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Endothelial Snail Regulates Capillary Branching Morphogenesis via Vascular Endothelial Growth Factor Receptor 3 Expression.

Park JA, Kim DY, Kim YM, Lee IK, Kwon YG - PLoS Genet. (2015)

Bottom Line: Results from in vitro functional studies demonstrate that Snail expression colocalized with VEGFR3 and upregulated VEGFR3 mRNA by directly binding to the VEGFR3 promoter via cooperating with early growth response protein-1.Snail knockdown in postnatal mice attenuated the formation of the deep capillary plexus, not only by impairing vertical sprouting vessels but also by downregulating VEGFR3 expression.Collectively, these data suggest that the Snail-VEGFR3 axis controls capillary extension, especially in vessels expressing VEGFR2 at low levels.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul, Korea.

ABSTRACT
Vascular branching morphogenesis is activated and maintained by several signaling pathways. Among them, vascular endothelial growth factor receptor 2 (VEGFR2) signaling is largely presented in arteries, and VEGFR3 signaling is in veins and capillaries. Recent reports have documented that Snail, a well-known epithelial-to-mesenchymal transition protein, is expressed in endothelial cells, where it regulates sprouting angiogenesis and embryonic vascular development. Here, we identified Snail as a regulator of VEGFR3 expression during capillary branching morphogenesis. Snail was dramatically upregulated in sprouting vessels in the developing retinal vasculature, including the leading-edged vessels and vertical sprouting vessels for capillary extension toward the deep retina. Results from in vitro functional studies demonstrate that Snail expression colocalized with VEGFR3 and upregulated VEGFR3 mRNA by directly binding to the VEGFR3 promoter via cooperating with early growth response protein-1. Snail knockdown in postnatal mice attenuated the formation of the deep capillary plexus, not only by impairing vertical sprouting vessels but also by downregulating VEGFR3 expression. Collectively, these data suggest that the Snail-VEGFR3 axis controls capillary extension, especially in vessels expressing VEGFR2 at low levels.

No MeSH data available.


