilinpathway (2a) and stimulates MLC phosphorylation (2b). TheLIMK/cofilin pathway may stabilize F-actin via decreased severing(Geneste et al., 2002). Enhanced MLC phosphorylation may contrib-ute to SMA expression by acting through various nonexclusivemechanisms: it might promote actin polymerization/stabilization(i), may directly participate in the nuclear translocation or retentionof MRTF (ii), or might be required for the internalization of cellcontact components (iii; Ivanov et al., 2004). Increased nuclear ac-cumulation of MRTF acts in concert with SRF through the CArGboxes. In addition, contact injury triggers the internalization of�-catenin, which—through yet unidentified mechanisms—potenti-ates the activation of the SMA promoter (Masszi et al., 2004). Finally,TGF-�1 activates a multitude of signaling pathways, which throughvarious transcription factors impact on the TCE and SBE cis ele-ments. TGF-�1 may also contribute to Rho activation; however, thiseffect in itself is insufficient to provoke long-lasting MLC phosphor-ylation, which has a permissive effect on the activation of the SMApromoter. The underlined processes or pathways are addressed inthe current study. Question marks denote potential mechanisms.The relative contribution of the primary, permissive, and potentiat-ing mechanisms remains to be defined.

L. Fan et al.


“steal away” monomeric actin from MRTF, or the formationof actin–myosin complexes may specifically reduce theMRTF-binding competent pool of actin. Indeed, blebbistatin,which inhibits basal myosin activity as well, promoted thedissociation of stress fibers. However we found no evidencethat blebbistatin would significantly decrease the total F-actin pool. Furthermore, using DN-MLC, we did not observeany major change in the actin skeleton organization andonly a modest reduction in F-actin. In this regard, it is worthpointing out that both the monomer-sequestering drug la-trunculin B and the barbed end capper cytochalasin D in-duce strong F-actin disassembly, yet the former inhibitswhile the latter triggers the nuclear translocation of MRTF(Miralles et al., 2003). This observation suggests that thecritical factor is not necessarily the content of F-actin or theF/G actin ratio, but rather the interaction of G-actin withinhibitors or proteins, which will determine whether G-actincan bind to MRTF. Taken together our observations suggestthat a gross alteration in the G/F actin ratio is unlikely to berequired for the inhibition of SMA expression by suppressedmyosin phosphorylation. Indeed, several observations sug-gest that Rho impacts on SRF signaling not exclusively viaactin polymerization: Certain Rho mutants stimulate SRFwithout inducing stress fibers formation (Sahai et al., 1998;Zohar et al., 1998), and a newly described Rho effectorhCNK1 activates SRF without promoting stress fiber assem-bly (Jaffe et al., 2004). Nonetheless our data do not rule outthe possibility that inhibition of myosin might interfere withstimulus-induced localized F-actin assembly. Clearly, futurestudies should directly address whether myosin activityimpacts on the formation of the actin/actin-binding pro-tein/MRTF complexes. 2) Another potential mechanism isthat myosin, as a force-generating protein, might be requiredfor the efficient nuclear import or retention of MRTF. Thereis accumulating evidence that both the microtubule and themicrofilament cytoskeleton is involved in the nuclear importof certain proteins (Campbell and Hope, 2003). Recently,endothelial MLC kinase has been shown to regulate thenuclear translocation of NF�B and the consequent reportergene activation (Wadgaonkar et al., 2005). Although theunderlying mechanism remains to be clarified, a similarprocess might participate in MRTF translocation as well,except in LLC-PK1 cells the predominant enzyme responsi-ble for MLC phosphorylation is ROK and not MLCK (Szasziet al., 2005). 3) Finally, myosin may affect other processes inaddition to MRTF translocation. We have previously shownthat upon contact injury E-cadherin is degraded allowingthe liberation of �-catenin, which potentiates the activationof the SMA promoter (Masszi et al., 2004). Importantly,Ivanov et al. (2004) have shown that myosin activity is es-sential for the contact disassembly–induced internalizationof E-cadherin, and blebbistatin maintains E-cadherin at thecell surface. Similarly, the Src-mediated delocalization ofE-cadherin from the AJ also requires MLC phosphorylation(Avizienyte et al., 2004). Taken together, junction stabiliza-tion by myosin inhibition may contribute to the inhibition ofthe SMA promoter.

MRTF and EMTThe present work identifies MRTF as a central factor inmesenchymal/myofibroblast transdifferentiation of epithe-lial cells. This conclusion is supported by the findings that 1)overexpression of MRTF is sufficient to induce SMA pro-moter activation and protein expression in tubular cells; 2)induction of robust actin polymerization induces nuclearaccumulation of MRTF concomitant with SMA expression,and 3) DN-myocardin prevents the Ca2� depletion–induced

promoter activation and the synergism between contact in-jury and TGF-�1. In nonstimulated LLC-PK1 cells endoge-nous MRTF (as visualized by the anti-BSAC antibody) wascytosolic. Interestingly, the heterologously expressed MRTF-Awas predominantly nuclear, whereas MRTF-B localizedmainly to the cytosol. In contrast, both MRTF-A and -B werecytosolic in fibroblasts, pointing to tissue-specific differencesin localization, presumably due to a different set of actin-binding proteins. Indeed, recently a striated muscle-specificactin- and MRTF-binding protein (STARS) has been identi-fied (Kuwahara et al., 2005). Further studies are warranted toinvestigate the contribution of endogenous MRTF isoformsto EMT.

We observed that TGF-�1 was unable to induce MRTFtranslocation in fully confluent layers, but it enhanced nu-clear accumulation after contact disassembly. This findingimplies that MRTF localization is one of the key targetmechanisms that underlies the synergy between TGF-�1 andcontact injury. Presumably, the strong, contact-dependentRho activation is indispensable for the efficient nuclear ac-cumulation of MRTF. On the other hand, moderate translo-cation of endogenous MRTF may not be sufficient to induceSMA expression, because cells adjacent to the wound are nottransformed in the absence of TGF-�1. The SMA promoterharbors several transcriptional regulatory elements, includ-ing the SRF/MRTF-binding CArG-boxes, the Kruppel fac-tor-binding TGF-� control element (TCE), and the TGF-�–responsive SBE (Hautmann et al., 1997; Hu et al., 2003; Liu etal., 2003b). Accordingly, the promoter can be collectivelyregulated by contact-dependent (Rho-mediated) and TGF-�1–dependent (partially Rho-independent) pathways (Fig-ure 10). Interestingly MRTF may have multiple roles: inaddition to forming a ternary complex with SRF and CArGboxes, it was found to bind to the SMAD proteins too, andthus it might facilitate transcription through the SBE (Qiu etal., 2005). Moreover SMADs were shown to associate with�-catenin as well (Tian and Phillips, 2002). These multipleinputs then can culminate in robust promoter activation.Finally, we observed that even under the maximally effec-tive two-hit conditions, MRTF accumulation in the nucleusis transient. Future studies should investigate the regulationof the nuclear export of MRTF.

In summary, we propose that the contact injury-induced,Rho-ROK-phospho-myosin–mediated MRTF translocationrepresents a central signaling pathway in transdifferentia-tion of epithelial cells, and thereby in the pathogenesis oforgan fibrosis.

ACKNOWLEDGMENTS

This work was supported by grants from the Kidney Foundation of Canadato A.K., by the Hungarian Science and Technology Foundation to A.K. andI.M., by the Canadian Institute of Health Research to K.S. and A.K., and by theOntario Heart and Stroke Foundation, T419 to C.A.M.

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