Abstract: The Snail transcription factor has been described recently as a strong repressor of E-cadherin in epithelial cell lines, where its stable expression leads to the loss of E-cadherin expression and induces epithelial-mesenchymal transitions and an invasive phenotype. The mechanisms regulating Snail expression in development and tumor progression are not yet known. We show here that transforming growth factor β-1 (TGFβ1) induces Snail expression in Madin-Darby canine kidney cells and triggers epithelial-mesenchymal transitions by a mechanism dependent on the MAPK signaling pathway. Furthermore, TGFβ1 induces the activity of Snail promoter, whereas fibroblast growth factor-2 has a milder effect but cooperates with TGFβ1 in the induction of Snail promoter. Interestingly, TGFβ1-mediated induction of Snail promoter is blocked by a dominant negative form of H-Ras (N17Ras), whereas oncogenic H-Ras (V12Ras) induces Snail promoter activity and synergistically cooperates with TGFβ1. The effects of TGFβ1 on Snail promoter are dependent of MEK1/2 activity but are apparently independent of Smad4 activity. In addition, H-Ras-mediated induction of Snail promoter, alone or in the presence of TGFβ1, depends on both MAPK and phosphatidylinositol 3-kinase activities. These data support that MAPK and phosphatidylinositol 3-kinase signaling pathways are implicated in TGFβ1-mediated induction of Snail promoter, probably through Ras activation and its downstream effectors. The Snail transcription factor has been described recently as a strong repressor of E-cadherin in epithelial cell lines, where its stable expression leads to the loss of E-cadherin expression and induces epithelial-mesenchymal transitions and an invasive phenotype. The mechanisms regulating Snail expression in development and tumor progression are not yet known. We show here that transforming growth factor β-1 (TGFβ1) induces Snail expression in Madin-Darby canine kidney cells and triggers epithelial-mesenchymal transitions by a mechanism dependent on the MAPK signaling pathway. Furthermore, TGFβ1 induces the activity of Snail promoter, whereas fibroblast growth factor-2 has a milder effect but cooperates with TGFβ1 in the induction of Snail promoter. Interestingly, TGFβ1-mediated induction of Snail promoter is blocked by a dominant negative form of H-Ras (N17Ras), whereas oncogenic H-Ras (V12Ras) induces Snail promoter activity and synergistically cooperates with TGFβ1. The effects of TGFβ1 on Snail promoter are dependent of MEK1/2 activity but are apparently independent of Smad4 activity. In addition, H-Ras-mediated induction of Snail promoter, alone or in the presence of TGFβ1, depends on both MAPK and phosphatidylinositol 3-kinase activities. These data support that MAPK and phosphatidylinositol 3-kinase signaling pathways are implicated in TGFβ1-mediated induction of Snail promoter, probably through Ras activation and its downstream effectors. The molecular mechanisms underlying local invasion and metastasis are still poorly understood, but evidence accumulated in the last years indicates the existence of common cellular mechanisms for the local invasive process that represent the first stage into the metastatic cascade of carcinomas (1Hanahan D. Weinberg R.A. Cell. 2000; 100: 57-70Abstract Full Text Full Text PDF PubMed Scopus (22406) Google Scholar, 2Stetler-Stevenson W.G. Aznavoorian S. Liotta L.A. Annu. Rev. Cell Biol. 1993; 9: 541-573Crossref PubMed Scopus (1523) Google Scholar). Among those, loss of expression or function of the E-cadherin cell-cell adhesion molecule has emerged as an important