Related in: MedlinePlus

Snail upregulates VEGFR3 transcripts via cooperating with Egr-1.(A) VEGFR3 promoter activity after the exposure of HRECs to immobilized FN. HRECs were transfected with the human VEGFR3 promoter_luciferase (hVEGFR3_Luc) reporter (wildR3) and then reseeded at a density of 2–2.5×104 cells/cm2 on FN-coated dishes. (B) Schematic illustration of the location of putative Snail and the Egr-1-binding site in the human VEGFR3 promoter. WildR3, human VEGFR3 promoter_luciferase (hVEGFR3_Luc) reporter; mutR3(Snail), mutation in the putative E-box. Broken line, Egr-1-binding elements; thick line, putative E-box; Luc, luciferase. (C) Western blot analysis showing the effect of Egr-1 knockdown on VEGFR3. HRECs were reseeded after transfections with siCon or siEgr-1 on FN-coated dishes. Arrow, an Egr-1 band; *, a non-specific band. (D) VEGFR3 promoter activity after the knockdown of Snail or Egr-1. HRECs were co-transfected with the indicated siRNA and the wildR3 reporter and then reseeded and cultured on FN-coated dishes for 16 h. (E) Mutant VEGFR3 promoter activity. HRECs were transfected with the indicated wildR3 and mutR3 (Snail) reporters and then reseeded on FN-coated dishes for 16 h. (F) Immunoprecipitation assay demonstrating the complex association between Snail and Egr-1. HRECs were seeded on FN-coated dishes. After 2 h, the cell lysates were immunoprecipitated (IP) with immunoglobulin G (IgG) or anti-Egr-1 antibody (α-Egr-1). (G) Chromatin immunoprecipitation analysis of the VEGFR3 promoter in HUVECs. HUVECs were transfected with flag-Snail (Snail) and immunoprecipitated using anti-Snail antibodies (α-Snail). PCR was performed to detect the VEGFR3 promoter region containing the putative E box.
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pgen.1005324.g004: Snail upregulates VEGFR3 transcripts via cooperating with Egr-1.(A) VEGFR3 promoter activity after the exposure of HRECs to immobilized FN. HRECs were transfected with the human VEGFR3 promoter_luciferase (hVEGFR3_Luc) reporter (wildR3) and then reseeded at a density of 2–2.5×104 cells/cm2 on FN-coated dishes. (B) Schematic illustration of the location of putative Snail and the Egr-1-binding site in the human VEGFR3 promoter. WildR3, human VEGFR3 promoter_luciferase (hVEGFR3_Luc) reporter; mutR3(Snail), mutation in the putative E-box. Broken line, Egr-1-binding elements; thick line, putative E-box; Luc, luciferase. (C) Western blot analysis showing the effect of Egr-1 knockdown on VEGFR3. HRECs were reseeded after transfections with siCon or siEgr-1 on FN-coated dishes. Arrow, an Egr-1 band; *, a non-specific band. (D) VEGFR3 promoter activity after the knockdown of Snail or Egr-1. HRECs were co-transfected with the indicated siRNA and the wildR3 reporter and then reseeded and cultured on FN-coated dishes for 16 h. (E) Mutant VEGFR3 promoter activity. HRECs were transfected with the indicated wildR3 and mutR3 (Snail) reporters and then reseeded on FN-coated dishes for 16 h. (F) Immunoprecipitation assay demonstrating the complex association between Snail and Egr-1. HRECs were seeded on FN-coated dishes. After 2 h, the cell lysates were immunoprecipitated (IP) with immunoglobulin G (IgG) or anti-Egr-1 antibody (α-Egr-1). (G) Chromatin immunoprecipitation analysis of the VEGFR3 promoter in HUVECs. HUVECs were transfected with flag-Snail (Snail) and immunoprecipitated using anti-Snail antibodies (α-Snail). PCR was performed to detect the VEGFR3 promoter region containing the putative E box.

Mentions: To explore whether Snail mediated VEGFR3 via the enhancement of VEGFR3 promoter activity, we employed the luciferase reporter system. Exposure of ECs to ECM components enhanced VEGFR3 promoter activity (Fig 4A). VEGFR3 promoter activity was downregulated and upregulated by Snail knockdown and ectopic Snail, respectively (Figs 4D, S4B and S4C). Because Notch activates VEGFR3 promoter activity [27], we examined whether the ECM-mediated increase in VEGFR3 was Notch dependent. Notch siRNA (siNotch) transfection slightly downregulated VEGFR3 promoter activity. A similar effect was observed with DAPT, which is an inhibitor of the γ-secretase and Notch response (S4D Fig). Therefore, the intracellular domain of Notch is unlikely to be a transcriptional regulator of VEGFR3 under the influence of ECM in our system. The Snail family is known to act as a transcriptional repressor for tight junction genes, polarity-related genes, and cell cycle regulators by directly binding to their conserved E-box element [10]. Nonetheless, many genes are also upregulated by the Snail family, which suggests that it functions as a transcriptional activator. Several reports indicated that Snail interacts and cooperates with the Egr-1/Sp1 complex to enhance the promoter activity of its target genes, and Egr-1 is implicated in several vascular disease states and fibroblast growth factor 2-mediated angiogenesis [28–30]. By screening the TRANSFAC MATRIX TABLE, we found that the promoter region of human VEGFR3 contained multiple conserved Sp1-binding sites, a nearby conserved Egr-binding element, and a putative E-box element located within approximately 200 bp upstream from the initiation of VEGFR3 mRNA (Fig 4B). Exposure of ECs to fibronectin induced Egr-1, Snail, and VEGFR3 (Figs 3A and 4C). Knockdown of Egr-1 decreased VEGFR3 protein expression and VEGFR3 promoter activity, suggesting the involvement of Egr-1 in VEGFR3 transcription (Figs 4C, 4D, and S4B).


