Role of c-Met/Phosphatidylinositol 3-Kinase (PI3k)/Akt Signaling in Hepatocyte Growth Factor (HGF)-mediated Lamellipodia Formation, Reactive Oxygen Species (ROS) Generation, and Motility of Lung Endothelial Cells

Vijay Mohan,Viswanathan Natarajan,Peter V. Usatyuk,Kishore K. Wary,Steven M. Dudek,Yulia Epshtein,Joe G.N. Garcia,Panfeng Fu,Vytas Bindokas,Ravi Salgia,Julian Gomez-Cambronero,Jeffrey R. Jacobson

doi:10.1074/jbc.m113.527556

Abstract

Hepatocyte growth factor (HGF) mediated signaling promotes cell proliferation and migration in a variety of cell types and plays a key role in tumorigenesis. As cell migration is important to angiogenesis, we characterized HGF-mediated effects on the formation of lamellipodia, a pre-requisite for migration using human lung microvascular endothelial cells (HLMVECs). HGF, in a dose-dependent manner, induced c-Met phosphorylation (Tyr-1234/1235, Tyr-1349, Ser-985, Tyr-1003, and Tyr-1313), activation of PI3k (phospho-Yp85) and Akt (phospho-Thr-308 and phospho-Ser-473) and potentiated lamellipodia formation and HLMVEC migration. Inhibition of c-Met kinase by SU11274 significantly attenuated c-Met, PI3k, and Akt phosphorylation, suppressed lamellipodia formation and endothelial cell migration. LY294002, an inhibitor of PI3k, abolished HGF-induced PI3k (Tyr-458), and Akt (Thr-308 and Ser-473) phosphorylation and suppressed lamellipodia formation. Furthermore, HGF stimulated p47(phox)/Cortactin/Rac1 translocation to lamellipodia and ROS generation. Moreover, inhibition of c-Met/PI3k/Akt signaling axis and NADPH oxidase attenuated HGF- induced lamellipodia formation, ROS generation and cell migration. Ex vivo experiments with mouse aortic rings revealed a role for c-Met signaling in HGF-induced sprouting and lamellipodia formation. Taken together, these data provide evidence in support of a significant role for HGF-induced c-Met/PI3k/Akt signaling and NADPH oxidase activation in lamellipodia formation and motility of lung endothelial cells.

Highlights

Lamellipodia structures provide a platform for the spatio-temporal localization of key components necessary for cell migration
Our results demonstrated that Hepatocyte growth factor (HGF)-induced c-Met activation as well as phosphorylation of downstream targets such as phosphatidylinositol 3-kinase (PI3k)/Akt kinases is required for lamellipodia formation and EC motility
Despite extensive actin polymerization at the cell periphery, stress fibers remained stable. These results suggest a key role for HGF in lamellipodia formation in human lung microvascular endothelial cells (HLMVECs)

Summary

Background

Lamellipodia structures provide a platform for the spatio-temporal localization of key components necessary for cell migration. Inhibition of c-Met/PI3k/Akt signaling axis and NADPH oxidase attenuated HGF- induced lamellipodia formation, ROS generation and cell migration. Ex vivo experiments with mouse aortic rings revealed a role for c-Met signaling in HGF-induced sprouting and lamellipodia formation Taken together, these data provide evidence in support of a significant role for HGF-induced c-Met/PI3k/ Akt signaling and NADPH oxidase activation in lamellipodia formation and motility of lung endothelial cells. Vascular endothelial cells provide a selective structural barrier between circulation and surrounding tissue, and regulate vascular permeability and blood flow [1, 2] They play a major role in angiogenesis, a process required for the generation of new blood vessels and repair of damaged vasculature [3]. The HGF/c-Met signaling axis stimulated ROS formation at the lamellipodia, and blocking activation of PI3k, Akt, and NADPH oxidase components (p47phox, and Rac1) attenuated HGF-induced lamellipodia formation and cell migration

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION