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The transcription factors Tbx18 and Wt1 control the epicardial epithelial-mesenchymal transition through bi-directional regulation of Slug in murine primary epicardial cells.

Takeichi M, Nimura K, Mori M, Nakagami H, Kaneda Y - PLoS ONE (2013)

Bottom Line: The expression of Slug but not Snail decreased as a result of Tbx18 knockdown, but Slug expression increased following knockdown of Wt1.Furthermore, in normal murine mammary gland-C7 (NMuMG-C7) cells, Tbx18 acted to increase Slug expression, while Wt1 acted to decrease Slug expression.These results provide new insights into the regulatory mechanisms that control the epicardial EMT.

View Article: PubMed Central - PubMed

Affiliation: Division of Gene Therapy Science, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan.

ABSTRACT
During cardiac development, a subpopulation of epicardial cells migrates into the heart as part of the epicardial epithelial-mesenchymal transition (EMT) and differentiates into smooth muscle cells and fibroblasts. However, the roles of transcription factors in the epicardial EMT are poorly understood. Here, we show that two transcription factors expressed in the developing epicardium, T-box18 (Tbx18) and Wilms' tumor 1 homolog (Wt1), bi-directionally control the epicardial EMT through their effects on Slug expression in murine primary epicardial cells. Knockdown of Wt1 induced the epicardial EMT, which was accompanied by an increase in the migration and expression of N-cadherin and a decrease in the expression of ZO-1 as an epithelial marker. By contrast, knockdown of Tbx18 inhibited the mesenchymal transition induced by TGFβ1 treatment and Wt1 knockdown. The expression of Slug but not Snail decreased as a result of Tbx18 knockdown, but Slug expression increased following knockdown of Wt1. Knockdown of Slug also attenuated the epicardial EMT induced by TGFβ1 treatment and Wt1 knockdown. Furthermore, in normal murine mammary gland-C7 (NMuMG-C7) cells, Tbx18 acted to increase Slug expression, while Wt1 acted to decrease Slug expression. Chromatin immunoprecipitation and promoter assay revealed that Tbx18 and Wt1 directly bound to the Slug promoter region and regulated Slug expression. These results provide new insights into the regulatory mechanisms that control the epicardial EMT.

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Tbx18 and Wt1 regulate Slug expression in NMuMG-C7 cells.(A) Representative images of NMuMG-C7 cells expressing EGFP, Tbx18, Wt1 or a combination of Tbx18 and Wt1. Scale bar: 100 µm. (B) Western blot analysis of transduced NMuMG-C7 cell lines with antibodies against Tbx18, Wt1, Snail and Slug. β-actin was used as a loading control. (C) The relative mRNA expression of Slug in transduced NMuMG-C7 cell lines. The results were normalized to Gapdh expression, and the relative expression is provided as a ratio to EGFP transduced cells. The data are presented as the mean ± SD; n = 3; *P<0.05 vs. EGFP transduced cells, †P<0.0001 vs. Tbx18 transduced cells.
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pone-0057829-g005: Tbx18 and Wt1 regulate Slug expression in NMuMG-C7 cells.(A) Representative images of NMuMG-C7 cells expressing EGFP, Tbx18, Wt1 or a combination of Tbx18 and Wt1. Scale bar: 100 µm. (B) Western blot analysis of transduced NMuMG-C7 cell lines with antibodies against Tbx18, Wt1, Snail and Slug. β-actin was used as a loading control. (C) The relative mRNA expression of Slug in transduced NMuMG-C7 cell lines. The results were normalized to Gapdh expression, and the relative expression is provided as a ratio to EGFP transduced cells. The data are presented as the mean ± SD; n = 3; *P<0.05 vs. EGFP transduced cells, †P<0.0001 vs. Tbx18 transduced cells.

Mentions: To further investigate the molecular functions of Tbx18 and Wt1, overexpression experiments were performed. Because our results raise the possibility that Tbx18 and Wt1 have opposing functions, we used NMuMG-C7 cells, which do not express endogenous Tbx18 or Wt1. NMuMG-C7 cells were derived using the limiting dilution technique from NMuMG cells [38]. NMuMG-C7 cell lines that overexpress Tbx18 or Wt1 were established by retroviral transduction. As shown in Figure 5A, Wt1-expressing cells did not exhibit any changes in cell-cell adhesion or epithelial morphology when compared to the EGFP-expressing control cells. By contrast, Tbx18-expressing cells exhibited impaired cell-cell interactions and a fibroblastic morphology (Figure 5A).


