Epithelial-mesenchymal transition (EMT), a key process in the tumor metastatic cascade, is characterized by the loss of cell-cell junctions and cell polarity, as well as by the acquisition of migratory and invasive properties. However, the precise molecular events that initiate this complex EMT process are poorly understood. Snail expression induces EMT in Madin-Darby canine kidney (MDCK) cells and the human epidermoid carcinoma cell line, A431. Snail is a zinc finger transcription factor and triggers EMT by suppressing E-cadherin expression. In the present study, to broaden our knowledge of Snail-induced EMT, we generated stable Snail transfectants using Madin-Darby bovine kidney (MDBK) cells. Contrary to the MDCK or A431 cells examined in our previous studies, the MDBK cells transfected with the Snail construct maintained an epithelial morphology and showed no sign of reduced cell-cell adhesiveness compared to the control cells. Consistent with these observations, the down-regulation of epithelial marker proteins, e.g. E-cadherin and desmoglein, and the upregulation of mesenchymal marker proteins, e.g., N-cadherin and fibronectin, were not detected. Furthermore, the E-cadherin promoter was not methylated. Therefore, in the MDBK cells, the ectopic expression of Snail failed to induce EMT. As previously demonstrated, in MDCK cells, Snail expression is accompanied by the increased expression of other EMT-inducing transcription factors, e.g., Slug and zinc finger E-box-binding homeobox 1 (ZEB1). However, the MDBK cells transfected with the Snail construct did not exhibit an increased expression of these factors. Thus, it is possible that the failure to upregulate other EMT-related transcription factors may explain the lack of Snail-mediated induction of EMT in MDBK cells.
Epithelial-mesenchymal transition (EMT) is a complex process through which epithelial cells lose their polarity and reorganize their cytoskeleton, while also acquiring a mesenchymal phenotype and increased motility (
Epithelial cells are connected by the epithelial junctional complex, which consists of tight junctions, adherens junctions and desmosomes. E-cadherin is a component of the adherens junction and is involved in the formation and maintenance of epithelial structures (
A hallmark of EMT is the loss of E-cadherin expression (
EMT is accompanied by epigenetic modifications, including DNA methylation (
The ectopic expression of Snail in several epithelial cells, including Madin-Darby canine kidney (MDCK) cells and the human epidermoid carcinoma cell line, A431, has been shown to result in EMT (
MDBK cells, a bovine kidney epithelial cell line and MDCK cells, a canine kidney epithelial cell line, provided by Dr Rolf Kemler (Max-Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany) and Dr Satoshi Daikuhara (Kagoshima University), respectively. They were grown and were transfected as previously described (
Mouse monoclonal antibodies (mAbs) against E-cadherin (Cat. no. 610182), p120 (Cat. no. 612537) and fibronectin (Cat. no. 610077) were purchased from BD Biosciences (Lexington, KY, USA). A mouse mAb against vimentin (Cat. no. 18-0052) was obtained from Zymed Laboratories (South San Francisco, CA, USA). Mouse mAbs recognizing Snail (Cat. no. 3895) and Slug (Cat. no. 9589) were purchased from Cell Signaling Technology (Danvers, MA, USA). A mAb against desmoglein 1 and 2 (Cat. no. 61002) was purchased from Progen Biotechnik GmbH (Heidelberg, Germany). A goat antibody recognizing ZEB1 (Cat. no. sc-5711) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). A mouse mAb recognizing vinculin (Cat. no. V9131) was purchased from Sigma-Aldrich (St. Louis, MO, USA). A rat mAb against hemagglutinin (HA; Cat. no. 11867423001) was purchased from Roche Applied Science (Mannheim, Germany). All secondary antibodies were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA, USA).
Total RNA was extracted and reverse transcribed as previously described (
Cell aggregation assays were performed as previously described (
For immunoblot analysis, proteins were separated by 8% polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. After blocking, the membranes were incubated with specific primary antibodies followed by treatment with peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories). After washing with phosphate-buffered saline (PBS) containing 0.1% Tween-20, the protein bands were visualized by enhanced chemiluminescence (ECL; Amersham International, Little Chalfont, UK) as previously described (
For immunofluorescence, the cells were grown on coverslips, fixed with 3% paraformaldehyde in PBS for 20 min at room temperature, and permeabilized with 0.1% Triton X-100. The coverslips were immunostained with primary and secondary antibodies as previously described (
Genomic DNA (~0.75
MDBK cells, a cell line derived from bovine kidney, display epithelial properties, including a brickstone morphology. We introduced a control empty vector containing a neomycin resistance gene or an expression vector encoding HA-tagged Snail protein into the MDBK cells and isolated stable transfectants, designated as neo or Snail cells, respectively. The Snail cells retained the same epithelial morphology as the control neo cells (
Cells undergoing EMT lose cell-cell adhesion. It is well known that cadherins at the cell surface resist tryptic digestion in the presence of Ca2+, but not in the absence of Ca2+ (
Next, we determined the expression levels of epithelial markers, E-cadherin and desmoglein, using immunoblot analysis (
Consistent with the observations that Snail expression did not alter cadherin-mediated cell-cell adhesion (
Previous analysis of the E-cadherin gene revealed that its proximal promoter contains CpG islands, which are targets for methylation during TGF-β-induced EMT (
As previously reported, the expression of lymphoid enhancer-binding factor 1 (LEF-1), an EMT-inducer, in MDCK cells resulted in the significantly increased expression of other EMT-inducing transcription factors, including Slug and ZEB1 (
In this study, we demonstrated that the ectopic expression of Snail in MDBK cells, a bovine kidney epithelial cell line, failed to induce changes that were characteristic of EMT. None of the following events were observed: i) epithelial to fibroblastic morphological changes; ii) reduced cell-cell adhesion; iii) the downregulation of the epithelial markers, E-cadherin and desmoglein; or iv) the upregulation of the mesenchymal markers, N-cadherin, vimentin and fibronectin. Although the downregulation of E-cadherin and desmoglein in human squamous cell carcinoma HSC-4 cells is not extensive (
The exogenous expression of Snail has been reported to suppress the activity of an E-cadherin promoter-reporter construct in MDCK cells, but not in mouse mammary epithelial NMuMG cells (
The levels of EMT-inducing transcription factors are under the control of microRNAs, which are regulated by wild-type p53 (
As previously reported, the expression of LEF-1, an EMT-inducer, in MDCK cells resulted in the significantly increased expression of other EMT-inducing transcription factors, e.g., Slug and ZEB1 (
It has been demonstrated that shRNA-mediated knockdown of E-cadherin induces EMT (
We would like thank Dr M. Sato (Kagoshima University) for his helpful discussion. This study was supported by the following grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan. We would also like to thank the Joint Research Laboratory at Kagoshima University Graduate School of Medical and Dental Sciences for the use of their facilities.
epithelial-mesenchymal transition
Madin-Darby bovine kidney
Madin-Darby canine kidney
Madin-Darby bovine kidney (MDBK) cells ectopically expressing Snail protein display characteristics of the epithelial phenotype. (A) Both the control MDBK cells transfected with an empty vector containing a neomycin resistance gene (neo) and MDBK cells transfected with an expression vector encoding HA-tagged Snail protein (Snail) displayed typical epithelial cell morphology. (B) Immunofluorescence staining with an anti-HA antibody revealed the protein expression of Snail in the nucleus, which was co-stained with DAPI. (C) Cell aggregation assays revealed that the cells ectopically expressing Snail protein had similar adhesive properties as the control (neo) cells; furthermore, the observed cell-cell adhesion was calcium-dependent, indicating that it was mediated by cadherins. Scale bars, 20
Epithelial-mesenchymal transition (EMT) is not induced in Madin-Darby bovine kidney (MDBK) cells ectopically expressing Snail. (A) Immunoblot analysis revealed that Snail expression in the MDBK cells did not decrease the expression of the epithelial markers, E-cadherin and desmoglein, and did not increase the expression of the mesenchymal markers, N-cadherin, vimentin and fibronectin. α-tubulin was used as a loading control. (B) The ectopic expression of Snail altered the splicing patterns of p120 in the Madin-Darby canine kidney (MDCK) cells, but not in the MDBK cells. (C) E-cadherin (E-cad) and N-cadherin (N-cad) were detected at the membrane of the control cells [transfected with a neomycin resistance gene (neo) cells] and in the cells ectopically expressing Snail protein (Snail). Cells were stained with the appropriate primary antibody followed by a rhodamine-labeled secondary antibody. DAPI was used to detect the nucleus. Scale bar, 20
Ectopic expression of Snail in Madin-Darby bovine kidney (MDBK) cells does not induce DNA methylation of the E-cadherin promoter. Diagram showing the position of 4 E-boxes (-403 to -398, -201 to -196, -151 to -146, and -100 to -95; red bars) and CpG dinucleotides within the E-cadherin promoter region (circles). Genomic DNA was isolated from the control cells [transfected with a neomycin resistance gene (neo) cells] and Snail cells (cells ectopically expressing Snail protein), and the methylation of the E-cadherin promoter was analyzed by bisulfite sequencing. Genomic DNA incubated with CpG methyl-transfease prior to bisulfite treatment was used as a positive control for methylated DNA. Methylated and unmethylated dinucleotides are indicated as filled and open circles, respectively.
(A) RT-PCR of Slug, Twist and ZEB1 mRNA in control cells [transfected with a neomycin resistance gene (neo) cells] and Snail cells (cells ectopically expressing Snail protein). β-actin was used as an internal control. No significant differences were observed between the control cells and the Snail cells with respect to the mRNA levels of these proteins. (B) Immunoblot analysis using anti-Slug and anti-ZEB1 antibodies. Vinculin served as a loading control. Ectopic Snail expression increased Slug and ZEB1 protein levels in Madin-Darby canine kidney (MDCK) cells, but not in Madin-Darby bovine kidney (MDBK) cells. Ectopic expression of Snail in MDBK cells slightly increased the expression level of ZEB1 protein, but the quantification of the blots using NIH ImageJ software revealed that the relative amounts of ZEB1 protein in Snail-MDBK cells were <20% of those in the Snail-MDCK cells.
Relative expression levels of epithelial and mesenchymal markers in MDBK cells ectopically expressing Snail protein.
E-cadherin | Desmoglein | N-cadherin | Fibronectin | Vimentin | |
---|---|---|---|---|---|
Ratios | 0.76±0.09 | 0.87±0.06 | 0.74±0.13 | 0.74±0.13 | 1.16±0.12 |
The expression levels were determined using ImageJ software (National Institutes of Health). The data are presented as the relative intensity of the bands in the Snail-transfected MDBK cell samples as compared to the control [transfected with a neomycin resistance gene (neo)] MDBK cell samples. Values are the means ± SE obtained from 3 independent clones. MDBK cells, Madin-Darby bovine kidney cells.