Introduction Epithelial cell-cell junctions provide tissue integrity and promote cellular polarity (Perez-Moreno et al., 2003). The junctional complex is composed of tight junctions, adherens junctions and desmosomes (Tsukita et al., 2001 Wheelock and Johnson, 2003b). The adherens junction plays a pivotal role in regulating the activity of the entire junctional complex, and the major adhesion molecules in the adherens junctions are the cadherins (Nagafuchi, 2001 Wheelock and Johnson, 2003a Wheelock and Johnson, 2003b Wheelock et al., 2001). In some situations, including the dynamic cellular rearrangements that are integral to embryonic development, tissue integrity must be disrupted so that cells can migrate from their original position to establish new structures (Pla et al., 2001 Savagner, 2001 Shook and Keller, 2003). For example, during Drosophila gastrulation, prospective mesodermal cells (which originally express E-cadherin and form adherens junctions) lose their expression of E-cadherin, become disperse and begin to migrate (Oda et al., 1998). Likewise, when epithelial cells change their relative position within a tissue, they convert to motile fibroblastic cells. This phenomenon is referred to as the epithelium-to-mesenchyme transition (EMT) (Affolter et al., 2003 Hay, 1995). During the EMT, cell-cell junctions are disrupted, the actin cytoskeleton is extensively reorganized and the cells acquire increased migratory characteristics (Boyer et al., 2000 Savagner, 2001). It has been proposed that, when cancer cells invade adjacent tissues, they use a mechanism much like the EMT that is involved in normal developmental processes (Gotzmann et al., 2004 Thiery, 2002 Thiery, 2003). Transforming growth factor �� (TGF-��) suppresses the growth of many normal epithelial cells. In some cancers, TGF-�� signaling is genetically disrupted, which might contribute to unregulated cell growth (Brattain et al., 1996 de Caestecker et al., 2000 Massague et al., 2000 Waite and Eng, 2003). Interestingly, TGF-�� also has tumor-promoting activity, owing to its ability to induce EMT in some cell types (Akhurst and Derynck, 2001 Cui et al., 1996 Derynck et al., 2001 Oft et al., 2002 Roberts and Wakefield, 2003 Siegel and Massague, 2003 Wakefield and Roberts, 2002). During TGF-��-mediated EMT, E-cadherin expression is decreased while N-cadherin expression is increased (Bhowmick et al., 2001 Grande et al., 2002 Miettinen et al., 1994 Piek et al., 1999). The expression of E-cadherin is regulated by several transcription factors, including snail, slug, E12/E47, SIP1 and delta EF1/ZEB-1, each of which has been reported to repress its transcription (Batlle et al., 2000 Bolos et al., 2003 Cano et al., 2000 Comijn et al., 2001 Conacci-Sorrell et al., 2003 Grooteclaes and Frisch, 2000 Hajra et al., 2002 Perez- Moreno et al., 2001 van Grunsven et al., 2003). Snail expression is induced by TGF-�� in rat hepatocytes (Gotzmann et al., 2002 Spagnoli et al., 2000 Valdes et al., 2002) and in Madin-Darby canine kidney (MDCK) cells (Peinado et al., 2003). However, SIP1 but not snail has been reported to be induced when mouse mammary epithelial 873 Epithelium-to-mesenchyme transitions (EMTs) are characterized by morphological and behavioral changes in cells. During an EMT, E-cadherin is downregulated while N-cadherin is upregulated. The goal of this study was to understand the role cadherin switching plays in EMT using a classical model system: transforming growth factor ��1 (TGF-��1)-mediated EMT in mammary epithelial cells. We showed that stress fibers and focal adhesions are increased, and cell-cell junctions are decreased in response to TGF- ��1. Moreover, these changes were reversible upon removal of TGF-��1. Downregulation of E-cadherin and upregulation of N-cadherin were both transcriptional. Neither experimental knockdown nor experimental overexpression of N-cadherin interfered with the morphological changes. In addition, the morphological changes associated with EMT preceded the downregulation of E-cadherin. Interestingly, TGF-��1-induced motility in N-cadherin-knockdown cells was significantly reduced. Together, these data suggest that cadherin switching is necessary for increased motility but is not required for the morphological changes that accompany EMT. Key words: Cadherin, TGF-��, Epithelium-to-mesenchyme transition, Motility Summary Cadherin switching: essential for behavioral but not morphological changes during an epithelium-to- mesenchyme transition Masato Maeda1, Keith R. Johnson1,2,3,4,5,6 and Margaret J. Wheelock1,2,3,4,5,6,* 1Department of Oral Biology (College of Dentistry), 2Department of Biochemistry and Molecular Biology, 3Department of Genetics, Cell Biology and Anatomy, 4Department of Pathology and Microbiology (College of Medicine), 5Eppley Institute for Research in Cancer and Allied Diseases, and 6Eppley Cancer Center, University of Nebraska Medical Center, Omaha, NE 68198-7696, USA *Author for correspondence (e-mail: mwheelock@unmc.edu) Accepted 4 November 2004 Journal of Cell Science 118, 873-887 Published by The Company of Biologists 2005 doi:10.1242/jcs.01634 Research Article Journal of Cell Science

874 (NMuMG) cells are treated with TGF-��1 (Comijn et al., 2001). In addition, overexpression of these transcriptional repressors alone induces EMT in some experimental systems (Batlle et al., 2000 Bolos et al., 2003 Cano et al., 2000 Comijn et al., 2001 Savagner et al., 1997). E-Cadherin is expressed by most epithelial tissues and some epithelium-derived cancer cells have been shown to lose E- cadherin expression (Batlle et al., 2000 Cano et al., 2000 Comijn et al., 2001 Nieman et al., 1999). N-Cadherin is typically expressed by mesenchymal cells. However, some cancer cells inappropriately express N-cadherin, which promotes motility and invasion (Hazan et al., 2000 Islam et al., 1996 Nieman et al., 1999). Thus, the loss of E-cadherin expression and gain of N-cadherin expression in cancer cells is reminiscent of the cadherin switching that is seen during normal embryonic EMT (Cavallaro et al., 2002 Christofori, 2003). One could hypothesize that the function of cadherin switching during EMT is to allow epithelial cells to separate from one another and to acquire a motile phenotype, as occurs during gastrulation, for example. Studies presented here were designed, in part, to address this hypothesis. To investigate the role of cadherin switching during EMT, we used the classical model system, TGF-��1-induced EMT in mammary epithelial cells. Several laboratories have investigated cadherin expression during TGF-��-mediated EMT in the mouse mammary epithelial cell line NMuMG, with conflicting results. Some studies reported that the expression level of E-cadherin did not change in response to TGF-��1 (Bakin et al., 2000 Bhowmick et al., 2001), whereas others showed that E-cadherin was downregulated (Miettinen et al., 1994 Piek et al., 1999). In addition, there are some reports that TGF-��1 induces EMT in MCF10A human mammary epithelial cells (Seton-Rogers et al., 2004 Tang et al., 2003). In the present study, we defined the role cadherin switching plays in the morphological and behavioral changes that are fundamental to EMT using these two model systems: NMuMG mouse mammary epithelial cells and MCF10A human mammary epithelial cells. Materials and Methods Reagents, antibodies and cultured cells All reagents were from Sigma-Aldrich (St Louis, MO) or Fisher Chemicals (Fairlawn, NJ) unless otherwise indicated. Mouse monoclonal antibody (mAb) against the extracellular portion of human E-cadherin (HECD1) (Shimoyama et al., 1989) was a kind gift from M. Takeichi (RIKEN Center for Developmental Biology, Kobe, Japan). Mouse mAbs against the cytoplasmic portion of E-cadherin (4A2), N-cadherin (13A9) and ��-catenin (6F9) have been described previously (Johnson et al., 1993 Knudsen et al., 1995). 4A2 was used to detect mouse E-cadherin and HECD1 was used to detect human E- cadherin. A rabbit polyclonal antibody (pAb) against E-cadherin (Wheelock et al., 1987) was used for double staining. Rat mAb against the extracellular portion of mouse E-cadherin (ECCD2), mouse anti- ��-catenin mAb, rabbit anti-ZO-1 pAb (Zymed, San Francisco, CA), rabbit anti fibronectin pAb (recognizes mouse fibronectin Sigma- Aldrich), mouse anti-human-fibronectin mAb (Takara, Madison, WI), mouse anti-glyceraldehyde-3-phosphate-dehydrogenase (anti- GAPDH) mAb (Abcam, Cambridge, MA) were used. Mouse NMuMG cells (CRL-1636) and human MCF10A cells (CRL-10317) were obtained from the American Type Culture Collection (ATCC, Manasass, VA). Subclones of NMuMG cells, clones NMuMG/E9 and NMuMG/E11 cells, were prepared by limiting dilution in flat- bottomed 96-well plates. NMuMG cells and their subclones were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) (Hyclone Laboratories, Logan, UT), 4.5 g l���1 glucose and 10 ��g ml���1 insulin. MCF10A cells were maintained in a 1:1 mixture of DME and Ham���s F12 medium supplemented with 5% horse serum (Invitrogen-Gibco, Carlsbad, CA), 20 ng ml���1 epidermal growth factor (EGF), 100 ng ml���1 cholera toxin, 10 ��g ml���1 insulin and 500 ng ml���1 hydrocortisone. TGF-��1 (R&D Systems, Minneapolis, MN) treatment was done without serum deprivation. For long-term treatment with TGF-��1 or TGF-��1+EGF, the supplements were replenished every 3 days. Manipulation of cadherin expression Myc-tagged human N-cadherin (Kim et al., 2000 Salomon et al., 1992) was subcloned into LZRS-MS-neo (Ireton et al., 2002) and used to generate virus. Retroviral production and infection was performed as described on the Nolan lab website (http://www.stanford.edu/group/nolan/). Briefly, Phoenix 293 cells (Grignani et al., 1998) were transfected using calcium phosphate (Stratagene, La Jolla, CA) and selected with puromycin. To produce N-cadherin-overexpressing NMuMG/E9 and MCF10A cells, freshly prepared conditioned medium containing the recombinant retrovirus and supplemented with 4 ��g ml���1 polybrene was added to the cells and infected cells were selected with G418 (Cellgro, Mediatech, Herndon, VA). Myc-tagged human E-cadherin, the gene encoding which was inserted into the pLKneo vector (Kim et al., 2000) was transfected into NMuMG/E9 cells and NMuMG/E11 cells using Lipofectamine 2000 Reagent (Life Technology, Rockville, MD) and selection was done with G418. Untagged human E-cadherin was transduced into NMuMG/E9 cells using the retrovirus system. N-cadherin-knockdown NMuMG/E9 cells (NMuMG/E9���Ncad cells) were generated by inserting 19 nucleotides of N-cadherin (GACTGGATTTCCTGAAGAT nucleotides 431-449 of mouse N- cadherin GenBank�� accession AB008811, which is identical to nucleotides 215-233 of human N-cadherin GenBank accession BC036470) into the pSilencer���1.0-U6 vector (Ambion, Austin, TX). The recombinant plasmid was co-transfected with a neomycin- resistance plasmid (using Lipofectamine 2000) into NMuMG/E9 cells and selection was done with G418. N-Cadherin-knockdown MCF10A cells were generated by inserting the same 19 nucleotides of N- cadherin into pSUPER.retro.puro vector (OligoEngine, Seattle, WA) and transfecting Phoenix 293 cells to produce retroviral particles. Infected MCF10A cells were selected with puromycin. As a control small interfering RNA (siRNA), 19 nucleotides of the gene encoding enhanced green fluorescent protein (eGFP) (CGATGC- CACCTACGGCAAG nucleotides 786-804 of eGFP GenBank accession U55762) were inserted into pSilencer���1.0-U6 vector and pSUPER.retro.puro vector. Transwell motility assays 2��105 MCF10A cells or 5��105 NMuMG/E9 cells were plated in the upper chamber of uncoated polyethylene tetraphthalate membranes (BD BioCoat��� control culture inserts, six-well plates, pore size of 8 ��m Becton Dickinson, San Jose, CA). When TGF-��1 was used, it was added to the stock culture 2 days before plating on the membranes and to both the upper and the lower chamber during the assay. After plating on membranes, the cells were incubated for 24 hours, and the cells that did not migrate through the membrane were removed with cotton swabs. Cells traversing the membrane were stained with eosin y-methylene blue using HEMA3. Cells in nine random fields of view at 100�� magnification were counted and expressed as the average number of cells per field of view. Three independent experiments were performed and the data were represented as an average with the Journal of Cell Science 118 (5) Journal of Cell Science

