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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

(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.
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f4-ijmm-36-01-0166: (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.

Mentions: 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 (31). Using an Agilent Whole Canine Genome microarray, we found that the ectopic expression of Snail in MDCK cells resulted in the increased expression of Slug and ZEB1 [Ozawa et al, (32)]. The upregulation of Twist and ZEB1 expression and the induction of EMT in human mammary epithelial HMLE cells upon Snail overexpression have been previously reported (33). Therefore, in this study, we used RT-PCR to compare the mRNA expression levels of Slug, Twist and ZEB1 in the neo cells and Snail cells. We observed no significant changes in the mRNA levels of these factors upon the ectopic expression of Snail (Fig. 4). Furthermore, immunoblot analysis revealed that MDCK cells expressing Snail presented with an increased Slug and ZEB1 production at the protein level, whereas the MDBK cells expressing Snail did not. Thus, our data suggest that the Snail-mediated upregulation of Slug and ZEB1 is required for the downregulation of E-cadherin expression and the induction of EMT.


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)

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

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

f4-ijmm-36-01-0166: (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.
Mentions: 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 (31). Using an Agilent Whole Canine Genome microarray, we found that the ectopic expression of Snail in MDCK cells resulted in the increased expression of Slug and ZEB1 [Ozawa et al, (32)]. The upregulation of Twist and ZEB1 expression and the induction of EMT in human mammary epithelial HMLE cells upon Snail overexpression have been previously reported (33). Therefore, in this study, we used RT-PCR to compare the mRNA expression levels of Slug, Twist and ZEB1 in the neo cells and Snail cells. We observed no significant changes in the mRNA levels of these factors upon the ectopic expression of Snail (Fig. 4). Furthermore, immunoblot analysis revealed that MDCK cells expressing Snail presented with an increased Slug and ZEB1 production at the protein level, whereas the MDBK cells expressing Snail did not. Thus, our data suggest that the Snail-mediated upregulation of Slug and ZEB1 is required for the downregulation of E-cadherin expression and the induction of EMT.

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