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Slug is a direct Notch target required for initiation of cardiac cushion cellularization.

Niessen K, Fu Y, Chang L, Hoodless PA, McFadden D, Karsan A - J. Cell Biol. (2008)

Bottom Line: Slug deficiency results in impaired cellularization of the cardiac cushion at embryonic day (E)-9.5 but is compensated by increased Snail expression at E10.5, which restores cardiac cushion EMT.We further demonstrate that Slug, but not Snail, is directly up-regulated by Notch in endothelial cells and that Slug expression is required for Notch-mediated repression of the vascular endothelial cadherin promoter and for promoting migration of transformed endothelial cells.Collectively, our data suggest that combined expression of Slug and Snail is required for EMT in cardiac cushion morphogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biophysics, British Columbia Cancer Agency, Vancouver V5Z 1L3, Canada.

ABSTRACT
Snail family proteins are key regulators of epithelial-mesenchymal transition, but their role in endothelial-to-mesenchymal transition (EMT) is less well studied. We show that Slug, a Snail family member, is expressed by a subset of endothelial cells as well as mesenchymal cells of the atrioventricular canal and outflow tract during cardiac cushion morphogenesis. Slug deficiency results in impaired cellularization of the cardiac cushion at embryonic day (E)-9.5 but is compensated by increased Snail expression at E10.5, which restores cardiac cushion EMT. We further demonstrate that Slug, but not Snail, is directly up-regulated by Notch in endothelial cells and that Slug expression is required for Notch-mediated repression of the vascular endothelial cadherin promoter and for promoting migration of transformed endothelial cells. In contrast, transforming growth factor beta (TGF-beta) induces Snail but not Slug. Interestingly, activation of Notch in the context of TGF-beta stimulation results in synergistic up-regulation of Snail in endothelial cells. Collectively, our data suggest that combined expression of Slug and Snail is required for EMT in cardiac cushion morphogenesis.

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Slug represses the endothelial phenotype and directly regulates the VE-cadherin promoter. (A) Analysis of endothelial marker expression by qRT-PCR in Slug-transduced HMEC (n = 3). *, P < 0.05. (B) Immunoblots for endothelial and mesenchymal markers in empty vector–, NotchICD-, and Slug-expressing HMEC. (C) EMSA using in vitro–translated luciferase (Luc) or Slug-FLAG protein and 32P-labeled double-stranded oligonucleotides for an E-box cis element (−97) or two putative Slug E2-box motifs (−306 and −379) in the human VE-cadherin promoter. Supershift assays with anti–FLAG-M2 or IgG control antibodies and competition assays with 50× wild-type (wt) or mutant probes are also shown. (D) Promoter activity in endothelial cells cotransfected with vector or Slug plasmids and wild-type, E2-box mutant, or E-box mutant mouse VE-cadherin promoter-luciferase constructs (n = 4; each experiment performed in triplicate). *, P < 0.05. Error bars show SEM.
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fig4: Slug represses the endothelial phenotype and directly regulates the VE-cadherin promoter. (A) Analysis of endothelial marker expression by qRT-PCR in Slug-transduced HMEC (n = 3). *, P < 0.05. (B) Immunoblots for endothelial and mesenchymal markers in empty vector–, NotchICD-, and Slug-expressing HMEC. (C) EMSA using in vitro–translated luciferase (Luc) or Slug-FLAG protein and 32P-labeled double-stranded oligonucleotides for an E-box cis element (−97) or two putative Slug E2-box motifs (−306 and −379) in the human VE-cadherin promoter. Supershift assays with anti–FLAG-M2 or IgG control antibodies and competition assays with 50× wild-type (wt) or mutant probes are also shown. (D) Promoter activity in endothelial cells cotransfected with vector or Slug plasmids and wild-type, E2-box mutant, or E-box mutant mouse VE-cadherin promoter-luciferase constructs (n = 4; each experiment performed in triplicate). *, P < 0.05. Error bars show SEM.

Mentions: Given our findings demonstrating the requirement of Slug in cardiac EMT, we sought to examine the role that Slug plays in modulating the endothelial phenotype. Enforced expression of Slug repressed expression of key endothelial genes such as VE-cadherin, CD31, and Tie2 as determined by qRT-PCR, immunoblotting, and immunofluorescence (Fig. 4, A and B; and Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200710067/DC1). However, in contrast to activated Notch, Slug did not induce the mesenchymal markers smooth muscle α-actin and h1-calponin (Fig. 4 B). These findings suggest that Slug expression promotes the initial phases of EMT associated with the loss of endothelial phenotype but is not sufficient to complete the transition into a mesenchymal cell that is mediated by Notch activation.


