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The ectopic expression of Snail in MDBK cells does not induce epithelial-mesenchymal transition.

Izawa G, Kobayashi W, Haraguchi M, Sudo A, Ozawa M - Int. J. Mol. Med. (2015)

Bottom Line: 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.Therefore, in the MDBK cells, the ectopic expression of Snail failed to induce EMT.However, the MDBK cells transfected with the Snail construct did not exhibit an increased expression of these factors.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544, Japan.

ABSTRACT
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 downregulation 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.

No MeSH data available.


Related in: MedlinePlus

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 µm.
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f2-ijmm-36-01-0166: 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 µm.

Mentions: Next, we determined the expression levels of epithelial markers, E-cadherin and desmoglein, using immunoblot analysis (Fig. 2). Although the Snail cells expressed exogenous Snail protein as detected by anti-HA antibodies, they showed essentially the same expression levels of E-cadherin and desmoglein as the control neo cells. The control neo cells also expressed the mesenchymal markers, N-cadherin, vimentin and fibronectin, and the expression levels of these proteins did not increase in the Snail cells (Fig. 2 and Table I). Thus, the ectopic expression of Snail in the MDBK cells did not lead to the downregulation of E-cadherin or desmoglein expression or to the upregulation of N-cadherin, vimentin or fibronectin expression. Furthermore, as previously reported (28), the expression of Snail altered the splicing patterns of p120 in the MDCK cells, but not in the MDBK cells (Fig. 2B).


The ectopic expression of Snail in MDBK cells does not induce epithelial-mesenchymal transition.

Izawa G, Kobayashi W, Haraguchi M, Sudo A, Ozawa M - Int. J. Mol. Med. (2015)

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 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4494602&req=5

f2-ijmm-36-01-0166: 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 µm.
Mentions: Next, we determined the expression levels of epithelial markers, E-cadherin and desmoglein, using immunoblot analysis (Fig. 2). Although the Snail cells expressed exogenous Snail protein as detected by anti-HA antibodies, they showed essentially the same expression levels of E-cadherin and desmoglein as the control neo cells. The control neo cells also expressed the mesenchymal markers, N-cadherin, vimentin and fibronectin, and the expression levels of these proteins did not increase in the Snail cells (Fig. 2 and Table I). Thus, the ectopic expression of Snail in the MDBK cells did not lead to the downregulation of E-cadherin or desmoglein expression or to the upregulation of N-cadherin, vimentin or fibronectin expression. Furthermore, as previously reported (28), the expression of Snail altered the splicing patterns of p120 in the MDCK cells, but not in the MDBK cells (Fig. 2B).

Bottom Line: 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.Therefore, in the MDBK cells, the ectopic expression of Snail failed to induce EMT.However, the MDBK cells transfected with the Snail construct did not exhibit an increased expression of these factors.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544, Japan.

ABSTRACT
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 downregulation 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.

No MeSH data available.


Related in: MedlinePlus