Endothelial Snail Regulates Capillary Branching Morphogenesis via Vascular Endothelial Growth Factor Receptor 3 Expression.

Park JA, Kim DY, Kim YM, Lee IK, Kwon YG - PLoS Genet. (2015)

Snail upregulates VEGFR3 transcripts via cooperating with Egr-1.(A) VEGFR3 promoter activity after the exposure of HRECs to immobilized FN. HRECs were transfected with the human VEGFR3 promoter_luciferase (hVEGFR3_Luc) reporter (wildR3) and then reseeded at a density of 2–2.5×104 cells/cm2 on FN-coated dishes. (B) Schematic illustration of the location of putative Snail and the Egr-1-binding site in the human VEGFR3 promoter. WildR3, human VEGFR3 promoter_luciferase (hVEGFR3_Luc) reporter; mutR3(Snail), mutation in the putative E-box. Broken line, Egr-1-binding elements; thick line, putative E-box; Luc, luciferase. (C) Western blot analysis showing the effect of Egr-1 knockdown on VEGFR3. HRECs were reseeded after transfections with siCon or siEgr-1 on FN-coated dishes. Arrow, an Egr-1 band; *, a non-specific band. (D) VEGFR3 promoter activity after the knockdown of Snail or Egr-1. HRECs were co-transfected with the indicated siRNA and the wildR3 reporter and then reseeded and cultured on FN-coated dishes for 16 h. (E) Mutant VEGFR3 promoter activity. HRECs were transfected with the indicated wildR3 and mutR3 (Snail) reporters and then reseeded on FN-coated dishes for 16 h. (F) Immunoprecipitation assay demonstrating the complex association between Snail and Egr-1. HRECs were seeded on FN-coated dishes. After 2 h, the cell lysates were immunoprecipitated (IP) with immunoglobulin G (IgG) or anti-Egr-1 antibody (α-Egr-1). (G) Chromatin immunoprecipitation analysis of the VEGFR3 promoter in HUVECs. HUVECs were transfected with flag-Snail (Snail) and immunoprecipitated using anti-Snail antibodies (α-Snail). PCR was performed to detect the VEGFR3 promoter region containing the putative E box.
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pgen.1005324.g004: Snail upregulates VEGFR3 transcripts via cooperating with Egr-1.(A) VEGFR3 promoter activity after the exposure of HRECs to immobilized FN. HRECs were transfected with the human VEGFR3 promoter_luciferase (hVEGFR3_Luc) reporter (wildR3) and then reseeded at a density of 2–2.5×104 cells/cm2 on FN-coated dishes. (B) Schematic illustration of the location of putative Snail and the Egr-1-binding site in the human VEGFR3 promoter. WildR3, human VEGFR3 promoter_luciferase (hVEGFR3_Luc) reporter; mutR3(Snail), mutation in the putative E-box. Broken line, Egr-1-binding elements; thick line, putative E-box; Luc, luciferase. (C) Western blot analysis showing the effect of Egr-1 knockdown on VEGFR3. HRECs were reseeded after transfections with siCon or siEgr-1 on FN-coated dishes. Arrow, an Egr-1 band; *, a non-specific band. (D) VEGFR3 promoter activity after the knockdown of Snail or Egr-1. HRECs were co-transfected with the indicated siRNA and the wildR3 reporter and then reseeded and cultured on FN-coated dishes for 16 h. (E) Mutant VEGFR3 promoter activity. HRECs were transfected with the indicated wildR3 and mutR3 (Snail) reporters and then reseeded on FN-coated dishes for 16 h. (F) Immunoprecipitation assay demonstrating the complex association between Snail and Egr-1. HRECs were seeded on FN-coated dishes. After 2 h, the cell lysates were immunoprecipitated (IP) with immunoglobulin G (IgG) or anti-Egr-1 antibody (α-Egr-1). (G) Chromatin immunoprecipitation analysis of the VEGFR3 promoter in HUVECs. HUVECs were transfected with flag-Snail (Snail) and immunoprecipitated using anti-Snail antibodies (α-Snail). PCR was performed to detect the VEGFR3 promoter region containing the putative E box.
Mentions: To explore whether Snail mediated VEGFR3 via the enhancement of VEGFR3 promoter activity, we employed the luciferase reporter system. Exposure of ECs to ECM components enhanced VEGFR3 promoter activity (Fig 4A). VEGFR3 promoter activity was downregulated and upregulated by Snail knockdown and ectopic Snail, respectively (Figs 4D, S4B and S4C). Because Notch activates VEGFR3 promoter activity [27], we examined whether the ECM-mediated increase in VEGFR3 was Notch dependent. Notch siRNA (siNotch) transfection slightly downregulated VEGFR3 promoter activity. A similar effect was observed with DAPT, which is an inhibitor of the γ-secretase and Notch response (S4D Fig). Therefore, the intracellular domain of Notch is unlikely to be a transcriptional regulator of VEGFR3 under the influence of ECM in our system. The Snail family is known to act as a transcriptional repressor for tight junction genes, polarity-related genes, and cell cycle regulators by directly binding to their conserved E-box element [10]. Nonetheless, many genes are also upregulated by the Snail family, which suggests that it functions as a transcriptional activator. Several reports indicated that Snail interacts and cooperates with the Egr-1/Sp1 complex to enhance the promoter activity of its target genes, and Egr-1 is implicated in several vascular disease states and fibroblast growth factor 2-mediated angiogenesis [28–30]. By screening the TRANSFAC MATRIX TABLE, we found that the promoter region of human VEGFR3 contained multiple conserved Sp1-binding sites, a nearby conserved Egr-binding element, and a putative E-box element located within approximately 200 bp upstream from the initiation of VEGFR3 mRNA (Fig 4B). Exposure of ECs to fibronectin induced Egr-1, Snail, and VEGFR3 (Figs 3A and 4C). Knockdown of Egr-1 decreased VEGFR3 protein expression and VEGFR3 promoter activity, suggesting the involvement of Egr-1 in VEGFR3 transcription (Figs 4C, 4D, and S4B).

Bottom Line: Results from in vitro functional studies demonstrate that Snail expression colocalized with VEGFR3 and upregulated VEGFR3 mRNA by directly binding to the VEGFR3 promoter via cooperating with early growth response protein-1.Snail knockdown in postnatal mice attenuated the formation of the deep capillary plexus, not only by impairing vertical sprouting vessels but also by downregulating VEGFR3 expression.Collectively, these data suggest that the Snail-VEGFR3 axis controls capillary extension, especially in vessels expressing VEGFR2 at low levels.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul, Korea.

ABSTRACT
Vascular branching morphogenesis is activated and maintained by several signaling pathways. Among them, vascular endothelial growth factor receptor 2 (VEGFR2) signaling is largely presented in arteries, and VEGFR3 signaling is in veins and capillaries. Recent reports have documented that Snail, a well-known epithelial-to-mesenchymal transition protein, is expressed in endothelial cells, where it regulates sprouting angiogenesis and embryonic vascular development. Here, we identified Snail as a regulator of VEGFR3 expression during capillary branching morphogenesis. Snail was dramatically upregulated in sprouting vessels in the developing retinal vasculature, including the leading-edged vessels and vertical sprouting vessels for capillary extension toward the deep retina. Results from in vitro functional studies demonstrate that Snail expression colocalized with VEGFR3 and upregulated VEGFR3 mRNA by directly binding to the VEGFR3 promoter via cooperating with early growth response protein-1. Snail knockdown in postnatal mice attenuated the formation of the deep capillary plexus, not only by impairing vertical sprouting vessels but also by downregulating VEGFR3 expression. Collectively, these data suggest that the Snail-VEGFR3 axis controls capillary extension, especially in vessels expressing VEGFR2 at low levels.

No MeSH data available.


Related in: MedlinePlus