Slug is a direct Notch target required for initiation of cardiac cushion cellularization.

Niessen K, Fu Y, Chang L, Hoodless PA, McFadden D, Karsan A - J. Cell Biol. (2008)

Slug represses the endothelial phenotype and directly regulates the VE-cadherin promoter. (A) Analysis of endothelial marker expression by qRT-PCR in Slug-transduced HMEC (n = 3). *, P < 0.05. (B) Immunoblots for endothelial and mesenchymal markers in empty vector–, NotchICD-, and Slug-expressing HMEC. (C) EMSA using in vitro–translated luciferase (Luc) or Slug-FLAG protein and 32P-labeled double-stranded oligonucleotides for an E-box cis element (−97) or two putative Slug E2-box motifs (−306 and −379) in the human VE-cadherin promoter. Supershift assays with anti–FLAG-M2 or IgG control antibodies and competition assays with 50× wild-type (wt) or mutant probes are also shown. (D) Promoter activity in endothelial cells cotransfected with vector or Slug plasmids and wild-type, E2-box mutant, or E-box mutant mouse VE-cadherin promoter-luciferase constructs (n = 4; each experiment performed in triplicate). *, P < 0.05. Error bars show SEM.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2483533&req=5

fig4: Slug represses the endothelial phenotype and directly regulates the VE-cadherin promoter. (A) Analysis of endothelial marker expression by qRT-PCR in Slug-transduced HMEC (n = 3). *, P < 0.05. (B) Immunoblots for endothelial and mesenchymal markers in empty vector–, NotchICD-, and Slug-expressing HMEC. (C) EMSA using in vitro–translated luciferase (Luc) or Slug-FLAG protein and 32P-labeled double-stranded oligonucleotides for an E-box cis element (−97) or two putative Slug E2-box motifs (−306 and −379) in the human VE-cadherin promoter. Supershift assays with anti–FLAG-M2 or IgG control antibodies and competition assays with 50× wild-type (wt) or mutant probes are also shown. (D) Promoter activity in endothelial cells cotransfected with vector or Slug plasmids and wild-type, E2-box mutant, or E-box mutant mouse VE-cadherin promoter-luciferase constructs (n = 4; each experiment performed in triplicate). *, P < 0.05. Error bars show SEM.
Mentions: Given our findings demonstrating the requirement of Slug in cardiac EMT, we sought to examine the role that Slug plays in modulating the endothelial phenotype. Enforced expression of Slug repressed expression of key endothelial genes such as VE-cadherin, CD31, and Tie2 as determined by qRT-PCR, immunoblotting, and immunofluorescence (Fig. 4, A and B; and Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200710067/DC1). However, in contrast to activated Notch, Slug did not induce the mesenchymal markers smooth muscle α-actin and h1-calponin (Fig. 4 B). These findings suggest that Slug expression promotes the initial phases of EMT associated with the loss of endothelial phenotype but is not sufficient to complete the transition into a mesenchymal cell that is mediated by Notch activation.

Bottom Line: Slug deficiency results in impaired cellularization of the cardiac cushion at embryonic day (E)-9.5 but is compensated by increased Snail expression at E10.5, which restores cardiac cushion EMT.We further demonstrate that Slug, but not Snail, is directly up-regulated by Notch in endothelial cells and that Slug expression is required for Notch-mediated repression of the vascular endothelial cadherin promoter and for promoting migration of transformed endothelial cells.Collectively, our data suggest that combined expression of Slug and Snail is required for EMT in cardiac cushion morphogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biophysics, British Columbia Cancer Agency, Vancouver V5Z 1L3, Canada.

ABSTRACT
Snail family proteins are key regulators of epithelial-mesenchymal transition, but their role in endothelial-to-mesenchymal transition (EMT) is less well studied. We show that Slug, a Snail family member, is expressed by a subset of endothelial cells as well as mesenchymal cells of the atrioventricular canal and outflow tract during cardiac cushion morphogenesis. Slug deficiency results in impaired cellularization of the cardiac cushion at embryonic day (E)-9.5 but is compensated by increased Snail expression at E10.5, which restores cardiac cushion EMT. We further demonstrate that Slug, but not Snail, is directly up-regulated by Notch in endothelial cells and that Slug expression is required for Notch-mediated repression of the vascular endothelial cadherin promoter and for promoting migration of transformed endothelial cells. In contrast, transforming growth factor beta (TGF-beta) induces Snail but not Slug. Interestingly, activation of Notch in the context of TGF-beta stimulation results in synergistic up-regulation of Snail in endothelial cells. Collectively, our data suggest that combined expression of Slug and Snail is required for EMT in cardiac cushion morphogenesis.

Show MeSH
Related in: MedlinePlus