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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 TGFβ1 is inhibited by knockdown of Tbx18 or Slug.(A) Representative images of epicardial cells treated with TGFβ1 and transfected with control siRNA (siControl) or siRNA directed against Tbx18 (siTbx18), Slug (siSlug) or Wt1 (siWt1). (B) Immunostaining for ZO-1 (green) and DAPI nuclear staining (blue) of primary epicardial cells transfected with siRNAs. (C) Percentage of cells categorized as “Enlarged” or “Cobblestone-like,” based on cellular morphology. (D) The relative mRNA expression of Tbx18, Wt1, Snail and Slug by real-time PCR analysis in TGFβ1-treated epicardial cells transfected with siControl, siTbx18, siSlug or siWt1 (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 TGFβ1+siControl. (E) Western blot performed with antibodies against Tbx18, Wt1, Snail and Slug. β-actin and histone H3 were used as loading controls. The data are presented as the mean ± SD. Scale bars: 100 µm.
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pone-0057829-g003: The epicardial EMT induced by TGFβ1 is inhibited by knockdown of Tbx18 or Slug.(A) Representative images of epicardial cells treated with TGFβ1 and transfected with control siRNA (siControl) or siRNA directed against Tbx18 (siTbx18), Slug (siSlug) or Wt1 (siWt1). (B) Immunostaining for ZO-1 (green) and DAPI nuclear staining (blue) of primary epicardial cells transfected with siRNAs. (C) Percentage of cells categorized as “Enlarged” or “Cobblestone-like,” based on cellular morphology. (D) The relative mRNA expression of Tbx18, Wt1, Snail and Slug by real-time PCR analysis in TGFβ1-treated epicardial cells transfected with siControl, siTbx18, siSlug or siWt1 (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 TGFβ1+siControl. (E) Western blot performed with antibodies against Tbx18, Wt1, Snail and Slug. β-actin and histone H3 were used as loading controls. The data are presented as the mean ± SD. Scale bars: 100 µm.

Mentions: We then performed knockdown experiments of Tbx18, Slug or Wt1 during the TGFβ1-induced epicardial EMT. TGFβ1 successfully induced the epicardial EMT in siControl cells under this experimental condition, as evidenced by cellular morphology and ZO-1 expression (Figure S6). Knockdown of Tbx18 or Slug (siSlug) attenuated the TGFβ1-induced morphological changes (Figure 3A). Slug knockdown restored ZO-1 localization at the cell-cell junction; however, the effect of Tbx18 knockdown was weaker than that of Slug knockdown, suggesting that Tbx18 only partially contributes to ZO-1 expression (Figure 3B). By contrast, Wt1 knockdown increased morphological changes, as evidenced by the increased number of cells with a spread shape and impaired cell-cell contacts (Figure 3A, 3B and 3C, and Figure S1B).


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 TGFβ1 is inhibited by knockdown of Tbx18 or Slug.(A) Representative images of epicardial cells treated with TGFβ1 and transfected with control siRNA (siControl) or siRNA directed against Tbx18 (siTbx18), Slug (siSlug) or Wt1 (siWt1). (B) Immunostaining for ZO-1 (green) and DAPI nuclear staining (blue) of primary epicardial cells transfected with siRNAs. (C) Percentage of cells categorized as “Enlarged” or “Cobblestone-like,” based on cellular morphology. (D) The relative mRNA expression of Tbx18, Wt1, Snail and Slug by real-time PCR analysis in TGFβ1-treated epicardial cells transfected with siControl, siTbx18, siSlug or siWt1 (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 TGFβ1+siControl. (E) Western blot performed with antibodies against Tbx18, Wt1, Snail and Slug. β-actin and histone H3 were used as loading controls. The data are presented as the mean ± SD. Scale bars: 100 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585213&req=5

pone-0057829-g003: The epicardial EMT induced by TGFβ1 is inhibited by knockdown of Tbx18 or Slug.(A) Representative images of epicardial cells treated with TGFβ1 and transfected with control siRNA (siControl) or siRNA directed against Tbx18 (siTbx18), Slug (siSlug) or Wt1 (siWt1). (B) Immunostaining for ZO-1 (green) and DAPI nuclear staining (blue) of primary epicardial cells transfected with siRNAs. (C) Percentage of cells categorized as “Enlarged” or “Cobblestone-like,” based on cellular morphology. (D) The relative mRNA expression of Tbx18, Wt1, Snail and Slug by real-time PCR analysis in TGFβ1-treated epicardial cells transfected with siControl, siTbx18, siSlug or siWt1 (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 TGFβ1+siControl. (E) Western blot performed with antibodies against Tbx18, Wt1, Snail and Slug. β-actin and histone H3 were used as loading controls. The data are presented as the mean ± SD. Scale bars: 100 µm.
Mentions: We then performed knockdown experiments of Tbx18, Slug or Wt1 during the TGFβ1-induced epicardial EMT. TGFβ1 successfully induced the epicardial EMT in siControl cells under this experimental condition, as evidenced by cellular morphology and ZO-1 expression (Figure S6). Knockdown of Tbx18 or Slug (siSlug) attenuated the TGFβ1-induced morphological changes (Figure 3A). Slug knockdown restored ZO-1 localization at the cell-cell junction; however, the effect of Tbx18 knockdown was weaker than that of Slug knockdown, suggesting that Tbx18 only partially contributes to ZO-1 expression (Figure 3B). By contrast, Wt1 knockdown increased morphological changes, as evidenced by the increased number of cells with a spread shape and impaired cell-cell contacts (Figure 3A, 3B and 3C, and Figure S1B).

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