The PDZ Domains of Zonula Occludens-1 Induce an Epithelial to Mesenchymal Transition of Madin-Darby Canine Kidney I Cells
2000; Elsevier BV; Volume: 275; Issue: 13 Linguagem: Inglês
10.1074/jbc.275.13.9492
ISSN1083-351X
AutoresManuela Reichert, Thomas Müller, Walter Hunziker,
Tópico(s)Hippo pathway signaling and YAP/TAZ
ResumoThe integrity of cell-cell contacts such as adherens junctions (AJ) and tight junctions (TJ) is essential for the function of epithelia. During carcinogenesis, the increased motility and invasiveness of tumor cells reflect the loss of characteristic epithelial features, including cell adhesion. While β-catenin, a component of AJ, plays a well characterized dual role in cell adhesion and signal transduction leading to epithelial cell transformation, little is known about possible roles of tight junction components in signaling processes. Here we show that mutants of the TJ protein zonula occludens protein-1 (ZO-1), which encode the PDZ domains (ZO-1 PDZ) but no longer localize at the plasma membrane, induce a dramatic epithelial to mesenchymal transition (EMT) of Madin-Darby canine kidney I (MDCKI) cells. The observed EMT of these MDCK-PDZ cells is characterized by a repression of epithelial marker genes, a restricted differentiation potential and a significantly induced tumorigenicity. Intriguingly, the β-catenin signaling pathway is activated in the cells expressing the ZO-1 PDZ protein. Ectopic expression of the adenomatous polyposis coli tumor suppressor gene, known to down-regulate activated β-catenin signaling, reverts the transformed fibroblastoid phenotype of MDCK-PDZ cells. Thus, cytoplasmic localization of the ZO-1 PDZ domains induces an EMT in MDCKI cells, most likely by modulating β-catenin signaling. The integrity of cell-cell contacts such as adherens junctions (AJ) and tight junctions (TJ) is essential for the function of epithelia. During carcinogenesis, the increased motility and invasiveness of tumor cells reflect the loss of characteristic epithelial features, including cell adhesion. While β-catenin, a component of AJ, plays a well characterized dual role in cell adhesion and signal transduction leading to epithelial cell transformation, little is known about possible roles of tight junction components in signaling processes. Here we show that mutants of the TJ protein zonula occludens protein-1 (ZO-1), which encode the PDZ domains (ZO-1 PDZ) but no longer localize at the plasma membrane, induce a dramatic epithelial to mesenchymal transition (EMT) of Madin-Darby canine kidney I (MDCKI) cells. The observed EMT of these MDCK-PDZ cells is characterized by a repression of epithelial marker genes, a restricted differentiation potential and a significantly induced tumorigenicity. Intriguingly, the β-catenin signaling pathway is activated in the cells expressing the ZO-1 PDZ protein. Ectopic expression of the adenomatous polyposis coli tumor suppressor gene, known to down-regulate activated β-catenin signaling, reverts the transformed fibroblastoid phenotype of MDCK-PDZ cells. Thus, cytoplasmic localization of the ZO-1 PDZ domains induces an EMT in MDCKI cells, most likely by modulating β-catenin signaling. adherens junction adenomatous polyposis coli epithelial-mesenchymal transition guanylate kinase hepatocyte growth factor Madin-Darby canine kidney cells MDCK type I cells MDCKI cells expressing ZO-1 MDCKI cells expressing the PDZ domains of ZO-1 MDCKI cells expressing a ZO-1 construct encoding the PDZ and SH3 domains MDCKI cells expressing a ZO-1 construct encoding the PDZ, SH3, and GUK domains PSD-95/disk large tumor suppressor proteinDlg/ZO-1 Src homology domain T cell factor/lymphocyte enhancer factor transepithelial electrical resistance tight junction zonula occludens polymerase