The transcription factors Tbx18 and Wt1 control the epicardial epithelial-mesenchymal transition through bi-directional regulation of Slug in murine primary epicardial cells.

Takeichi M, Nimura K, Mori M, Nakagami H, Kaneda Y - PLoS ONE (2013)

Tbx18 and Wt1 regulate Slug expression in NMuMG-C7 cells.(A) Representative images of NMuMG-C7 cells expressing EGFP, Tbx18, Wt1 or a combination of Tbx18 and Wt1. Scale bar: 100 µm. (B) Western blot analysis of transduced NMuMG-C7 cell lines with antibodies against Tbx18, Wt1, Snail and Slug. β-actin was used as a loading control. (C) The relative mRNA expression of Slug in transduced NMuMG-C7 cell lines. The results were normalized to Gapdh expression, and the relative expression is provided as a ratio to EGFP transduced cells. The data are presented as the mean ± SD; n = 3; *P<0.05 vs. EGFP transduced cells, †P<0.0001 vs. Tbx18 transduced cells.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585213&req=5

pone-0057829-g005: Tbx18 and Wt1 regulate Slug expression in NMuMG-C7 cells.(A) Representative images of NMuMG-C7 cells expressing EGFP, Tbx18, Wt1 or a combination of Tbx18 and Wt1. Scale bar: 100 µm. (B) Western blot analysis of transduced NMuMG-C7 cell lines with antibodies against Tbx18, Wt1, Snail and Slug. β-actin was used as a loading control. (C) The relative mRNA expression of Slug in transduced NMuMG-C7 cell lines. The results were normalized to Gapdh expression, and the relative expression is provided as a ratio to EGFP transduced cells. The data are presented as the mean ± SD; n = 3; *P<0.05 vs. EGFP transduced cells, †P<0.0001 vs. Tbx18 transduced cells.
Mentions: To further investigate the molecular functions of Tbx18 and Wt1, overexpression experiments were performed. Because our results raise the possibility that Tbx18 and Wt1 have opposing functions, we used NMuMG-C7 cells, which do not express endogenous Tbx18 or Wt1. NMuMG-C7 cells were derived using the limiting dilution technique from NMuMG cells [38]. NMuMG-C7 cell lines that overexpress Tbx18 or Wt1 were established by retroviral transduction. As shown in Figure 5A, Wt1-expressing cells did not exhibit any changes in cell-cell adhesion or epithelial morphology when compared to the EGFP-expressing control cells. By contrast, Tbx18-expressing cells exhibited impaired cell-cell interactions and a fibroblastic morphology (Figure 5A).

Bottom Line: The expression of Slug but not Snail decreased as a result of Tbx18 knockdown, but Slug expression increased following knockdown of Wt1.Furthermore, in normal murine mammary gland-C7 (NMuMG-C7) cells, Tbx18 acted to increase Slug expression, while Wt1 acted to decrease Slug expression.These results provide new insights into the regulatory mechanisms that control the epicardial EMT.

View Article: PubMed Central - PubMed

Affiliation: Division of Gene Therapy Science, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan.

ABSTRACT
During cardiac development, a subpopulation of epicardial cells migrates into the heart as part of the epicardial epithelial-mesenchymal transition (EMT) and differentiates into smooth muscle cells and fibroblasts. However, the roles of transcription factors in the epicardial EMT are poorly understood. Here, we show that two transcription factors expressed in the developing epicardium, T-box18 (Tbx18) and Wilms' tumor 1 homolog (Wt1), bi-directionally control the epicardial EMT through their effects on Slug expression in murine primary epicardial cells. Knockdown of Wt1 induced the epicardial EMT, which was accompanied by an increase in the migration and expression of N-cadherin and a decrease in the expression of ZO-1 as an epithelial marker. By contrast, knockdown of Tbx18 inhibited the mesenchymal transition induced by TGFβ1 treatment and Wt1 knockdown. The expression of Slug but not Snail decreased as a result of Tbx18 knockdown, but Slug expression increased following knockdown of Wt1. Knockdown of Slug also attenuated the epicardial EMT induced by TGFβ1 treatment and Wt1 knockdown. Furthermore, in normal murine mammary gland-C7 (NMuMG-C7) cells, Tbx18 acted to increase Slug expression, while Wt1 acted to decrease Slug expression. Chromatin immunoprecipitation and promoter assay revealed that Tbx18 and Wt1 directly bound to the Slug promoter region and regulated Slug expression. These results provide new insights into the regulatory mechanisms that control the epicardial EMT.

Show MeSH
Related in: MedlinePlus