Growth arrest-specific 6 regulates thrombin-induced expression of vascular cell adhesion molecule-1 through forkhead box O1 in endothelial cells

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Abstract

Background:

Growth arrest-specific 6 (Gas6)-deficient mice are protected against venous thromboembolism (VTE), suggesting a role for Gas6 in this disorder. We previously demonstrated that Gas6 induces forkhead box O1 (FoxO-1) phosphorylation through the phosphoinositide 3-kinase–Akt pathway. FoxO-1 regulates the expression of vascular cell adhesion molecule-1 (VCAM-1), a molecule that has been implicated in VTE.

Objectives:

To assess the role of FoxO-1 in Gas6-dependent VCAM-1 expression.

Methods:

Thrombin was used to stimulate endothelial cells (ECs). Wild-type (WT) and Gas6–/– ECs were transfected with small interfering RNA targeting Axl or FoxO-1, a luciferase-coupled plasmid containing the FoxO-1 consensus sequence, and a phosphorylation-resistant FoxO-1 mutant, or treated with an Akt inhibitor. VCAM-1 mRNA expression was measured by real time-qPCR. VCAM-1 protein expression and FoxO-1 and Akt phosphorylation were assessed by western blot analysis. FoxO-1 localization was assessed by immunofluorescence. Adhesion of bone marrow mononuclear cells (BM-MCs) on ECs was assessed by fluorescence.

Results and conclusions:

Thrombin induces both VCAM-1 expression and FoxO-1 phosphorylation and nuclear exclusion in WT ECs only. Silencing of FoxO-1 enhances VCAM-1 expression in both WT and Gas6–/– ECs. Inhibition of Akt or FoxO-1 phosphorylation prevents VCAM-1 expression in WT ECs. These data show that Gas6 induces FoxO-1 phosphorylation, leading to derepression of VCAM-1 expression. BM-MC–EC adhesion is increased by thrombin in WT ECs. BM-MC–EC adhesion is further increased when FoxO-1 is silenced, but decreased when FoxO-1 phosphorylation is inhibited. These results demonstrate that the Gas6–FoxO-1 signaling axis plays an important role in VCAM-1 expression in the context of VTE by promoting BM-MC–EC adhesion.

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