chain reaction reverse transcription Dulbecco's modified Eagle's medium polyacrylamide gel electrophoresis base pair(s) 2-[bis(2-hydroxyethyl)amino]ethanesulfonic acid phosphate-buffered saline glutathione S-transferase membrane-associated guanylate kinase multimerized wild-type Lef binding sites multimerized mutant Lef binding sites An essential role of epithelial monolayers is the formation of cellular barriers that allow the generation and maintenance of compartments important for the physiological function of organs. In epithelial monolayers, individual cells are joined to each other by specialized structures including gap junctions, desmosomes, adherens junctions (AJ)1 and tight junctions (TJ). In addition to mediating cell-cell adhesion, TJ regulate the paracellular diffusion across epithelial monolayers and the maintenance of the asymmetric distribution of proteins and lipids to the apical and basolateral plasma membrane domains of epithelial cells (1.Stevenson B.R. Keon B.H. Annu. Rev. Cell Dev. Biol. 1998; 14: 89-109Crossref PubMed Scopus (219) Google Scholar, 2.Mitic L.L. Anderson J.M. Annu. Rev. Physiol. 1998; 60: 121-142Crossref PubMed Scopus (628) Google Scholar, 3.Cereijido M. Valdes J. Shoshani L. Contreras R.G. Annu. Rev. Physiol. 1998; 60: 161-177Crossref PubMed Scopus (221) Google Scholar). The tight junction protein zonula occludens protein 1 (ZO-1) is part of a multi-protein complex and binds directly to the integral TJ proteins occludin and to members of the claudin family (4.Itoh M. Furuse M. Morita K. Kubota K. Saitou M. Tsukita S. J. Cell Biol. 1999; 147: 1351-1363Crossref PubMed Scopus (915) Google Scholar), thereby linking the TJ to the cytoskeleton via a direct or indirect interaction with actin (5.Fanning A.S. Jameson B.J. Jesaitis L.A. Anderson J.M. J. Biol. Chem. 1998; 273: 29745-29753Abstract Full Text Full Text PDF PubMed Scopus (1114) Google Scholar). ZO-1 belongs to the membrane-associated guanylate kinase (MAGUK) protein family and contains three PDZ domains (6.Fanning A.S. Anderson J.M. Curr. Biol. 1996; 6: 1385-1388Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar), an Src homology 3 (SH3) domain, a guanylate kinase (GUK) homology domain, and a proline rich C-terminal region (see Fig. 1). Since occludin lacks PDZ-binding motifs, binding between occludin and ZO-1 probably does not involve the PDZ domains. The function of the GUK domain, which lacks kinase activity in the MAGUK proteins analyzed so far, is not known but has been suggested to be important for binding of ZO-1 to occludin (5.Fanning A.S. Jameson B.J. Jesaitis L.A. Anderson J.M. J. Biol. Chem. 1998; 273: 29745-29753Abstract Full Text Full Text PDF PubMed Scopus (1114) Google Scholar). ZO-2 and ZO-3, two additional members of the MAGUK protein family present in TJ, show extensive homology to each other and to ZO-1. ZO-3 interacts with ZO-1 and the cytoplasmic C-terminal tail of occludin, but does not bind ZO-2 (7.Haskins J. Gu L. Wittchen E.S. Hibbard J. Stevenson B.R. J. Cell Biol. 1998; 141: 199-208Crossref PubMed Scopus (495) Google Scholar). ZO-2 binds directly to ZO-1 and occludin. Actin cosedimentation studies showed that ZO-2, ZO-3, and occludin all interact directly with F-actinin vitro and colocalize with actin aggregates at cell boarders in cytocholasin D-treated MDCK cells. The suggested model at the moment is that two independent complexes comprising ZO-1-ZO-2 and ZO-1-ZO-3 exist (rather than a three-member complex, ZO-1-ZO-2-ZO-3), and that these complexes link the tight junction to the actin cytoskeleton (8.Wittchen E., S. Haskins J. Stevenson B., R. J. Biol. Chem. 1999; 274: 35179-35185Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar). Several other proteins have been described to interact with