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VE-PTP regulates VEGFR2 activity in stalk cells to establish endothelial cell polarity and lumen formation.

Hayashi M, Majumdar A, Li X, Adler J, Sun Z, Vertuani S, Hellberg C, Mellberg S, Koch S, Dimberg A, Koh GY, Dejana E, Belting HG, Affolter M, Thurston G, Holmgren L, Vestweber D, Claesson-Welsh L - Nat Commun (2013)

Bottom Line: Vessels in ve-ptp(-/-) teratomas also show increased VEGF receptor-2 activity and loss of endothelial polarization.Moreover, the zebrafish VE-PTP orthologue ptp-rb is essential for polarization and lumen formation in intersomitic vessels.We conclude that the role of Tie2 in maintenance of vascular quiescence involves VE-PTP-dependent dephosphorylation of VEGF receptor-2, and that VEGF receptor-2 activity regulates VE-cadherin tyrosine phosphorylation, endothelial cell polarity and lumen formation.

View Article: PubMed Central - PubMed

Affiliation: Uppsala University, Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Dag Hammarskjölds v. 20, 751 85 Uppsala, Sweden.

ABSTRACT
Vascular endothelial growth factor (VEGF) guides the path of new vessel sprouts by inducing VEGF receptor-2 activity in the sprout tip. In the stalk cells of the sprout, VEGF receptor-2 activity is downregulated. Here, we show that VEGF receptor-2 in stalk cells is dephosphorylated by the endothelium-specific vascular endothelial-phosphotyrosine phosphatase (VE-PTP). VE-PTP acts on VEGF receptor-2 located in endothelial junctions indirectly, via the Angiopoietin-1 receptor Tie2. VE-PTP inactivation in mouse embryoid bodies leads to excess VEGF receptor-2 activity in stalk cells, increased tyrosine phosphorylation of VE-cadherin and loss of cell polarity and lumen formation. Vessels in ve-ptp(-/-) teratomas also show increased VEGF receptor-2 activity and loss of endothelial polarization. Moreover, the zebrafish VE-PTP orthologue ptp-rb is essential for polarization and lumen formation in intersomitic vessels. We conclude that the role of Tie2 in maintenance of vascular quiescence involves VE-PTP-dependent dephosphorylation of VEGF receptor-2, and that VEGF receptor-2 activity regulates VE-cadherin tyrosine phosphorylation, endothelial cell polarity and lumen formation.

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VE-PTP dephosphorylates VEGFR2 in a Tie2-dependent manner.HEK 293 T cells, PAE/VEGFR2 or HUVECs treated as indicated with VEGF (20 ng ml−1, 15 min) or Ang1 (200 ng ml−1, 30 min) or VEGF+Ang1 (Ang 1, 200 ng ml−1, 15 min, followed by addition of VEGF 20 ng ml−1, 15 min). Analyses were performed by immunoblotting (blot) on total cell lysates or on immunoprecipitations (IPs) as indicated. All experiments were repeated at least twice. (a) HEK 293T cells expressing FLAG-tagged Tie2 (Tie2-FLAG) and WT or D/A mutant V5-tagged VE-PTP (VE-PTP-V5), treated with Ang1. (b) PAE/VEGFR2 cells expressing WT or D/A mutant VE-PTP-V5, treated with VEGF. (c) PAE/VEGFR2 cells expressing VE-PTP-V5 and Tie2-FLAG, treated with VEGF and Ang1. (d) PAE/VEGFR2 cells expressing WT VE-PTP-V5, D/A VE-PTP-V5 and Tie2-FLAG, treated with VEGF and Ang1. (e) HUVECs transfected with control or VE-PTP siRNAs, treated with VEGF and Ang1. Quantification of pVEGFR/VEGFR2 levels (right). (f) HUVECs transfected with control or Tie2 siRNAs, treated with VEGF. (g) HUVECs transfected with control siRNA or with two different Tie1 siRNAs (#1 and #2), and treated with VEGF. Samples were run on the same gel/blot. (h) PAE/VEGFR3 cells expressing VE-PTP-V5 and Tie2-FLAG, treated with 200 ng ml−1 VEGFC, for 15 min. (i) PAE/VEGFR2 cells expressing VE-PTP-V5 and Tie2-FLAG (Tie2-F), treated with VEGF and Ang1 individually and in combination. Sequential IP was performed first with anti-FLAG antibodies followed by low pH elution and re-IP with anti-V5 antibodies (resulting in VE-PTP enriched samples) as outlined to the upper right. The supernatants (VE-PTP-depleted samples) were used for re-IP with anti-VEGFR2 antibodies. Immunoblotting was performed of total cell lysates (Sample 1; left panel), VE-PTP-V5-enriched samples (Sample 2; middle panel) and VE-PTP-depleted samples (Sample 3; right panel). For the VE-PTP enriched and depleted samples, lanes 1–5 show the results of IP with specific antibodies; control IgG was used for IP of sample 6.
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f2: VE-PTP dephosphorylates VEGFR2 in a Tie2-dependent manner.HEK 293 T cells, PAE/VEGFR2 or HUVECs treated as indicated with VEGF (20 ng ml−1, 15 min) or Ang1 (200 ng ml−1, 30 min) or VEGF+Ang1 (Ang 1, 200 ng ml−1, 15 min, followed by addition of VEGF 20 ng ml−1, 15 min). Analyses were performed by immunoblotting (blot) on total cell lysates or on immunoprecipitations (IPs) as indicated. All experiments were repeated at least twice. (a) HEK 293T cells expressing FLAG-tagged Tie2 (Tie2-FLAG) and WT or D/A mutant V5-tagged VE-PTP (VE-PTP-V5), treated with Ang1. (b) PAE/VEGFR2 cells expressing WT or D/A mutant VE-PTP-V5, treated with VEGF. (c) PAE/VEGFR2 cells expressing VE-PTP-V5 and Tie2-FLAG, treated with VEGF and Ang1. (d) PAE/VEGFR2 cells expressing WT VE-PTP-V5, D/A VE-PTP-V5 and Tie2-FLAG, treated with VEGF and Ang1. (e) HUVECs transfected with control or VE-PTP siRNAs, treated with VEGF and Ang1. Quantification of pVEGFR/VEGFR2 levels (right). (f) HUVECs transfected with control or Tie2 siRNAs, treated with VEGF. (g) HUVECs transfected with control siRNA or with two different Tie1 siRNAs (#1 and #2), and treated with VEGF. Samples were run on the same gel/blot. (h) PAE/VEGFR3 cells expressing VE-PTP-V5 and Tie2-FLAG, treated with 200 ng ml−1 VEGFC, for 15 min. (i) PAE/VEGFR2 cells expressing VE-PTP-V5 and Tie2-FLAG (Tie2-F), treated with VEGF and Ang1 individually and in combination. Sequential IP was performed first with anti-FLAG antibodies followed by low pH elution and re-IP with anti-V5 antibodies (resulting in VE-PTP enriched samples) as outlined to the upper right. The supernatants (VE-PTP-depleted samples) were used for re-IP with anti-VEGFR2 antibodies. Immunoblotting was performed of total cell lysates (Sample 1; left panel), VE-PTP-V5-enriched samples (Sample 2; middle panel) and VE-PTP-depleted samples (Sample 3; right panel). For the VE-PTP enriched and depleted samples, lanes 1–5 show the results of IP with specific antibodies; control IgG was used for IP of sample 6.

Mentions: To compare VE-PTP’s effect on VEGFR2 and Tie2, we employed a substrate-trapping, phosphatase-dead mutant of VE-PTP (D/A VE-PTP; aspartic acid 1180 in the catalytic domain exchanged for alanine). Substrate-trapping mutants bind their substrates without dephosphorylation25. Accordingly, expression of D/A VE-PTP allowed co-immunoprecipitation of pY992Tie2 with VE-PTP in Ang1-treated cells (Fig. 2a). Tie2 was co-immunoprecipiated also with WT VE-PTP, which was enzymatically active towards a standard substrate, Src optimal peptide (Supplementary Fig. S2a). The pY992Tie2 signal in response to Ang1 was weaker in cells expressing WT VE-PTP than D/A VE-PTP, indicating dephosphorylation of phosphorylated Tie2 by WT VE-PTP (Fig. 2a). Tie2 was also dephosphorylated in vitro using a purified VE-PTP catalytic domain fragment (Supplementary Fig. S2b).