875 Role of cadherin switching in EMT standard deviation. The statistical analysis was by one-way analysis of variance followed by Scheffe���s F test. In vitro wound healing assays Cells were plated on a dish with a 2 mm grid 3 days before the initiation of the assay so that they would be confluent on the day of the experiment. TGF-��1 treatment was initiated 2 days before the assay. Monolayers of confluent cultures were lightly scratched with a pipette tip and were observed at timed intervals for up to 27 hours. Quantification was done by measuring the number of pixels in the wound area using Adobe�� Photoshop�� and calculating the decrease in the area by subtracting the number of pixels from the number of pixels in the corresponding wound area at the 0 hour time point. Statistical analysis was done as above. Detergent extraction, SDS-PAGE and immunoblots Monolayers of cultured cells were washed with ice-cold PBS and extracted on ice with TNE buffer (10 mM Tris-HCl, pH 8.0, 0.5% Nonidet P-40, 1 mM EDTA, 2 mM phenylmethylsulfonyl fluoride). Extracts were centrifuged at 20,000 g for 15 minutes at 4��C and the supernatant was collected. Protein concentration was determined using the DC protein assay (Bio-Rad, Hercules CA). Cell extracts were resolved by sodium-dodecyl-sulfate polyacrylamide- gel electrophoresis (SDS-PAGE) and immunoblotted as described previously (Johnson et al., 1993). Immunoprecipitation A 500 ��l aliquot of cell extract (500 ��g) was incubated with 300 ��l 4A2 (against the E-cadherin cytoplasmic domain) hybridoma supernatant for 30 minutes at 4��C. 50 ��l packed anti-mouse IgG affinity gel (ICN Pharmaceuticals, CostaMesa, CA) was added, and mixing continued for 30 minutes. Immune complexes were washed with TBST (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20). After the final wash, the packed beads were resuspended in 2�� sample buffer and boiled for 5 minutes, and the proteins were resolved by SDS-PAGE and immunoblotted. Immunofluorescence microscopy Cells were processed as previously described (Kim et al., 2000). When double staining included TRITC-conjugated phalloidin, the cells were fixed in 3.7% formaldehyde for 15 minutes, permeabilized with 0.2% Triton X-100 in PBS for 15 minutes and blocked in 10% goat serum in PBS. To confirm E-cadherin expression on the cell surface after TGF- ��1 treatment, the cells were fixed in 3.7% formaldehyde for 15 minutes without permeabilization and probed with the ECCD2 antibody, which recognizes the extracellular portion of E- cadherin. Cells were examined on a Zeiss Axiovert 200M microscope (G��ttingen, Germany) equipped with an ORCA-ER digital camera (Hamamatsu, Houston, TX). Images were collected and processed using OpenLab software (Improvision, Boston, MA). Conventional RT-PCR Total RNA was extracted with TRI reagent and analysed by reverse- transcription polymerase chain reaction (RT-PCR) using a TITANIUM One-Step RT-PCR kit (Becton Dickinson, San Jose, CA) and previously reported forward and reverse primers for snail (Gotzmann et al., 2002), SIP1 (Cacheux et al., 2001), slug (Zhao et al., 2002), E12/E47 (Perez-Moreno et al., 2001), E-cadherin (Tegoshi et al., 2000), N-cadherin (Chung et al., 1998) and GAPDH (Xu et al., 2000). ��-Actin primers were supplied with the kit. The conditions for PCR reactions were: 94��C for 30 seconds, 65��C for 30 seconds and 68��C for 1 minute for 28-30 cycles for snail, SIP1, slug, E12/E47, E- cadherin and ��-actin 94��C for 1 minute, 62��C for 2 minutes and 72��C Fig. 1. Subcloning NMuMG cells. (A) Parental NMuMG cells (a-c), clone NMuMG/E9 (d,e) and clone NMuMG/E11 (f,g) were stained for N-cadherin (a,d,f) and E-cadherin (b,e,g). (c) A merged picture of a,b. (a-c) Photographs were taken using a 10�� objective scale bar, 50 ��m. (d-g) Photographs were taken using a 40�� dry objective scale bar, 10 ��m. (B) TNE extracts of NMuMG/E9 (lane 1) and NMuMG/E11 (lane 2) were analysed for N-cadherin (top) and E-cadherin (bottom) by immunoblots. (C) Both clones underwent morphological changes (elongation of cell shape, b,d) in response to TGF- ��1 [T��1 5 ng ml���1 for 1 day (d)]. Photographs were taken of living cells using a 10�� objective scale bar, 50 ��m. Journal of Cell Science