Nerve growth factor induces endothelial cell invasion and cord formation by promoting matrix metalloproteinase-2 expression through the phosphatidylinositol 3-kinase/Akt signaling pathway and AP-2 transcription factor

Abstract

Nerve growth factor (NGF) is a well characterized neurotrophic agonist in the nervous system that triggers angiogenesis. In this study, we investigated the signaling mechanisms involved in NGF-induced angiogenesis. NGF stimulated endothelial cell invasion and cord formation on Matrigel in vitro but had marginal effect on proliferation and migration of these cells. NGF stimulated matrix metalloproteinase (MMP)-2 mRNA expression and protein secretion in human umbilical vein endothelial cells. Using synthetic and endogenous inhibitors of MMP-2 and MMP-2 small interfering RNA suppressed NGF-induced invasion and cord formation. We demonstrated that NGF-induced MMP-2 secretion, invasion, and cord formation are regulated via activation of the NGF receptor, TrkA, phosphatidylinositol 3-kinase (PI3K), and Akt using various pharmacological inhibitors. Specifically, NGF enhanced TrkA phosphorylation, PI3K activity, and Akt phosphorylation. Introduction of NGF-neutralizing antibodies, dominant-negative Akt, or wild-type PTEN effectively inhibited NGF-induced MMP-2 secretion and cord formation. Deletion and site-directed mutagenesis analysis of the MMP-2 promoter demonstrated that the AP-2-binding site is critical for NGF-induced MMP-2 promoter activity. NGF increased the DNA binding activity of AP-2, which was suppressed by inhibitors of TrkA and PI3K. Furthermore, transfection of AP-2 small interfering RNA effectively blocked NGF-induced MMP-2 secretion and cord formation. Finally, NGF promoted neovessel formation in Matrigel plugs in vivo, which was significantly inhibited by K252a and LY294002, but it failed to promote angiogenesis using MMP-2 knock-out mice. Our data collectively suggest that NGF stimulates endothelial cell invasion and cord formation by augmenting MMP-2 via the PI3K/Akt signaling pathway and AP-2 transcription factor, which may be responsible for triggering angiogenesis.