VE-PTP regulates VEGFR2 activity in stalk cells to establish endothelial cell polarity and lumen formation.

Hayashi M, Majumdar A, Li X, Adler J, Sun Z, Vertuani S, Hellberg C, Mellberg S, Koch S, Dimberg A, Koh GY, Dejana E, Belting HG, Affolter M, Thurston G, Holmgren L, Vestweber D, Claesson-Welsh L - Nat Commun (2013)

VE-PTP dephosphorylates VEGFR2 in a Tie2-dependent manner.HEK 293 T cells, PAE/VEGFR2 or HUVECs treated as indicated with VEGF (20 ng ml−1, 15 min) or Ang1 (200 ng ml−1, 30 min) or VEGF+Ang1 (Ang 1, 200 ng ml−1, 15 min, followed by addition of VEGF 20 ng ml−1, 15 min). Analyses were performed by immunoblotting (blot) on total cell lysates or on immunoprecipitations (IPs) as indicated. All experiments were repeated at least twice. (a) HEK 293T cells expressing FLAG-tagged Tie2 (Tie2-FLAG) and WT or D/A mutant V5-tagged VE-PTP (VE-PTP-V5), treated with Ang1. (b) PAE/VEGFR2 cells expressing WT or D/A mutant VE-PTP-V5, treated with VEGF. (c) PAE/VEGFR2 cells expressing VE-PTP-V5 and Tie2-FLAG, treated with VEGF and Ang1. (d) PAE/VEGFR2 cells expressing WT VE-PTP-V5, D/A VE-PTP-V5 and Tie2-FLAG, treated with VEGF and Ang1. (e) HUVECs transfected with control or VE-PTP siRNAs, treated with VEGF and Ang1. Quantification of pVEGFR/VEGFR2 levels (right). (f) HUVECs transfected with control or Tie2 siRNAs, treated with VEGF. (g) HUVECs transfected with control siRNA or with two different Tie1 siRNAs (#1 and #2), and treated with VEGF. Samples were run on the same gel/blot. (h) PAE/VEGFR3 cells expressing VE-PTP-V5 and Tie2-FLAG, treated with 200 ng ml−1 VEGFC, for 15 min. (i) PAE/VEGFR2 cells expressing VE-PTP-V5 and Tie2-FLAG (Tie2-F), treated with VEGF and Ang1 individually and in combination. Sequential IP was performed first with anti-FLAG antibodies followed by low pH elution and re-IP with anti-V5 antibodies (resulting in VE-PTP enriched samples) as outlined to the upper right. The supernatants (VE-PTP-depleted samples) were used for re-IP with anti-VEGFR2 antibodies. Immunoblotting was performed of total cell lysates (Sample 1; left panel), VE-PTP-V5-enriched samples (Sample 2; middle panel) and VE-PTP-depleted samples (Sample 3; right panel). For the VE-PTP enriched and depleted samples, lanes 1–5 show the results of IP with specific antibodies; control IgG was used for IP of sample 6.
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f2: VE-PTP dephosphorylates VEGFR2 in a Tie2-dependent manner.HEK 293 T cells, PAE/VEGFR2 or HUVECs treated as indicated with VEGF (20 ng ml−1, 15 min) or Ang1 (200 ng ml−1, 30 min) or VEGF+Ang1 (Ang 1, 200 ng ml−1, 15 min, followed by addition of VEGF 20 ng ml−1, 15 min). Analyses were performed by immunoblotting (blot) on total cell lysates or on immunoprecipitations (IPs) as indicated. All experiments were repeated at least twice. (a) HEK 293T cells expressing FLAG-tagged Tie2 (Tie2-FLAG) and WT or D/A mutant V5-tagged VE-PTP (VE-PTP-V5), treated with Ang1. (b) PAE/VEGFR2 cells expressing WT or D/A mutant VE-PTP-V5, treated with VEGF. (c) PAE/VEGFR2 cells expressing VE-PTP-V5 and Tie2-FLAG, treated with VEGF and Ang1. (d) PAE/VEGFR2 cells expressing WT VE-PTP-V5, D/A VE-PTP-V5 and Tie2-FLAG, treated with VEGF and Ang1. (e) HUVECs transfected with control or VE-PTP siRNAs, treated with VEGF and Ang1. Quantification of pVEGFR/VEGFR2 levels (right). (f) HUVECs transfected with control or Tie2 siRNAs, treated with VEGF. (g) HUVECs transfected with control siRNA or with two different Tie1 siRNAs (#1 and #2), and treated with VEGF. Samples were run on the same gel/blot. (h) PAE/VEGFR3 cells expressing VE-PTP-V5 and Tie2-FLAG, treated with 200 ng ml−1 VEGFC, for 15 min. (i) PAE/VEGFR2 cells expressing VE-PTP-V5 and Tie2-FLAG (Tie2-F), treated with VEGF and Ang1 individually and in combination. Sequential IP was performed first with anti-FLAG antibodies followed by low pH elution and re-IP with anti-V5 antibodies (resulting in VE-PTP enriched samples) as outlined to the upper right. The supernatants (VE-PTP-depleted samples) were used for re-IP with anti-VEGFR2 antibodies. Immunoblotting was performed of total cell lysates (Sample 1; left panel), VE-PTP-V5-enriched samples (Sample 2; middle panel) and VE-PTP-depleted samples (Sample 3; right panel). For the VE-PTP enriched and depleted samples, lanes 1–5 show the results of IP with specific antibodies; control IgG was used for IP of sample 6.
Mentions: To compare VE-PTP’s effect on VEGFR2 and Tie2, we employed a substrate-trapping, phosphatase-dead mutant of VE-PTP (D/A VE-PTP; aspartic acid 1180 in the catalytic domain exchanged for alanine). Substrate-trapping mutants bind their substrates without dephosphorylation25. Accordingly, expression of D/A VE-PTP allowed co-immunoprecipitation of pY992Tie2 with VE-PTP in Ang1-treated cells (Fig. 2a). Tie2 was co-immunoprecipiated also with WT VE-PTP, which was enzymatically active towards a standard substrate, Src optimal peptide (Supplementary Fig. S2a). The pY992Tie2 signal in response to Ang1 was weaker in cells expressing WT VE-PTP than D/A VE-PTP, indicating dephosphorylation of phosphorylated Tie2 by WT VE-PTP (Fig. 2a). Tie2 was also dephosphorylated in vitro using a purified VE-PTP catalytic domain fragment (Supplementary Fig. S2b).

