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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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Knockdown of Wt1 and Tbx18 in primary epicardial cells.(A) Representative images of primary epicardial cells transfected with control siRNA (siControl) or siRNA directed against Tbx18 (siTbx18) or Wt1 (siWt1). Scale bar: 200 µm. (B) Immunostaining for ZO-1 or N-cadherin (green) and DAPI nuclear staining (blue) of primary epicardial cells transfected with siRNAs. Scale bars: 100 µm. (C) Percentage of cells categorized as “Enlarged” or “Cobblestone-like,” based on the cellular morphology of primary epicardial cells transfected with siRNAs. (D) Representative images of primary epicardial cells transfected with siRNAs at 0 and 14 hr after the scratch was made. Scale bar: 200 µm. (E) Quantification of migration distance, given as a ratio to the siControl (n = 4; *P<0.01 vs. siControl). (F) The relative mRNA expression of Tbx18, Wt1, Snail and Slug by real-time PCR analysis (n = 3; *P<0.05 vs. siControl). The results were normalized to Gapdh expression, and the relative expression level is given as a ratio to the siControl. (G) Western blot performed with antibodies against Tbx18, Wt1, adhesion molecules (E-cadherin and N-cadherin) and EMT regulators (Snail and Slug). β-actin and histone H3 were used as loading controls. The data are presented as the mean ± SD.
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pone-0057829-g002: Knockdown of Wt1 and Tbx18 in primary epicardial cells.(A) Representative images of primary epicardial cells transfected with control siRNA (siControl) or siRNA directed against Tbx18 (siTbx18) or Wt1 (siWt1). Scale bar: 200 µm. (B) Immunostaining for ZO-1 or N-cadherin (green) and DAPI nuclear staining (blue) of primary epicardial cells transfected with siRNAs. Scale bars: 100 µm. (C) Percentage of cells categorized as “Enlarged” or “Cobblestone-like,” based on the cellular morphology of primary epicardial cells transfected with siRNAs. (D) Representative images of primary epicardial cells transfected with siRNAs at 0 and 14 hr after the scratch was made. Scale bar: 200 µm. (E) Quantification of migration distance, given as a ratio to the siControl (n = 4; *P<0.01 vs. siControl). (F) The relative mRNA expression of Tbx18, Wt1, Snail and Slug by real-time PCR analysis (n = 3; *P<0.05 vs. siControl). The results were normalized to Gapdh expression, and the relative expression level is given as a ratio to the siControl. (G) Western blot performed with antibodies against Tbx18, Wt1, adhesion molecules (E-cadherin and N-cadherin) and EMT regulators (Snail and Slug). β-actin and histone H3 were used as loading controls. The data are presented as the mean ± SD.

Mentions: Tbx18 and Wt1 are transcription factors that are expressed in the embryonic epicardium. To investigate the functions of Tbx18 and Wt1 in the epicardial EMT, we performed knockdown experiments of Tbx18 and Wt1 in primary embryonic epicardial cells. Cells treated with control siRNA (siControl) maintained a cobblestone-like cell shape, although a slight morphological change was observed at the edge of the colony after several days in culture (Figure 2A). ZO-1, an epicardial and epithelial adhesion molecule, was localized at the cell-cell junction, and N-cadherin, a mesenchymal adhesion molecule, was not observed in the siControl cells (Figure 2B). Knockdown of Wt1 (siWt1) induced significant morphological changes, including impaired cell-cell contacts and a larger cell size (Figure 2A). In Wt1-knockdown cells, ZO-1 expression was decreased; however, N-cadherin was increased at the cell-cell interfaces, suggesting that the Wt1 knockdown induced the mesenchymal transition (Figure 2B). Knockdown of Tbx18 (siTbx18) did not affect cell-cell interactions or ZO-1 localization at the cell-cell junctions, and all cells, including those at the edge of the colony, maintained a more compact cell shape when compared to control cells (Figure 2A and 2B). Furthermore, knockdown of Wt1 but not Tbx18 significantly increased the proportion of enlarged cells (Figure 2C and Figure S1A).