event for local invasion of epithelial tumor cells, leading to the consideration of E-cadherin as an invasion-suppressor gene (3Berx G. Cleton-Jansen A.M. Nollet F. de Leeuw W.J. van de Vijver M. Cornelisse C. van Roy F. EMBO J. 1995; 14: 6107-6115Crossref PubMed Scopus (658) Google Scholar, 4Birchmeier W. Behrens J. Biochim. Biophys Acta. 1994; 1198: 11-26Crossref PubMed Scopus (933) Google Scholar, 5Christofori G. Semb H. Trends Biochem. Sci. 1999; 24: 73-76Abstract Full Text Full Text PDF PubMed Scopus (650) Google Scholar). The process of invasion is frequently associated with the loss of other epithelial markers and the acquisition of mesenchymal markers and a migratory and motility behavior, collectively known as epithelial-mesenchymal transitions (EMTs) 1The abbreviations used are: EMTs, epithelial-mesenchymal transitions; AP, activator protein; ERK, extracellular signal-regulated kinase; FGF, fibroblast growth factor; MAPK, mitogen-activated protein kinase; MDCK, Madin-Darby canine kidney; MEK, MAPK/ERK kinase; PI3K, phosphatidylinositol 3-kinase; RT, reverse transcription; TGFβ, transforming growth factor β; FBS, fetal bovine serum. (see Ref. 6Thiery J.P. Nat. Rev. Cancer. 2002; 2: 442-454Crossref PubMed Scopus (5489) Google Scholar for a recent review). EMTs also occur during normal embryonic development in a strict spatio-temporal control, and they are required at specific stages, such as during gastrulation, formation of the neural crest cells, and other morphogenetic processes (6Thiery J.P. Nat. 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EMBO J. 1995; 14: 6107-6115Crossref PubMed Scopus (658) Google Scholar, 11Becker K.F. Atkinson M.J. Reich U. Becker I. Nekarda H. Siewert J.R. Hofler H. Cancer Res. 1994; 54: 3845-3852PubMed Google Scholar, 12Guilford P. Hopkins J. Harraway J. McLeod M. McLeod N. Harawira P. Taite H. Scoular R. Miller A. Reeve A.E. Nature. 1998; 392: 402-405Crossref PubMed Scopus (1379) Google Scholar), whereas the majority of carcinomas with down-regulated E-cadherin maintain an intact E-cadherin locus. Hypermethylation of the E-cadherin promoter and transcriptional alterations have emerged as the main mechanisms responsible for E-cadherin down-regulation in most carcinomas (5Christofori G. Semb H. Trends Biochem. Sci. 1999; 24: 73-76Abstract Full Text Full Text PDF PubMed Scopus (650) Google Scholar, 13Cheng C.W. Wu P.E. Yu J.C. Huang C.S. Yue C.T. Wu C.W. Shen C.Y. Oncogene. 2001; 20: 3814-3823Crossref PubMed Scopus (196) Google Scholar). Several transcriptional repressors of E-cadherin have been isolated recently, including the zinc finger factors Snail (14Cano A. Pérez-Moreno M.A. Rodrigo I. Locascio A. Blanco M.J. del Barrio M.G. Portillo F. Nieto M.A. Nat. Cell Biol. 2000; 2: 76-83Crossref PubMed Scopus (2931) Google Scholar, 15Batlle E. Sancho E. Franci C. Dominguez D. Monfar M. Baulida J. Garcia De Herreros A. Nat. Cell Biol. 2000; 2: 84-89Crossref PubMed Scopus (2172) Google Scholar) and Slug (16Hajra K.M. Chen D.Y. Fearon E.R. Cancer Res. 2002; 62: 1613-1618PubMed Google Scholar, 17Bolós V Peinado H. Pérez M oreno M.A. Fraga M.F. Esteller M. Cano A. J. Cell Sci. 2003; 116: 499-511Crossref PubMed Scopus (931) Google Scholar), the two-handed zinc factors ZEB-1 and SIP-1 (18Comijn J. Berx G. Vermassen P. Verschueren K. van Grunsven L. Bruyneel E. Mareel M. Huylebroeck D. van Roy F. Mol. Cell. 2001; 7: 1267-1278Abstract Full Text Full Text PDF PubMed Scopus (1155) Google Scholar, 19Grooteclaes M.L. Frisch S.M. Oncogene. 