Bottom Line: Vessels in ve-ptp(-/-) teratomas also show increased VEGF receptor-2 activity and loss of endothelial polarization.Moreover, the zebrafish VE-PTP orthologue ptp-rb is essential for polarization and lumen formation in intersomitic vessels.We conclude that the role of Tie2 in maintenance of vascular quiescence involves VE-PTP-dependent dephosphorylation of VEGF receptor-2, and that VEGF receptor-2 activity regulates VE-cadherin tyrosine phosphorylation, endothelial cell polarity and lumen formation.

View Article: PubMed Central - PubMed

Affiliation: Uppsala University, Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Dag Hammarskjölds v. 20, 751 85 Uppsala, Sweden.

ABSTRACT
Vascular endothelial growth factor (VEGF) guides the path of new vessel sprouts by inducing VEGF receptor-2 activity in the sprout tip. In the stalk cells of the sprout, VEGF receptor-2 activity is downregulated. Here, we show that VEGF receptor-2 in stalk cells is dephosphorylated by the endothelium-specific vascular endothelial-phosphotyrosine phosphatase (VE-PTP). VE-PTP acts on VEGF receptor-2 located in endothelial junctions indirectly, via the Angiopoietin-1 receptor Tie2. VE-PTP inactivation in mouse embryoid bodies leads to excess VEGF receptor-2 activity in stalk cells, increased tyrosine phosphorylation of VE-cadherin and loss of cell polarity and lumen formation. Vessels in ve-ptp(-/-) teratomas also show increased VEGF receptor-2 activity and loss of endothelial polarization. Moreover, the zebrafish VE-PTP orthologue ptp-rb is essential for polarization and lumen formation in intersomitic vessels. We conclude that the role of Tie2 in maintenance of vascular quiescence involves VE-PTP-dependent dephosphorylation of VEGF receptor-2, and that VEGF receptor-2 activity regulates VE-cadherin tyrosine phosphorylation, endothelial cell polarity and lumen formation.

Show MeSH
Related in: MedlinePlus