ZO-1, but the domains involved in binding or the physiological relevance of the interactions are, in most cases, unknown. Carcinogenesis is a multistep process well characterized in human colon cancer. Mutations in the adenomatous polyposis coli (APC) gene are thought to initiate the process, leading to aberrant crypt foci that develop into areas of benign epithelial hyperplasia or dysplasia and adenomas. Progression of these areas to carcinomas in situand malignant tumors depends on further changes in the transformed cells such as mutations in p21 Ras and p53 and the gradual loss of a number of characteristic features of differentiated epithelial cells. This process, also known as epithelial to mesenchymal transition (EMT), includes the disruption of apical-basolateral polarity, the disassembly of AJ and TJ, and the ability of the cells to degrade the basement membrane, to migrate, and to form metastases at distant sites. A reduced intercellular adhesion is a requisite for the higher motility and invasiveness of tumor cells and the expression or integrity of several components of AJ (i.e. α-catenin, β-catenin, γ-catenin/plakoglobin, E-cadherin) is altered or lost in different types of carcinoma (9.Morin P.J. Sparks A.B. Korinek V. Barker N. Clevers H. Vogelstein B. Kinzler K.W. Science. 1997; 275: 1787-1790Crossref PubMed Scopus (3517) Google Scholar). β-Catenin plays a dual role as a structural component of AJ (10.Yap A.S. Brieher W.M. Gumbiner B.M. Annu. Rev. Cell Dev. Biol. 1997; 13: 119-146Crossref PubMed Scopus (688) Google Scholar) and as a signaling molecule in the Wnt signaling pathway (11.Wodarz A. Nusse R. Annu. Rev. Cell Dev. Biol. 1998; 14: 59-88Crossref PubMed Scopus (1743) Google Scholar). In the absence of Wnt glycoproteins, the Ser/Thr-specific glycogen synthase kinase 3β phosphorylates β-catenin, APC, and axin/conductin (12.Behrens J. Jerchow B., A. Wurtele M. Grimm J. Asbrand C. Wirtz R. Kuhl M. Wedlich D. Birchmeier W. Science. 1998; 280: 596-599Crossref PubMed Scopus (1118) Google Scholar, 13.Hart M.J. de los Santos R. Albert I.N. Rubinfeld B. Polakis P. Curr. Biol. 1998; 8: 573-581Abstract Full Text Full Text PDF PubMed Google Scholar), which are present as a multi-protein complex in the cytosol. Phosphorylated β-catenin is rapidly ubiquitinated and degraded by the proteosomal pathway (14.Maniatis T. Genes Dev. 1999; 13: 505-510Crossref PubMed Scopus (369) Google Scholar). Binding of Wnt glycoproteins to the Frizzled family of receptors results in the inactivation of glycogen synthase kinase 3β and thereby to an enhanced stability of β-catenin. Stabilized β-catenin can translocate into the nucleus where, in association with members of the Tcf/Lef transcription factor family, it regulates gene expression (15.Eastman Q. Grosschedl R. Curr. Opin. Cell Biol. 1999; 11: 233-240Crossref PubMed Scopus (474) Google Scholar, 16.Aoki M. Hecht A. Kruse U. Kemler R. Vogt P.K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 139-144Crossref PubMed Scopus (155) Google Scholar), probably by recruiting the basal transcription machinery to promoter regions of Wnt target genes such as cyclin D1 (17.Shtutman M. Zhurinsky J. Simcha I. Albanese C. D'Amico M. Pestell R. Ben-Ze'ev A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5522-5527Crossref PubMed Scopus (1926) Google Scholar, 18.Tetsu O. McCormick F. Nature. 1999; 398: 422-426Crossref PubMed Scopus (3265) Google Scholar). Oncogenic transformation of mammalian cells is closely linked to the signaling function of β-catenin (19.Smits R. Kielman M.F. Breukel C. Zurcher C. Neufeld K. Jagmohan-Changur S. Hofland N. van Dijk J. White R. Edelmann W. Kucherlapati R. Khan P., M. Fodde R. Genes Dev. 1999; 13: 1309-1321Crossref PubMed Scopus (192) Google Scholar). Intestinal cells carrying mutations in APC that activate the β-catenin/Tcf/Lef signaling pathway develop into adenomas and adenocarcinomas (20.Korinek V. Barker N. Morin P.J. van Wichen D. de Weger R. Kinzler K.W. Vogelstein B. Clevers H. Science. 