Angiopoietin-1/Tie2 Signal Augments Basal Notch Signal Controlling Vascular Quiescence by Inducing Delta-Like 4 Expression through AKT-mediated Activation of β-Catenin

Jianghui Zhang,Shigetomo Fukuhara,Keisuke Sako,Takato Takenouchi,Hiroshi Kitani,Tsutomu Kume,Gou Young Koh,Naoki Mochizuki

doi:10.1074/jbc.m110.192641

Abstract

Angiopoietin-1 (Ang1) regulates both vascular quiescence and angiogenesis through the receptor tyrosine kinase Tie2. We and another group previously showed that Ang1 and Tie2 form distinct signaling complexes at cell-cell and cell-matrix contacts. We further demonstrated that the former up-regulates Notch ligand delta-like 4 (Dll4) only in the presence of cell-cell contacts. Because Dll4/Notch signal restricts sprouting angiogenesis and promotes vascular stabilization, we investigated the mechanism of how the Ang1/Tie2 signal induces Dll4 expression to clarify the role of the Dll4/Notch signal in Ang1/Tie2 signal-mediated vascular quiescence. Under confluent endothelial cells, the basal Notch signal was observed. Ang1, moreover, induced Dll4 expression and production of the Notch intracellular domain (NICD). Ang1 stimulated transcriptional activity of β-catenin through phosphoinositide 3-kinase (PI3K)/AKT-mediated phosphorylation of glycogen synthase kinase 3β (GSK3β). Correspondingly, the GSK3β inhibitor up-regulated Dll4, whereas depletion of β-catenin by siRNA blocked Ang1-induced Dll4 expression, indicating the indispensability of β-catenin in Ang1-mediated up-regulation of Dll4. In addition, Dll4 expression by the GSK3β inhibitor was only observed in confluent cells, and was impeded by DAPT, a γ-secretase inhibitor, implying requirement of the Notch signal in β-catenin-dependent Dll4 expression. Consistently, we found that either Ang1 or NICD up-regulates Dll4 through the RBP-J binding site within intron 3 of the DLL4 gene and that β-catenin forms a complex with NICD/RBP-J to enhance Dll4 expression. Ang1 induced the deposition of extracellular matrix that is preferable for basement membrane formation through Dll4/Notch signaling. Collectively, the Ang1/Tie2 signal potentiates basal Notch signal controlling vascular quiescence by up-regulating Dll4 through AKT-mediated activation of β-catenin.

Highlights

Ang1 or Notch intracellular domain (NICD) up-regulates Dll4 through the RBP-J binding site within intron 3 of the DLL4 gene and that ␤-catenin forms a complex with NICD/RBP-J to enhance Dll4 expression
Because Dll4/Notch signal restricts sprouting angiogenesis and promotes vascular stabilization, we investigated the mechanism of how the Ang1/Tie2 signal induces Dll4 expression to clarify the role of the Dll4/Notch signal in Ang1/Tie2 signal-mediated vascular quiescence
We found that Ang1 induces activation of ␤-catenin through phosphoinositide 3-kinase (PI3K)/AKT pathway-mediated inhibition of glycogen synthase kinase 3␤ (GSK3␤) and that the stabilized ␤-catenin subsequently enhances Notch signal-induced Dll4 expression by forming a complex with NICD/RBP-J on the Dll4 intron3 enhancer, thereby potentiating the Dll4/ Notch signal leading to vascular stabilization

Summary

The abbreviations used are

Angiopoietin-1; Dll, delta-like 4; VEGF, vascular endothelial growth factor; NICD, Notch intracellular domain; GSK3␤, glycogen synthase kinase 3␤; Ang, angiopoietin-2; COMP, cartilage oligomeric matrix protein; DAPT, N-(N-(3,5-difluorophenacetyl)-L-alanyl)-S-phenylglycine t-butyl ester; HUVECs, human umbilical vein endothelial cells; CA-␤Cat, constitutively active mutant of ␤-catenin; CA-AKT, constitutively active mutant of AKT. VEGF up-regulates Dll expression in endothelial tip cells, which in turn leads to Notch activation in adjacent stalk cells. Tie is activated in the endothelium of quiescent adult vasculature, and is believed to be involved in the maintenance of vascular quiescence (6, 32) Both Ang1/Tie and Dll4/ Notch signaling promote recruitment of mural cells to the vessel wall and induce deposition of basement membrane proteins around the vessels, both of which are important for vascular stabilization (26, 33–37). We found that Ang induces activation of ␤-catenin through PI3K/AKT pathway-mediated inhibition of glycogen synthase kinase 3␤ (GSK3␤) and that the stabilized ␤-catenin subsequently enhances Notch signal-induced Dll expression by forming a complex with NICD/RBP-J on the Dll intron enhancer, thereby potentiating the Dll4/ Notch signal leading to vascular stabilization

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION