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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 are bound to the Slug promoter region and regulate the activity of the Slug promoter.(A) Immunoblot performed with an anti-Tbx18 antibody and an anti-Wt1 antibody. Tbx18 was immunoprecipitated with an anti-FLAG antibody (FLAG) or control IgG (IgG) in NMuMG-C7 cells expressing 3×FLAG-tagged Tbx18, and Wt1 was immunoprecipitated with an anti-Wt1 antibody (Wt1) or control IgG (IgG) in NMuMG-C7 cells expressing Wt1. (B) Direct binding of Tbx18 or Wt1 near the transcription start site (TSS) of the Slug gene in NMuMG-C7 cells, as assessed by ChIP. DNA fragments co-precipitated with Tbx18 or Wt1 were quantified by real-time PCR. The data are presented as the mean ± SD; n = 3; *P<0.05 vs. control IgG. (C) The relative luciferase activity of a reporter construct carrying the Slug promoter in primary epicardial cells. The data are provided as ratios to siControl and are presented as the mean ± SD; n = 4; *P<0.01 vs. siControl. (D) The relative luciferase activity of a reporter construct carrying the Slug promoter (Full) or the Slug promoter lacking the region around −200 from the TSS (−200 del) or +350 from the TSS (+350 del) in primary epicardial cells. The data are provided as ratios to Full and are presented as the mean ± SD; n = 4; *P<0.05 vs. Full.
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pone-0057829-g006: Tbx18 and Wt1 are bound to the Slug promoter region and regulate the activity of the Slug promoter.(A) Immunoblot performed with an anti-Tbx18 antibody and an anti-Wt1 antibody. Tbx18 was immunoprecipitated with an anti-FLAG antibody (FLAG) or control IgG (IgG) in NMuMG-C7 cells expressing 3×FLAG-tagged Tbx18, and Wt1 was immunoprecipitated with an anti-Wt1 antibody (Wt1) or control IgG (IgG) in NMuMG-C7 cells expressing Wt1. (B) Direct binding of Tbx18 or Wt1 near the transcription start site (TSS) of the Slug gene in NMuMG-C7 cells, as assessed by ChIP. DNA fragments co-precipitated with Tbx18 or Wt1 were quantified by real-time PCR. The data are presented as the mean ± SD; n = 3; *P<0.05 vs. control IgG. (C) The relative luciferase activity of a reporter construct carrying the Slug promoter in primary epicardial cells. The data are provided as ratios to siControl and are presented as the mean ± SD; n = 4; *P<0.01 vs. siControl. (D) The relative luciferase activity of a reporter construct carrying the Slug promoter (Full) or the Slug promoter lacking the region around −200 from the TSS (−200 del) or +350 from the TSS (+350 del) in primary epicardial cells. The data are provided as ratios to Full and are presented as the mean ± SD; n = 4; *P<0.05 vs. Full.

Mentions: According to our experiments with primary epicardial cells and NMuMG-C7 cells, Slug is a potential downstream target of Tbx18 and Wt1. Thus, we investigated the direct binding of these transcription factors to the Slug promoter region in NMuMG-C7 cells by ChIP. In NMuMG-C7 cells expressing 3×FLAG-tagged Tbx18, we observed Tbx18 protein bound to the first intron of the Slug gene (Figure 6A and 6B). In addition, in NMuMG-C7 cells expressing Wt1, we detected Wt1 bound near the transcription start site (TSS) (Figure 6A and 6B).


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 are bound to the Slug promoter region and regulate the activity of the Slug promoter.(A) Immunoblot performed with an anti-Tbx18 antibody and an anti-Wt1 antibody. Tbx18 was immunoprecipitated with an anti-FLAG antibody (FLAG) or control IgG (IgG) in NMuMG-C7 cells expressing 3×FLAG-tagged Tbx18, and Wt1 was immunoprecipitated with an anti-Wt1 antibody (Wt1) or control IgG (IgG) in NMuMG-C7 cells expressing Wt1. (B) Direct binding of Tbx18 or Wt1 near the transcription start site (TSS) of the Slug gene in NMuMG-C7 cells, as assessed by ChIP. DNA fragments co-precipitated with Tbx18 or Wt1 were quantified by real-time PCR. The data are presented as the mean ± SD; n = 3; *P<0.05 vs. control IgG. (C) The relative luciferase activity of a reporter construct carrying the Slug promoter in primary epicardial cells. The data are provided as ratios to siControl and are presented as the mean ± SD; n = 4; *P<0.01 vs. siControl. (D) The relative luciferase activity of a reporter construct carrying the Slug promoter (Full) or the Slug promoter lacking the region around −200 from the TSS (−200 del) or +350 from the TSS (+350 del) in primary epicardial cells. The data are provided as ratios to Full and are presented as the mean ± SD; n = 4; *P<0.05 vs. Full.
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pone-0057829-g006: Tbx18 and Wt1 are bound to the Slug promoter region and regulate the activity of the Slug promoter.(A) Immunoblot performed with an anti-Tbx18 antibody and an anti-Wt1 antibody. Tbx18 was immunoprecipitated with an anti-FLAG antibody (FLAG) or control IgG (IgG) in NMuMG-C7 cells expressing 3×FLAG-tagged Tbx18, and Wt1 was immunoprecipitated with an anti-Wt1 antibody (Wt1) or control IgG (IgG) in NMuMG-C7 cells expressing Wt1. (B) Direct binding of Tbx18 or Wt1 near the transcription start site (TSS) of the Slug gene in NMuMG-C7 cells, as assessed by ChIP. DNA fragments co-precipitated with Tbx18 or Wt1 were quantified by real-time PCR. The data are presented as the mean ± SD; n = 3; *P<0.05 vs. control IgG. (C) The relative luciferase activity of a reporter construct carrying the Slug promoter in primary epicardial cells. The data are provided as ratios to siControl and are presented as the mean ± SD; n = 4; *P<0.01 vs. siControl. (D) The relative luciferase activity of a reporter construct carrying the Slug promoter (Full) or the Slug promoter lacking the region around −200 from the TSS (−200 del) or +350 from the TSS (+350 del) in primary epicardial cells. The data are provided as ratios to Full and are presented as the mean ± SD; n = 4; *P<0.05 vs. Full.
Mentions: According to our experiments with primary epicardial cells and NMuMG-C7 cells, Slug is a potential downstream target of Tbx18 and Wt1. Thus, we investigated the direct binding of these transcription factors to the Slug promoter region in NMuMG-C7 cells by ChIP. In NMuMG-C7 cells expressing 3×FLAG-tagged Tbx18, we observed Tbx18 protein bound to the first intron of the Slug gene (Figure 6A and 6B). In addition, in NMuMG-C7 cells expressing Wt1, we detected Wt1 bound near the transcription start site (TSS) (Figure 6A and 6B).

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