C5b-9-induced Endothelial Cell Proliferation and Migration Are Dependent on Akt Inactivation of Forkhead Transcription Factor FOXO1

Matthew Fosbrink,Florin Niculescu,Violeta Rus,Moon L Shin,Horea Rus

doi:10.1074/jbc.m602055200

Matthew Fosbrink, Florin Niculescu + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m602055200

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Abstract

Migration and proliferation of aortic endothelial cells (AEC) are critical processes involved in angiogenesis, atherosclerosis, and postangioplasty restenosis. Activation of complement and assembly of the C5b-9 complement complex have been implicated in the pre-lesional stage of atherogenesis and progression of the atherosclerotic lesion. We have shown that C5b-9 induces proliferation and activates phosphatidylinositol 3-kinase (PI3K), but it is unknown whether this can lead to activation of Akt in AEC, a major downstream target of PI3K, or if C5b-9 can induce the migration of AEC, a critical step in angiogenesis. In this study, we show that C5b-9 induces AEC proliferation and migration and also activates the PI3K/Akt pathway. C5b-9 activates Akt as shown by in vitro kinase assay and phosphorylation of Ser-473. C5b-9-induced cell cycle activation was inhibited by pretreatment with LY294002 (PI3K inhibitor), SH-5 (Akt inhibitor), or transfection with Akt siRNA. These data suggests that the PI3K/Akt pathway is required for C5b-9-induced cell cycle activation. FOXO1, a member of forkhead transcription factor family, was phosphorylated at Ser-256 and inactivated after C5b-9 stimulation as shown by a decrease in DNA binding and cytoplasmic relocalization. Cytoplasmic relocalization was significantly reduced after pretreatment with LY294002, SH-5, or transfection with Akt siRNA. Silencing FOXO1 expression using siRNA stimulated AEC proliferation and regulated angiogenic factor release. Our data indicate that C5b-9 regulation of the cell cycle activation in AEC through Akt pathway is dependent on inactivation of FOXO1.

Highlights

Endothelial cell activation and proliferation play a prominent role in angiogenesis, atherogenesis, postangioplasty restenosis, and other inflammatory vascular diseases [1,2,3]
We show that phosphatidylinositol 3-kinase (PI3K)/Akt is required for endothelial cell cycle activation by C5b-9, and this leads to the inactivation of FOXO1
We report for the first time that C5b-9-induced cell cycle activation in aortic endothelial cells (AEC) is mediated through Akt-induced inactivation of FOXO1

Summary

Introduction

Endothelial cell activation and proliferation play a prominent role in angiogenesis, atherogenesis, postangioplasty restenosis, and other inflammatory vascular diseases [1,2,3]. Akt itself phosphorylates a multitude of substrates involved in cell cycle activation and cell survival functions (18 –21) One such target is the FOXO transcription factor family. FOXO members bind to the consensus DNA sequence of TTGTTTAC [27] Through their function as transcription factors, FOXO proteins regulate the expression of cell cycle regulators such as the cyclin-dependent kinase inhibitor, p27 (28 –30), and pro-apoptotic genes, such as FasL and Bim [28, 31]. For C5b-9-induced endothelial cell proliferation, we studied the requirement of the PI3K/Akt pathway and the role of FOXO1. We show that PI3K/Akt is required for endothelial cell cycle activation by C5b-9, and this leads to the inactivation of FOXO1. We show that C5b-9 induces AEC proliferation and migration through PI3K/Akt-dependent phosphorylation and inactivation of FOXO transcription factors

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