2000; 19: 3823-3828Crossref PubMed Scopus (268) Google Scholar), and the bHLH factor E12/E47 (20Pérez-Moreno M.A. Locascio A. Rodrigo I. Dhondt G. Portillo F. Nieto M.A. Cano A. J. Biol. Chem. 2001; 276: 27424-27431Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar). Snail family factors are in fact involved in EMTs when overexpressed in epithelial cell lines (14Cano A. Pérez-Moreno M.A. Rodrigo I. Locascio A. Blanco M.J. del Barrio M.G. Portillo F. Nieto M.A. Nat. Cell Biol. 2000; 2: 76-83Crossref PubMed Scopus (2931) Google Scholar, 15Batlle E. Sancho E. Franci C. Dominguez D. Monfar M. Baulida J. Garcia De Herreros A. Nat. Cell Biol. 2000; 2: 84-89Crossref PubMed Scopus (2172) Google Scholar, 17Bolós V Peinado H. Pérez M oreno M.A. Fraga M.F. Esteller M. Cano A. J. Cell Sci. 2003; 116: 499-511Crossref PubMed Scopus (931) Google Scholar), as well as in embryonic development (reviewed in Ref. 21Nieto M.A. Nat. Rev. Mol. Cell. Biol. 2002; 3: 155-166Crossref PubMed Scopus (1421) Google Scholar), and are proposed to act as inducers of the invasion process (14Cano A. Pérez-Moreno M.A. Rodrigo I. Locascio A. Blanco M.J. del Barrio M.G. Portillo F. Nieto M.A. Nat. Cell Biol. 2000; 2: 76-83Crossref PubMed Scopus (2931) Google Scholar, 22Blanco M.J. Moreno-Bueno G. Sarrio D. Locascio A. Cano A. Palacios J. Nieto M.A. Oncogene. 2002; 21: 3241-3246Crossref PubMed Scopus (487) Google Scholar). Generation of Snail knockout mice has further established the role of this factor in EMT and as the E-cadherin gene repressor. The null Snail embryos die at gastrulation as they fail to undergo a complete EMT process, forming an altered mesodermal layer that maintains the expression of E-cadherin (23Carver E.A. Jiang R. Lan Y. Oram K.F. Gridley T. Mol. Cell. Biol. 2001; 21: 8184-8188Crossref PubMed Scopus (513) Google Scholar). Nevertheless, the mechanisms that regulate the expression of Snail factors are still poorly understood (6Thiery J.P. Nat. Rev. Cancer. 2002; 2: 442-454Crossref PubMed Scopus (5489) Google Scholar, 21Nieto M.A. Nat. Rev. Mol. Cell. Biol. 2002; 3: 155-166Crossref PubMed Scopus (1421) Google Scholar). Different growth factors and cytokines have also been implicated in the process of EMTs in both epithelial cell systems and in embryonic development. Studies on development have indicated the participation of several members of the transforming growth factor (TGFβ)/bone morphogenetic family of growth factors in specific EMT processes in different species (24Liem Jr., K.F. Tremml G. Roelink H. Jessell T.M. Cell. 1995; 82: 969-979Abstract Full Text PDF PubMed Scopus (916) Google Scholar, 25Romano L.A. Runyan R.B. Dev. Biol. 2000; 223: 91-102Crossref PubMed Scopus (150) Google Scholar), whereas fibroblast growth factor (FGF) signaling has been reported recently (26Ciruna B. Rossant J. Dev. Cell. 2001; 1: 37-49Abstract Full Text Full Text PDF PubMed Scopus (513) Google Scholar) as a determinant for mesoderm cell fate specification in the mouse embryo. Several studies have also indicated that a multiple cross-talk among TGFβ/bone morphogenetics, FGF, and Wnt signals could be required for some EMTs in development (26Ciruna B. Rossant J. Dev. Cell. 2001; 1: 37-49Abstract Full Text Full Text PDF PubMed Scopus (513) Google Scholar, 27Dorsky R.I. Moon R.T. Raible D.W. Nature. 1998; 396: 370-373Crossref PubMed Scopus (416) Google Scholar, 28LaBonne C. Bronner-Fraser M. Development. 1998; 125: 2403-2414Crossref PubMed Google Scholar). In epithelial cell systems, several growth factors have been widely studied and reported to induce a scattering phenotype or a complete EMT depending on the specific cell system analyzed (reviewed in Refs. 6Thiery J.P. Nat. Rev. Cancer. 2002; 2: 442-454Crossref PubMed Scopus (5489) Google Scholar and 29Boyer B. Valles A.M. Edme N. Biochem. Pharmacol. 2000; 60: 1091-1099Crossref PubMed Scopus (383) Google Scholar). Among them, TGFβ has been identified as an important molecular player of EMT both in vitro and in vivo (30Caulín C. Scholl F.G. Frontelo P. Gamallo C. Quintanilla M. Cell Growth Differ. 1995; 6: 1027-1035PubMed Google Scholar, 31Cui W. Fowlis D.J. Bryson S. Duffie E. Ireland H. Balmain A. Akhurst R.J. Cell. 1996; 86: 531-542Abstract Full Text Full Text PDF PubMed Scopus (539) Google Scholar, 32Miettinen P.J. Ebner R. Lopez A.R. Derynck R. J. Cell Biol. 1994; 127: 2021-2036Crossref PubMed Scopus (796) Google Scholar, 33Oft M. Peli J. Rudaz C. Schwarz H. Beug H. Reichmann E. Genes Dev. 1996; 10: 2462-2477Crossref PubMed Scopus (565) Google Scholar, 34Portella G. Cumming S.A. Liddell J. Cui W. Ireland H. Akhurst R.J. Balmain A. Cell Growth Differ. 1998; 9: 393-404PubMed Google Scholar). In some cell systems, a synergistic cooperation between H-Ras activation and TGFβ signaling appears to be required for induction of a complete EMT (33Oft M. Peli J. Rudaz C. Schwarz H. Beug H. Reichmann E. Genes Dev. 1996; 10: 2462-2477Crossref PubMed Scopus (565) Google Scholar, 35Gotzmann J. Huber H. Thallinger C. Wolschek M. Jansen B. Schulte-Hermann R. Beug H. Mikulits W. J. Cell Sci. 2002; 115: 1189-1202PubMed Google Scholar, 36Janda E. Lehmann K. Killisch I. Jechlinger M. Herzig M. Downward J. Beug H. Grunert S. J. Cell Biol. 2002; 156: 299-313Crossref PubMed Scopus (613) Google Scholar). Recently, TGFβ has been reported to induce the expression of Snail in fetal and in immortalized murine hepatocytes and in human mesothelial cells (37Spagnoli F.M. Cicchini C. Tripodi M. Weiss M.C. J. Cell Sci. 2000; 113: 3639-3647PubMed Google Scholar, 38Valdes F. Alvarez A.M. Locascio A. Vega S. Herrera B. Fernandez M. Benito M. Nieto M.A. Fabregat I. Mol. Cancer Res. 2002; 1: 68-78PubMed Google Scholar, 39Yanez-Mo M. Lara-Pezzi E. Selgas R. Ramirez-Huesca M. Dominguez-Jimenez C. Jimenez-Heffernan J.A. Aguilera A. Sanchez-Tomero J.A. Bajo M.A. Alvarez V. Castro M.A. del Peso G. Cirujeda A. Gamallo C. Sanchez-Madrid F. Lopez-Cabrera M. N. Engl. J. Med. 2003; 348: 403-413Crossref PubMed Scopus (623) Google Scholar), but whether this is a direct or indirect effect has not yet been established. The participation of specific signaling pathways activated by TGFβ and/or H-Ras activation in EMTs has been analyzed previously with somewhat contradictory results as regard to the specific implication of Smad, mitogen-activated protein kinase (MAPK) and/or phosphatidylinositol 3-kinase (PI3K) pathways (36Janda E. Lehmann K. Killisch I. Jechlinger M. Herzig M. Downward J. Beug H. Grunert S. J. Cell Biol. 2002; 156: 299-313Crossref PubMed Scopus (613) Google Scholar, 40Bakin A.V. Tomlinson A.K. Bhowmick N.A. Moses H.L. Arteaga C.L. J. Biol. Chem. 2000; 275: 36803-36810Abstract Full Text Full Text PDF PubMed Scopus (830) Google Scholar, 41Chen Y. Lu Q. Schneeberger E.E. Goodenough D.A. Mol. Biol. Cell. 2000; 11: 849-862Crossref PubMed Scopus (241) Google Scholar, 42Montesano R. Soriano J.V. Hosseini G. Pepper M.S. Schramek H. Cell Growth Differ. 1999; 10: 317-332PubMed Google Scholar, 43Potempa S. Ridley A.J. Mol. Biol. Cell. 1998; 9: 2185-2200Crossref PubMed Scopus (300) Google Scholar). The issue has been unraveled recently (36Janda E. Lehmann K. Killisch I. Jechlinger M. Herzig M. Downward J. Beug H. Grunert S. J. Cell Biol. 2002; 156: 299-313Crossref PubMed Scopus (613) Google Scholar) in the EpRas model with the implication of MAPK in TGFβ-induced EMT, tumorigenesis, and metastasis, whereas PI3K is involved in cell scattering and resistance to TGF-β induced apoptosis. It remains to be established, however, if the same situation applies to other systems and, more importantly, the identification of the target genes involved in the specific growth factor signaling leading to EMTs. We have used the prototypic epithelial MDCK cells to further analyze the process of EMT induced by TGFβ and FGF. We have previously used this cell system to show that Snail overexpression leads to the full repression of E-cadherin expression and induction of a complete EMT (14Cano A. Pérez-Moreno M.A. Rodrigo I. Locascio A. Blanco M.J. del Barrio M.G. Portillo F. Nieto M.A. Nat. Cell Biol. 2000; 2: 76-83Crossref PubMed Scopus (2931) Google Scholar). In the present work we have investigated the ability of TGFβ1 and FGF2 to induce an EMT in MDCK cells and ask whether Snail is a target gene of this process. We present evidence that TGFβ1 treatment induces an EMT process linked to Snail induction in MDCK cells. Analysis of the mouse Snail promoter indicates that it is directly induced by TGFβ1 and that FGF2 and activated H-Ras cooperate with TGFβ1 in induction of the Snail promoter. Our results also indicate that the MAPK and PI3K pathways are involved in the TGFβ1- and H-Ras-mediated induction of Snail promoter. These results strongly support that Snail is a direct target of TGFβ1 and oncogenic H-Ras and open the way for future studies on the molecular mechanisms and targets of EMTs and the invasion process. Cell Culture and Treatments—MDCK-II cells were grown in Dulbecco's modified Eagle's medium and MCA3D and PDV cells in Ham's F-12 medium, in the presence of 10% FBS, 10 mm glutamine (Invitrogen), and 100 μg/ml ampicillin, 32 μg/ml gentamicin (Sigma). Cells were grown at 37 °C in a humidified CO2 atmosphere. All the transfections and treatments were done in FBS-free culture medium. For the indicated treatments, 10 μg/ml stocks of recombinant TGFβ1 (BioNova Corp.) and 100 μg/ml of FGF2 (Peprotech) were prepared according to manufacturer's instructions and added to the indicated concentrations. The PI3K and MEK1/2 inhibitors, LY294002 and PD98059 (Calbiochem), respectively, were kept as 30–10 mm stocks in Me2SO, which was used as vehicle control in all the inhibitor treatments. RT-PCR Analyses—Total RNA was isolated from the different cell lines, and RT-PCR analyses were carried out as described previously (14Cano A. Pérez-Moreno M.A. Rodrigo I. Locascio A. Blanco M.J. del Barrio M.G. Portillo F. Nieto M.A. Nat. Cell Biol. 2000; 2: 76-83Crossref PubMed Scopus (2931) Google Scholar, 17Bolós V Peinado H. Pérez M oreno M.A. Fraga M.F. Esteller M. Cano A. J. Cell Sci. 2003; 116: 499-511Crossref PubMed Scopus (931) Google Scholar, 20Pérez-Moreno M.A. Locascio A. Rodrigo I. Dhondt G. Portillo F. Nieto M.A. Cano A. J. Biol. Chem. 2001; 276: 27424-27431Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar). Canine PCR products were obtained after 30–35 cycles of amplification with an annealing temperature of 60–65 °C. Primer sequences were as follows: for canine E-cadherin (sequence kindly provided by Y. Chen, Harvard Medical School), forward: 5′-GGAATCCTTGGAGGGATCCTC-3′; reverse: 5′-GTCGTCCTCGC-CACCGCCGTACAT-3′ (amplifies a fragment of 560 bp); for canine Snail, forward: 5′-CCCAAGCCCAGCCGATGAG-3′; reverse: 5′-CTTGGCCACGGAGAGCCC-3′ (amplifies a fragment of 200 bp); and for canine glyceralde-hyde-3-phosphate dehydrogenase, forward: 5′-TGAAGGTCGGT-GTGAACGGATTTGGC-3′; reverse: 5′-CATGTAGGCCATGAGGTCCACCAC-3′ (amplifies a fragment of 900 bp). 3TP-Lux, E-cadherin, and Snail Promoter Analyses—For 3TP-Lux assays a reporter construct containing the 12-O-tetradecanoylphorbol-13-acetate and TGFβ response elements fused to the Luciferase reporter gene (44Lagna G. Hata A. Hemmati-Brivanlou A. Massague J. Nature. 1996; 383: 832-836Crossref PubMed Scopus (810) Google Scholar) was used. The generation of mouse E-cadherin promoter constructs containing -178/+92 sequences in its wild-type or mutant Epal fused to Luciferase has been reported previously (17Bolós V Peinado H. Pérez M oreno M.A. Fraga M.F. Esteller M. Cano A. J. Cell Sci. 2003; 116: 499-511Crossref PubMed Scopus (931) Google Scholar). Generation of full-length mouse Snail promoter construct (-900 bp) has also been described recently (17Bolós V Peinado H. Pérez M oreno M.A. Fraga M.F. Esteller M. Cano A. J. Cell Sci. 2003; 116: 499-511Crossref PubMed Scopus (931) Google Scholar). Deletion constructs of the Snail promoter mutants were obtained by PCR amplification from the full-length -900 bp promoter using appropriate primers containing BamHI and KpnI restriction sites and the corresponding PCR products cloned into the same restriction sites in the pXP1-Luciferase vector. To determine the activity of 3TP-Lux and the Snail promoter 2 × 105 cells grown in 24-well plates were transiently transfected with 200 to 500 ng of the indicated reporter constructs and 20 ng of TK-Renilla construct (Promega) as a control of transfection efficiency. Luciferase and renilla activities were measured using a dual-luciferase reporter assay kit (Promega), and after normalization the results were referred to the wild-type promoter activity detected in mock-transfected cells. Results represent the mean ± S.D. of at least two independent experiments performed in duplicate samples. For the cotransfection experiments 500 ng of the following plasmids were used: pSmad4 DN (1–514) in pCMV5 vector (provided by J. Massagué, Sloan-Kettering Memorial Cancer Center) (44Lagna G. Hata A. Hemmati-Brivanlou A. Massague J. Nature. 1996; 383: 832-836Crossref PubMed Scopus (810) Google Scholar); pLXSNHRasV12, pLXSNHRasN17, and the different mutants of HRasV12 (pLXSNHRasV12S35, pLXSNHRasV12C40, and pHras-LXSNRasV12G37) in the pLXSN vector (a gift of P. Rodriguez-Viciana, University of California Cancer Research Institute) (45Rodriguez-Viciana P. Warne P.H. Khwaja A. Marte B.M. Pappin D. Das P. Waterfield M.D. Ridley A. Downward J. Cell. 1997; 89: 457-467Abstract Full Text Full Text PDF PubMed Scopus (960) Google Scholar); β-catenin S33Y (provided by A. Ben-Ze'ev, Weizmann Institute) and Lef-1 (provided by H. Clevers, Utrecht University Hospital) cloned in pcDNA3. The corresponding empty vectors, pLXSN, pCMV5, or pcDNA3 were used in control transfections and for normalization of the total amount of DNA. Immunofluorescence and Western Blot Analyses—For immunofluorescence staining cells grown on coverslips were fixed in methanol (-20 °C, 30 s) and stained for E-cadherin, vimentin, cytokeratin-8, and fibronectin as described previously (14Cano A. Pérez-Moreno M.A. Rodrigo I. Locascio A. Blanco M.J. del Barrio M.G. Portillo F. Nieto M.A. Nat. Cell Biol. 2000; 2: 76-83Crossref PubMed Scopus (2931) Google Scholar, 17Bolós V Peinado H. Pérez M oreno M.A. Fraga M.F. Esteller M. Cano A. J. Cell Sci. 2003; 116: 499-511Crossref PubMed Scopus (931) Google Scholar, 20Pérez-Moreno M.A. Locascio A. Rodrigo I. Dhondt G. Portillo F. Nieto M.A. Cano A. J. Biol. Chem. 2001; 276: 27424-27431Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar). For F-actin stain, cells were fixed in 3.7% formaldehyde, 0.5% Triton X-100 for 30 min at room temperature, stained with tetramethyl rhodamine isothiocyanate-conjugated phalloidin (Sigma) and washed four times in phosphate-buffered saline. The cells were mounted on Mowiol, and the preparations were visualized using a Leica confocal TCS SP2 microscope. For Western blot, whole cell extracts of control and treated cells were obtained in radioimmune precipitation assay buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1% Nonidet P-40, 5% deoxycholate, 0.1% SDS) and analyzed for the indicated molecules by Western blot and enhanced chemiluminescence detection as described previously (14Cano A. Pérez-Moreno M.A. Rodrigo I. Locascio A. Blanco M.J. del Barrio M.G. Portillo F. Nieto M.A. Nat. Cell Biol. 2000; 2: 76-83Crossref PubMed Scopus (2931) Google Scholar, 17Bolós V Peinado H. Pérez M oreno M.A. Fraga M.F. Esteller M. Cano A. J. Cell Sci. 2003; 116: 499-511Crossref PubMed Scopus (931) Google Scholar, 20Pérez-Moreno M.A. Locascio A. Rodrigo I. Dhondt G. Portillo F. Nieto M.A. Cano A. J. Biol. Chem. 2001; 276: 27424-27431Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar). Primary anti-bodies included rat monoclonal anti-E-cadherin ECCD-2 (1:100) (provided by M. Takeichi, Kyoto University), mouse monoclonal antivimentin (1:200) (Dako), mouse monoclonal anti-cytokeratin 8 (1:200) (Progen), rabbit polyclonal anti-fibronectin (1:100), and mouse monoclonal anti-α-tubulin (1:1000) (Sigma). For cell signaling analysis, Western blots were carried out on cell extracts obtained by lysis in Buffer A (20 mm Hepes, pH 7.5, 10 mm EGTA, 40 mm β-glycerophosphate, 2.5 mm MgCl2, 1% Nonidet P-40, 1 mm dithiothreitol), containing the appropriate protease and phosphatase inhibitors, during 30 min. at 4 °C. Primary antisera included goat anti-AKT (1:1000) (Santa Cruz Biotechnology, Inc.), rabbit anti-phospho (Ser-473)-AKT (1:500), rabbit anti-ERK1/2 and anti-phospho (Thr-202/Tyr-204)-ERK1/2 (1:1000) (Cell Signaling Technology), and rabbit anti-Smad2/3 and rabbit anti-phospho (Ser-465/Ser-467)-Smad2/3 (1:500) (Upstate Biotechnology). Secondary antibodies were BODIPY-conjugated goat anti-rat, anti-mouse and anti-rabbit IgG (Molecular Probes), and horseradish peroxidase-conjugated sheep anti-mouse (1:1000) (Amersham Biosciences), donkey anti-goat (1:1000) (Santa Cruz Biotechnology, Inc.), goat anti-rat (1:10,000) (Pierce), and goat anti-rabbit (1:4000) (Nordic) IgG. Cell Proliferation Assays—The indicated number of cells (2.5 × 105 or 5 × 105) were seeded in triplicate samples in 92 plates and grown in complete medium for 3 h. After washing in phosphate-buffered saline, TGFβ1 (10 ng/ml) in FBS-free medium was added, and the cells were grown for an additional 24 h. [3H]Thymidine was added during the last 5 h of treatment. The cells were collected using a cell harvester device, and [3H]thymidine incorporation was determined in a scintillation counter. The values, representing the mean ± S.D., were normalized to those obtained in control untreated cells. Migration Assays—The migratory/motility behavior of MDCK cells was analyzed in in vitro wound healing assays as described previously (14Cano A. Pérez-Moreno M.A. Rodrigo I. Locascio A. Blanco M.J. del Barrio M.G. Portillo F. Nieto M.A. Nat. Cell Biol. 2000; 2: 76-83Crossref PubMed Scopus (2931) Google Scholar, 17Bolós V Peinado H. Pérez M oreno M.A. Fraga M.F. Esteller M. Cano A. J. Cell Sci. 2003; 116: 499-511Crossref PubMed Scopus (931) Google Scholar). Monolayers of confluent cultures were lightly scratched with a Gilson pipette tip and, after washing to remove detached cells, treated with TGFβ1 (10 ng/ml) and/or PD98059 (10 μm), as indicated. Cultures were observed at timely intervals for up to 36 h post-incision. TGFβ1 Induces Cell Scattering and Increased Motility in MDCK Cells Dependent on MEK1/2 Activity—Some previous studies have related the TGFβ/bone morphogenetic signaling pathway to the regulation of EMT both during embryonic development (24Liem Jr., K.F. Tremml G. Roelink H. Jessell T.M. Cell. 1995; 82: 969-979Abstract Full Text PDF PubMed Scopus (916) Google Scholar, 25Romano L.A. Runyan R.B. Dev. Biol. 2000; 223: 91-102Crossref PubMed Scopus (150) Google Scholar) and in some epithelial cell lines (30Caulín C. Scholl F.G. Frontelo P. Gamallo C. Quintanilla M. Cell Growth Differ. 1995; 6: 1027-1035PubMed Google Scholar, 32Miettinen P.J. Ebner R. Lopez A.R. Derynck R. J. Cell Biol. 1994; 127: 2021-2036Crossref PubMed Scopus (796) Google Scholar, 33Oft M. Peli J. Rudaz C. Schwarz H. Beug H. Reichmann E. Genes Dev. 1996; 10: 2462-2477Crossref PubMed Scopus (565) Google Scholar, 37Spagnoli F.M. Cicchini C. Tripodi M. Weiss M.C. J. Cell Sci. 2000; 113: 3639-3647PubMed Google Scholar), whereas others have potentially implicated a similar function for FGFs (46Migdal M. Soker S. Yarden Y. Neufeld G. J. Cell. Physiol. 1995; 162: 266-276Crossref PubMed Scopus (10) Google Scholar, 47Strutz F. Zeisberg M. Ziyadeh F.N. Yang C.Q. Kalluri R. Muller G.A. Neilson E.G. Kidney Int. 2002; 61: 1714-1728Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar, 48Valles A.M. Boyer B. Badet J. Tucker G.C. Barritault D. Thiery J.P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1124-1128Crossref PubMed Scopus (158) Google Scholar). To get further insights into the regulation of EMT by both kinds of growth factors, we choose the prototypic epithelial MDCK cell line. Twenty-four h of treatment with TGFβ1 (10 ng/ml) (Fig. 1A, b) or a combination of TGFβ1 (10 ng/ml) and FGF2 (100 ng/ml) (Fig. 1A, c) induced a dramatic change of the cellular phenotype; MDCK cells became dissociated with reduced cell-cell contacts and acquired a more spindle phenotype. Lower concentrations of TGFβ1 (1–5 ng) induced a milder effect, and treatment with FGF2 alone did not affect the phenotype of MDCK cells (data not shown). The phenotypic changes induced by TGFβ1 were also associated to increased cell motility, as ascertained by in vitro wound healing assays (Fig. 1B). Eight h after incision of the wound, MDCK cells growing in the presence of TGFβ1 started to colonize the wound surface, whereas control untreated cells hardly started to migrate (data not shown). The differences in cell motility were evident 24 h after incision when TGFβ1-treated cells colonized about 70–80% of the wound surface in a random fashion (Fig. 1B, g), in contrast to untreated cells that had only colonized 20–30% of the wound surface by unidirectional migration (Fig. 1B, e). The increased motility induced by TGFβ1 treatment is not because of increased cell proliferation. Analysis of [3H]thymidine incorporation showed that MDCK cells treated with TGFβ1 exhibited an 80% reduction of their proliferation potential, as compared with control untreated cells (Fig. 2A). After 3–4 days of TGFβ1 treatment MDCK cells started to show signs of apoptosis, and most cells died after 7 days of treatment (data not shown). The sensitivity of MDCK cells to TGFβ1 was also evidenced by the quick induction of the responsive 3TP-Lux promoter in the presence of the growth factor (Fig. 2B).Fig. 2TGFβ1 treatment induces proliferation arrest and transcriptional responses in MDCK cells.A, [3H]thymidine incorporation