1997; 275: 1784-1787Crossref PubMed Scopus (2950) Google Scholar). In addition, human colorectal neoplasms expressing a wild-type APC often show mutations in β-catenin that activate its signaling capacity (9.Morin P.J. Sparks A.B. Korinek V. Barker N. Clevers H. Vogelstein B. Kinzler K.W. Science. 1997; 275: 1787-1790Crossref PubMed Scopus (3517) Google Scholar). Furthermore, mice expressing a dominant allele of the β-catenin gene develop adenomatous intestinal polyps and nascent microadenomas, providing further evidence that activated β-catenin signaling contributes to cancer development (21.Harada N. Tamai Y. Ishikawa T. Sauer B. Takaku K. Oshima M. Taketo M.M. EMBO J. 1999; 18: 5931-5942Crossref PubMed Scopus (981) Google Scholar). A few observations suggested that TJ components, in addition to their structural role, may also be involved in signaling events. ZO-1 is related to the Drosophila discs-large tumor suppressor (Dlg-A), a component of septate junctions inDrosophila implicated in signaling during mitosis. Dlg proteins with mutations in the PDZ and SH3 domains cause neoplastic overgrowth of larval imaginal disc epithelial cells (22.Woods D.F. Hough C. Peel D. Callaini G. Bryant P.J. J. Cell Biol. 1996; 134: 1469-1482Crossref PubMed Scopus (358) Google Scholar). TheDrosophila orthologue of ZO-1, tamou, has been implicated in regulating the expression of extramacrochaetae (23.Takahisa M. Togashi S. Suzuki T. Kobayashi M. Murayama A. Kondo K. Miyake T. Ueda R. Genes Dev. 1996; 10: 1783-1795Crossref PubMed Scopus (61) Google Scholar), the fly orthologue of the inhibitor of differentiation protein. ZO-1 itself has been found in the nucleus of migrating epithelial cells at the edge of wounded monolayers or in epithelial cells induced to migrate by HGF (24.Gottardi C. Arpin M. Fanning S. Louvard D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10779-10784Crossref PubMed Scopus (305) Google Scholar). To explore possible additional functions of ZO-1 besides its role as a structural component of TJ, we expressed progressive C-terminal deletion mutants of the protein in epithelial MDCKI cells. Surprisingly, mutants encoding only the N terminus including the PDZ domains no longer localized at the plasma membrane and induced a dramatic loss of the epithelial phenotype of MDCKI cells. This EMT included changes in the differentiation potential and tumorigenicity of the cells, together with a repression of epithelial (e.g.E-cadherin) and an induction of mesenchymal (e.g.fibronectin) marker genes. Interestingly, β-catenin/Tcf/Lef signaling was activated in MCDK-PDZ cells, indicating an involvement of β-catenin/Tcf/Lef signaling in the induction of the observed EMT. Thus, our results show that the cytosolic localization of the PDZ domains of ZO-1 leads to the transformation of MDCKI cells, most likely through a direct or indirect modulation of the β-catenin/Tcf/Lef signaling pathway. Human ZO-1 cDNA (GenBank™accession no. L14837) was kindly provided by J. Anderson. The following deletion mutants (see Fig. 1) were created by the PCR technique usingPwo polymerase (Roche): ZO-1-PDZ (amino acids 1–568), ZO-1-PS (amino acids 1–759), ZO-1-PSG (amino acids 1–798), and wild-type ZO-1. A FLAG epitope tag (5′-gattacaaagacgatgacgataaa-3′) was introduced into each 3′ oligonucleotide to generate a ZO-1 fusion protein with the FLAG tag at the C terminus. The different PCR products were cloned into the pCRblunt vector (Invitrogen), cut out withEcoRV, and cloned into the eukaryotic retroviral expression vector pLNCX (CLONTECH), cut with HpaI. Full-length human APC cDNA (GenBank™ accession no.M74088) was kindly provided by B. Vogelstein, and a myc tag (5′-gaacaaaaactcatctcagaagaggatctgaat-3′) was introduced at the 3′ end by PCR. The neomycin resistance gene in the retroviral pLNCX vector was replaced by a hygromycin-thymidine kinase fusion cDNA (kindly provided by C. Karreman) to yield the pLHygTkCX vector, into which the myc-tagged APC cDNA was cloned. The plasmid coding for the GST-E-cadherin cytoplasmic fusion protein was kindly provided by A. Ullrich and described elsewhere in detail (25.Müller T. Choidas A. Reichmann E. Ullrich A. J. Biol. Chem. 1999; 274: 10173-10183Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar). MDCKI cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS) (Life Technologies, Inc.), 100 units/ml penicillin and streptomycin, and 2 mm glutamine. MDCKI cells were plated at a density of 1 × 104 cells/cm2 on 10-cm plates (Nunc) the day before transfection using the calcium phosphate technique as described (26.Chen C. Okayama H. Mol. Cell. Biol. 1987; 7: 2745-2752Crossref PubMed Scopus (4824) Google Scholar). Briefly, 16 μg of plasmid DNA (Nucleobond purified) were mixed with 40 μl of 2.5 m CaCl2 to a final volume of 400 μl. An equal volume of 2× BBS (50 mmBES (Sigma), 280 mm NaCl, 1.5 mmNa2HPO4, pH 6.5) was added, and after 10 min at room temperature the mixture was added to the cells. The cells were incubated overnight at 3%CO2, washed twice with PBS, and incubated at 5% CO2. For stable transfections, 750 μg/ml G418 or 350 μg/ml hygromycin, respectively (Life Technologies, Inc.), were added 24 h after transfection. Single cell clones were established using either limited dilution or cloning rings. Transepithelial electrical resistance (TER) was measured 3 days after 5 × 104 cells/cm2 had been plated on Transwell filters (Costar). TER was determined by applying an AC square wave current of 620 mA at 12.5 Hz across a cell monolayer plated on a 6.5-mm diameter Transwell™ filter. The voltage deflection was measured with a pair of Ag/AgCl voltage sensors (EVOM, World Precision Instruments). TER values were calculated by subtracting the blank values from the filter and the medium, and were normalized to the area of the monolayer (filter). 5 × 104cells/cm2 were grown on coverslips to confluence, washed twice with PBS, and fixed with 3% freshly prepared paraformaldehyde for 25 min at room temperature. The cells were washed and permeabilized with 0.5% Triton X-100 10 min at room temperature. Unspecific binding was blocked with 10% goat serum in PBS for 1 h at room temperature, and primary antibodies (M2 monoclonal α-FLAG antibody; Eastman Kodak Co.) or fluorescein isothiocyanate-labeled phalloidin (Sigma; diluted 1:100 in 10% goat serum) were added for 2 h at room temperature. After washing the cells several times with PBS, bound primary antibodies were detected with fluorochrome-coupled isotype-specific secondary antibodies (Alexa). Coverslips were mounted (16.7% Mowiol, 33% glycerol in 120 mm Tris-HCl, pH 8.5) and viewed with a conventional fluorescence microscope (Leica). Cells were trypsinized, and 2 × 104 cells were added to 800 μg of collagen type I (Promocell) in 10× DMEM to yield a final volume of 2 ml in 1× DMEM, 10% FCS and plated in 24-well plates (Costar). The plates were incubated 10 min at room temperature and 45 min at 37 °C before 1 ml of DMEM including 10% FCS was added. Where indicated, HGF (40 ng/ml, kindly provided by W. Birchmeier) was added to the medium and incubation was continued until a ductlike morphology became visible. Cells were fixed with 3% freshly prepared paraformaldehyde, permeabilized with 0.5% Triton X-100, and stained with 0.2% Carmin-Hemalaun in H2O overnight. Cells were resuspended at a cell density of 2 × 106 cells in 50 μl of DMEM and injected subcutaneously into the flank region of Swiss nude mice (IGR Villejuif, Paris, France) using five animals per cell line. Tumor formation was monitored by measuring the width (W) and length (L) of the tumors with W <L. The tumor volume was calculated according to the formula (W 2 × L × π/6). Cells were plated at a density of 1 × 104 cells/cm2 in six-well plates (Costar) the day before transfection. Transfection was performed using the calcium-phosphate technique as described above with 4 μg of luciferase reporter constructs containing either multimerized wild-type (TOP-FLASH) or mutant (FOP-FLASH) Tcf/Lef binding sites (kindly provided by H. Clevers). As a control for transfection efficiency, 1 μg of β-galactosidase construct under the control of the simian virus 40 promoter was included in each transfection. Cells were washed 24 h after transfection, and extracts were prepared in 400 μl of reporter lysis buffer (Promega). Luciferase and β-galactosidase activity were assayed according to the manufacturer's protocol using the luciferase assay kit from Promega. The relative luciferase units corresponding to the enzymatic luciferase activities obtained for the TOP or FOP reporter gene transcription, respectively, were normalized to the relative β-galactosidase activity. To allow easier comparison of the transcriptional activities, the background transcriptional activity represented by the FOP values was subtracted from the TOP values. Each transfection was done in triplicate, and the luciferase and β-galactosidase activities of each sample were measured in triplicate. The assay was performed in three independent experiments. To analyze the expression of proteins or to determine the free pool of β-catenin, cells were washed with ice-cold PBS and scraped from the plate in ice-cold lysis buffer (20 mm imidazole-HCl, pH 6.8, 100 mm KCl, 2 mm MgCl2 20 mm EDTA, 300 mm sucrose, 0.1 mm sodium orthovanadate, 1 mm NaF, 0.2% Triton X-100, and freshly added protease inhibitors (25 μg/ml aprotinin, 25 μg/ml leupeptin, 1 mm phenylmethylsulfonyl fluoride; Sigma). The lysates were spun at 15,000 rpm in a table-top centrifuge and the supernatant (Triton X-100-soluble fraction) was snap-frozen in liquid nitrogen and stored at −80 °C. The pellet (Triton X-100-insoluble fraction) was re-extracted in RIPA buffer (10 mm Tris-HCl, pH 7.4, 150 mm NaCl, 0.1% SDS, 1% Triton X-100, 10% glycerol) for 30 min at 4 °C. The extract was spun at 15,000 rpm, and the supernatant was also snap-frozen in liquid nitrogen. Protein concentration was determined according to Bradford. To analyze the distribution of the different ZO-1 proteins to membrane and cytosol fractions, cells were washed twice with ice-cold PBS and scraped into homogenization buffer (25 mm Tris-HCl, pH 7.4, 2 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 10 mm β-mercaptoethanol, 10% glycerol, 1× protease inhibitor mixture (Roche Molecular Biochemicals)). After incubating for 10 min on ice, the cells were homogenized in a Dounce homogenizer and insoluble material was pelleted in a low speed centrifugation (5 min, 500 × g). The supernatant was centrifuged at 100,000 × g for 90 min at 4 °C to obtain a membrane pellet and a cytosol fraction. The membrane pellet was extracted with homogenization buffer containing 1% Triton X-100 for 30 min at 4 °C, and insoluble material was pelleted for 30 min at 4 °C and 100,000 × g. The cytosol and the Triton X-100-soluble membrane fraction were analyzed by Western blot to detect the different ZO-1 proteins. Equal amounts of proteins were separated by SDS-PAGE and transferred onto nitrocellulose membrane (BDH Laboratories) using a semidry blotting apparatus (Bio-Rad). Unspecific antibody binding was blocked with 10% nonfat milk powder in PBS. The membrane was probed with primary antibodies (anti-Flag M2 (Kodak), anti- α, β -catenin, anti-plakoglobin (Transduction Laboratories), anti-E-cadherin (kindly provided by Axel Ullrich), anti-ZO-1 (Zymed Laboratories Inc.), or anti-pan cytokeratins (Sigma)) overnight at 4 °C. The membranes were washed several times with PBS, 0.5% Tween20 (Sigma), and immunoreactive bands were visualized with the enhanced chemiluminescence detection system (Pierce) using horseradish peroxidase-coupled secondary antibodies (Jackson). Equal amounts of cell lysates were precleared with glutathione-Sepharose for 30 min at 4 °C and incubated with 5 μg of purified GST-E-cadherin cytoplasmic fusion protein or a 3-fold molar excess of GST immobilized on glutathione-Sepharose (Amersham Pharmacia Biotech). The resulting complexes were washed three times with 20 mm HEPES, pH 7.5, 150 mm NaCl, 10 mm pyrophosphate, 10 mm NaF, 0.2 mm ammonium molybdate, 10% glycerol, 0.1% Triton X-100, 2 mm sodium orthovanadate; bound complexes were separated by SDS-PAGE and transferred onto nitrocellulose membranes, and β-catenin was visualized by immunodetection. Total RNA was prepared as described previously (27.Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63232) Google Scholar). Briefly, the cells were washed with PBS and lysed in 4 m guanidine thiocyanate (Fulka), 100 mm Tris-HCl, pH 7.5, and 1% β- mercaptoethanol (Sigma). After the solution had been passed several times through a sterile needle, an equal volume of water-saturated phenol:chloroform (1:1) was added and vortexed for at least 10 s. After centrifugation, the total cellular RNA of the aqueous phase was precipitated with an equal volume of isopropanol. The RNA was recovered by centrifugation, the pellet was resuspended in sterile water, and the RNA was separated on a denaturing formaldehyde-agarose gel to visualize the integrity of the RNA. For semiquantitative analysis, 0.5 μg of total RNA was used for RT-PCR using the TITAN One-Tube RT-PCR kit (Roche Molecular Biochemicals) according to the manufacturer's protocol. The reverse transcriptase reaction was performed for 45 min at 50 °C or 40 °C. PCR cycles used to amplify the cDNA were as follows: denaturing step: 45 s 94 °C, annealing step: 45 s at 40–50 °C, and elongation step: 68 °C, 1 min per 1000 bp to be amplified; 25 cycles were used. RT-PCR products were analyzed on 0.5–2% agarose gels. The following primer pairs were used: gapdh, 5′-gggctccttctgctgat-3′ and 5′-gggctgggtggcagtgat-3′; ZO-1, 5′-ggttcttcgagaagctgga-3′ and 5′-ggtttgtctggcctctgccc-3′; E-cadherin, 5′-ccgaattctcccgatgaaattggaaat-3′ and 5′-cgcggatccctagtcgtcctcgccgcc-3′; occludin, 5′-ggggaattcacttcaggcagcctcg-3′ and 5′-gggaagcttttctatgttttctgtcta-3′; fibronectin, 5′-ggtatgaagtgagtgtctatgctct-3′ and 5′-ggttctgccactgttctcc-3′; vimentin, 5′-ggggtgtcctcgtcctcctac-3′ and 5′-ggggaggtcaggcttggaaac-3′. ZO-1 wild-type and deletion mutants encoding the PDZ domains (PDZ), the PDZ and the SH3 domains (PS) or the PDZ, SH3 and GUK domains (PSG) were constructed, each carrying a C-terminal FLAG epitope tag (Fig.1). The different cDNAs were subcloned into the pLNCX expression vector and transfected into MDCKI epithelial cells. These cells possess characteristics similar to the principal cells of the collecting systems in the kidney and polarize under appropriate conditions. Compared with the MDCKII cell line, MDCKI cells show a significantly increased TER (28.Nakazato Y. Suzuki H. Saruta T. Biochim. Biophys. Acta. 1989; 1014: 57-65Crossref PubMed Scopus (23) Google Scholar,29.Hansson G.C. Simons K. van Meer G. EMBO J. 1986; 5: 483-489Crossref PubMed Scopus (94) Google Scholar), 2M. Reichert and