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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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The epicardial EMT induced by Wt1 knockdown is inhibited by knockdown of Tbx18 or Slug.(A) Representative images of Wt1-knockdown epicardial cells co-transfected with siTbx18 or siSlug. Epicardial cells transfected with siControl or siWt1 were used as controls. (B) Immunostaining for ZO-1 (green) and DAPI nuclear staining (blue) of primary epicardial cells transfected with siRNA. (C) Percentage of cells categorized as “Enlarged” or “Cobblestone-like,” based on cellular morphology. (D) The relative mRNA expression of Tbx18, Wt1 and Slug by real-time PCR in Wt1-knockdown epicardial cells co-transfected with siTbx18 or siSlug, in comparison to siControl-transfected epicardial cells (n = 3; *P<0.05 vs. siControl, †P<0.05 vs. siWt1). The results were normalized to Gapdh expression and the relative expression level is provided as a ratio to the siControl. The data are presented as the mean ± SD. Scale bars: 100 µm.
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pone-0057829-g004: The epicardial EMT induced by Wt1 knockdown is inhibited by knockdown of Tbx18 or Slug.(A) Representative images of Wt1-knockdown epicardial cells co-transfected with siTbx18 or siSlug. Epicardial cells transfected with siControl or siWt1 were used as controls. (B) Immunostaining for ZO-1 (green) and DAPI nuclear staining (blue) of primary epicardial cells transfected with siRNA. (C) Percentage of cells categorized as “Enlarged” or “Cobblestone-like,” based on cellular morphology. (D) The relative mRNA expression of Tbx18, Wt1 and Slug by real-time PCR in Wt1-knockdown epicardial cells co-transfected with siTbx18 or siSlug, in comparison to siControl-transfected epicardial cells (n = 3; *P<0.05 vs. siControl, †P<0.05 vs. siWt1). The results were normalized to Gapdh expression and the relative expression level is provided as a ratio to the siControl. The data are presented as the mean ± SD. Scale bars: 100 µm.

Mentions: Because Slug is a key molecule in the epicardial EMT, we hypothesized that Tbx18 and Wt1 coordinately determine epicardial cell fate through the bi-directional regulation of Slug expression. To assess this hypothesis, we examined whether the mesenchymal transition induced by Wt1 knockdown is inhibited following knockdown of Tbx18 or Slug. Wt1-knockdown cells exhibited a mesenchymal morphology and decreased ZO-1 expression at the cell-cell junction, as shown in Figure 4A and 4B. These changes induced by Wt1 knockdown were inhibited by the co-transfection of an siRNA directed against Tbx18 or Slug (Figure 4A, 4B and 4C, and Figure S1C). Furthermore, the increase in Slug mRNA expression caused by Wt1 knockdown was reduced upon knockdown of Tbx18 (Figure 4D). Taken together, these results suggest that Tbx18 promotes the mesenchymal transition through the upregulation of Slug, and that Wt1 maintains the epicardial cell fate through the downregulation of Slug.


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)

The epicardial EMT induced by Wt1 knockdown is inhibited by knockdown of Tbx18 or Slug.(A) Representative images of Wt1-knockdown epicardial cells co-transfected with siTbx18 or siSlug. Epicardial cells transfected with siControl or siWt1 were used as controls. (B) Immunostaining for ZO-1 (green) and DAPI nuclear staining (blue) of primary epicardial cells transfected with siRNA. (C) Percentage of cells categorized as “Enlarged” or “Cobblestone-like,” based on cellular morphology. (D) The relative mRNA expression of Tbx18, Wt1 and Slug by real-time PCR in Wt1-knockdown epicardial cells co-transfected with siTbx18 or siSlug, in comparison to siControl-transfected epicardial cells (n = 3; *P<0.05 vs. siControl, †P<0.05 vs. siWt1). The results were normalized to Gapdh expression and the relative expression level is provided as a ratio to the siControl. The data are presented as the mean ± SD. Scale bars: 100 µm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585213&req=5

pone-0057829-g004: The epicardial EMT induced by Wt1 knockdown is inhibited by knockdown of Tbx18 or Slug.(A) Representative images of Wt1-knockdown epicardial cells co-transfected with siTbx18 or siSlug. Epicardial cells transfected with siControl or siWt1 were used as controls. (B) Immunostaining for ZO-1 (green) and DAPI nuclear staining (blue) of primary epicardial cells transfected with siRNA. (C) Percentage of cells categorized as “Enlarged” or “Cobblestone-like,” based on cellular morphology. (D) The relative mRNA expression of Tbx18, Wt1 and Slug by real-time PCR in Wt1-knockdown epicardial cells co-transfected with siTbx18 or siSlug, in comparison to siControl-transfected epicardial cells (n = 3; *P<0.05 vs. siControl, †P<0.05 vs. siWt1). The results were normalized to Gapdh expression and the relative expression level is provided as a ratio to the siControl. The data are presented as the mean ± SD. Scale bars: 100 µm.
Mentions: Because Slug is a key molecule in the epicardial EMT, we hypothesized that Tbx18 and Wt1 coordinately determine epicardial cell fate through the bi-directional regulation of Slug expression. To assess this hypothesis, we examined whether the mesenchymal transition induced by Wt1 knockdown is inhibited following knockdown of Tbx18 or Slug. Wt1-knockdown cells exhibited a mesenchymal morphology and decreased ZO-1 expression at the cell-cell junction, as shown in Figure 4A and 4B. These changes induced by Wt1 knockdown were inhibited by the co-transfection of an siRNA directed against Tbx18 or Slug (Figure 4A, 4B and 4C, and Figure S1C). Furthermore, the increase in Slug mRNA expression caused by Wt1 knockdown was reduced upon knockdown of Tbx18 (Figure 4D). Taken together, these results suggest that Tbx18 promotes the mesenchymal transition through the upregulation of Slug, and that Wt1 maintains the epicardial cell fate through the downregulation of Slug.

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