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)

Knockdown of Wt1 and Tbx18 in primary epicardial cells.(A) Representative images of primary epicardial cells transfected with control siRNA (siControl) or siRNA directed against Tbx18 (siTbx18) or Wt1 (siWt1). Scale bar: 200 µm. (B) Immunostaining for ZO-1 or N-cadherin (green) and DAPI nuclear staining (blue) of primary epicardial cells transfected with siRNAs. Scale bars: 100 µm. (C) Percentage of cells categorized as “Enlarged” or “Cobblestone-like,” based on the cellular morphology of primary epicardial cells transfected with siRNAs. (D) Representative images of primary epicardial cells transfected with siRNAs at 0 and 14 hr after the scratch was made. Scale bar: 200 µm. (E) Quantification of migration distance, given as a ratio to the siControl (n = 4; *P<0.01 vs. siControl). (F) The relative mRNA expression of Tbx18, Wt1, Snail and Slug by real-time PCR analysis (n = 3; *P<0.05 vs. siControl). The results were normalized to Gapdh expression, and the relative expression level is given as a ratio to the siControl. (G) Western blot performed with antibodies against Tbx18, Wt1, adhesion molecules (E-cadherin and N-cadherin) and EMT regulators (Snail and Slug). β-actin and histone H3 were used as loading controls. The data are presented as the mean ± SD.
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pone-0057829-g002: Knockdown of Wt1 and Tbx18 in primary epicardial cells.(A) Representative images of primary epicardial cells transfected with control siRNA (siControl) or siRNA directed against Tbx18 (siTbx18) or Wt1 (siWt1). Scale bar: 200 µm. (B) Immunostaining for ZO-1 or N-cadherin (green) and DAPI nuclear staining (blue) of primary epicardial cells transfected with siRNAs. Scale bars: 100 µm. (C) Percentage of cells categorized as “Enlarged” or “Cobblestone-like,” based on the cellular morphology of primary epicardial cells transfected with siRNAs. (D) Representative images of primary epicardial cells transfected with siRNAs at 0 and 14 hr after the scratch was made. Scale bar: 200 µm. (E) Quantification of migration distance, given as a ratio to the siControl (n = 4; *P<0.01 vs. siControl). (F) The relative mRNA expression of Tbx18, Wt1, Snail and Slug by real-time PCR analysis (n = 3; *P<0.05 vs. siControl). The results were normalized to Gapdh expression, and the relative expression level is given as a ratio to the siControl. (G) Western blot performed with antibodies against Tbx18, Wt1, adhesion molecules (E-cadherin and N-cadherin) and EMT regulators (Snail and Slug). β-actin and histone H3 were used as loading controls. The data are presented as the mean ± SD.
Mentions: Tbx18 and Wt1 are transcription factors that are expressed in the embryonic epicardium. To investigate the functions of Tbx18 and Wt1 in the epicardial EMT, we performed knockdown experiments of Tbx18 and Wt1 in primary embryonic epicardial cells. Cells treated with control siRNA (siControl) maintained a cobblestone-like cell shape, although a slight morphological change was observed at the edge of the colony after several days in culture (Figure 2A). ZO-1, an epicardial and epithelial adhesion molecule, was localized at the cell-cell junction, and N-cadherin, a mesenchymal adhesion molecule, was not observed in the siControl cells (Figure 2B). Knockdown of Wt1 (siWt1) induced significant morphological changes, including impaired cell-cell contacts and a larger cell size (Figure 2A). In Wt1-knockdown cells, ZO-1 expression was decreased; however, N-cadherin was increased at the cell-cell interfaces, suggesting that the Wt1 knockdown induced the mesenchymal transition (Figure 2B). Knockdown of Tbx18 (siTbx18) did not affect cell-cell interactions or ZO-1 localization at the cell-cell junctions, and all cells, including those at the edge of the colony, maintained a more compact cell shape when compared to control cells (Figure 2A and 2B). Furthermore, knockdown of Wt1 but not Tbx18 significantly increased the proportion of enlarged cells (Figure 2C and Figure S1A).

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