Abstract
The class II α-isoform of phosphatidylinositol 3-kinase (PI3K-C2α) plays a crucial role in angiogenesis at least in part through participating in endocytosis and, thereby, endosomal signaling of several cell surface receptors including VEGF receptor-2 and TGFβ receptor in vascular endothelial cells (ECs). The Notch signaling cascade regulates many cellular processes including cell proliferation, cell fate specification and differentiation. In the present study, we explored a role of PI3K-C2α in Delta-like 4 (Dll4)-induced Notch signaling in ECs. We found that knockdown of PI3K-C2α inhibited Dll4-induced generation of the signaling molecule Notch intracellular domain 1 (NICD1) and the expression of Notch1 target genes including HEY1, HEY2 and NOTCH3 in ECs but not in vascular smooth muscle cells. PI3K-C2α knockdown did not inhibit Dll4-induced endocytosis of cell surface Notch1. In contrast, PI3K-C2α knockdown as well as clathrin heavy chain knockdown impaired endocytosis of Notch1-cleaving protease, γ-secretase complex, with the accumulation of Notch1 at the perinuclear endolysosomes. Pharmacological blockage of γ-secretase also induced the intracellular accumulation of Notch1. Taken together, we conclude that PI3K-C2α is required for the clathrin-mediated endocytosis of γ-secretase complex, which allows for the cleavage of endocytosed Notch1 by γ-secretase complex at the endolysosomes to generate NICD1 in ECs.
Highlights
Phosphatidylinositol 3-kinases (PI3Ks) are a family of lipid kinases which phosphorylate membrane phosphoinositides at the 3′-position of their inositol rings
Defective angiogenesis that PI3K-C2α is required for ligand-induced endocytosis of vascular endothelial growth factor (VEGF) receptor-2 (VEGFR2)[5], sphingosine 1-phosphate receptor-1 (S1P1)[6] and transforming growth factor β (TGFβ) r eceptor[7] in vascular endothelial cells (ECs), and subsequent endosomal signaling including the activation of Rho, Rac and Smads
We found that Delta-like 4 (Dll4)- and Jag1-induced Notch intracellular domain 1 (NICD1) production and its target gene expression were dependent on PI3K-C2α in ECs, but not in vascular smooth muscle cells (SMCs)
Summary
Phosphatidylinositol 3-kinases (PI3Ks) are a family of lipid kinases which phosphorylate membrane phosphoinositides at the 3′-position of their inositol rings. We explored possible involvement of PI3K-C2α-mediated endocytosis in Notch signaling in ECs. We found that Dll4- and Jag1-induced NICD1 production and its target gene expression were dependent on PI3K-C2α in ECs, but not in vascular smooth muscle cells (SMCs). Knockdown of PI3K-C2α as well as clathrin heavy chain (CHC), inhibited the internalization of γ-secretase complex from the cell surface and resulted in the accumulation of Notch[1] at the endolysosomal compartment, suggesting that PI3K-C2α is involved in clathrin-dependent endocytosis of γ-secretase complex and subsequent Notch[1] cleavage by γ-secretase complex at the endolysosomal compartments. These observations show that PI3K-C2α is required for Notch signaling in ECs through the involvement in clathrin-mediated endocytosis of γ-secretase complex
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