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.
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.
Park
Myung-Jin
MJ
Laboratory of Functional Genomics, Korea Institute of Radiological and Medical Sciences, 215-4 Gongneung-dong, Nowon-gu, Seoul 139-706, Korea.
Kwak
Hee-Jin
HJ
Lee
Hyung-Chahn
HC
Yoo
Doo-Hyun
DH
Park
In-Chul
IC
Kim
Mi-Suk
MS
Lee
Seung-Hoon
SH
Rhee
Chang Hun
CH
Hong
Seok-Il
SI
D000818:Animals; D002465:Cell Movement / Q000187:drug effects* / Q000502:physiology; D002478:Cells, Cultured; D002867:Chromones / Q000494:pharmacology; D042783:Endothelial Cells / Q000201:enzymology*; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D017353:Gene Deletion; D006801:Humans; D020778:Matrix Metalloproteinase 2 / Q000235:genetics / Q000378:metabolism*; D061965:Matrix Metalloproteinase Inhibitors; D051379:Mice; D018345:Mice, Knockout; D009025:Morpholines / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D018919:Neovascularization, Physiologic / Q000187:drug effects / Q000502:physiology; D020932:Nerve Growth Factor / Q000494:pharmacology*; D009570:Nitriles / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011759:Pyrrolidines / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000494:pharmacology; D020917:Receptor, trkA / Q000037:antagonists & inhibitors / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D050656:Transcription Factor AP-2 / Q000378:metabolism*; D014471:Umbilical Veins / Q000166:cytology / Q000201:enzymology*
D002867:Chromones; D004791:Enzyme Inhibitors; D020778:Matrix Metalloproteinase 2; D061965:Matrix Metalloproteinase Inhibitors; D009025:Morpholines; D020932:Nerve Growth Factor; D009570:Nitriles; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D051057:Proto-Oncogene Proteins c-akt; D011759:Pyrrolidines; D034741:RNA, Small Interfering; D020917:Receptor, trkA; D050656:Transcription Factor AP-2; C494668:(S)-1-(4-methyl-1-(2-pyrimidinyl)-4-piperidylamino)acetyl-2-pyrrolidinecarbonitrile; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C522361:MMP2 protein, human; C522362:Mmp2 protein, mouse