W. Hunziker, unpublished observations. indicating the formation of well established TJ. After transfection, G418-resistant cells were selected, pooled, and used for further analysis. Immunofluorescence experiments using M2 anti-Flag antibodies showed that the cells in a given pooled population of G418-resistant cells homogeneously expressed the different ZO-1 proteins (data not shown, see below). To characterize the subcellular distribution of the different ZO-1 proteins, cells were grown to confluence and the localization of the tagged proteins was detected by indirect immunofluorescence (Fig. 2 a). In MDCK-ZO-1 cells, the transfected FLAG-tagged wild-type ZO-1 localized at the plasma membrane to regions of cell-cell contact (Fig2 a, panel A), probably the TJ since these cells displayed an increased TER (see below). Similarly, in MDCKI cells transfected with the PSG construct, the expressed protein was present at regions of cell-cell contact (panel C). In contrast, however, deletion mutants lacking the GUK domain no longer localized at the plasma membrane in transfected MDCK-PS cells (data not shown) or MDCK-PDZ cells (panel B). The apparent cytosolic localization of the ZO-1 PDZ construct observed by immunofluorescence (Fig. 2 a, panel B) was confirmed biochemically. MDCK-PDZ and MDCK-PSG cells were homogenized in the absence of detergent; cytosol and membrane fractions were prepared as described under “Materials and Methods” and analyzed by Western blot. As shown in Fig. 2 b, the transfected ZO-1 PSG protein was almost exclusively detected in the membrane fraction. In contrast, the ZO-1 PDZ protein was mostly recovered in the cytosolic fraction. This experiment thus confirms the immunofluorescence data and shows that the ZO-1 PDZ protein no longer localizes at the plasma membrane. These results are consistent with previous data (5.Fanning A.S. Jameson B.J. Jesaitis L.A. Anderson J.M. J. Biol. Chem. 1998; 273: 29745-29753Abstract Full Text Full Text PDF PubMed Scopus (1114) Google Scholar) indicating that the GUK domain may be critical for the localization of ZO-1 at the plasma membrane, presumably by binding to occludin. Neither MDCKI cells expressing the wild-type ZO-1 nor cells transfected with the empty pLNCX vector (MDCK-pLNCX) showed apparent changes in morphology when compared with the parental cell line, and the morphology of MDCK-PSG cells was also not altered. Surprisingly, however, the expression of proteins encoding the PDZ domains but lacking the GUK domain led to a dramatic change in the morphology of MDCK-PDZ and MDCK-PS cells (panel B and data not shown). The cells lost their epithelial phenotype and instead displayed a fibroblast-like morphology with long lamellipodia. Alterations in the morphology of MDCK-PDZ cells correlated with changes in the organization of the cytoskeleton. The typical cortical actin ring of polarized epithelial cells was observed in control MDCK-pLNCX cells (Fig. 2 a, panel F). In contrast, MDCK-PDZ cells displayed actin stress fibers normally absent from polarized epithelial cells but characteristic for fibroblastoid or migrating epithelial cells (panel E). Thus, MDCKI cells expressing the PDZ domains of ZO-1 in the cytosol (i.e. ZO-1 PDZ or ZO-PS) underwent profound changes in morphology and cytoskeletal organization. This loss of epithelial polarity and gain of mesenchymal properties was characterized in more detail using MDCK-PDZ cells. To determine whether MDCK-PDZ cells were still able to form monolayers with functional tight junctions, cells were grown on permeable filter supports. As shown in Fig.3 a, control MDCK-pLNCX cells formed tight monolayers as evidenced by the establishment of a TER characteristic for MDCKI cells. MD
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