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

• Published in: Journal of thrombosis and haemostasis 2015-12, Vol.13 (12), p.2260-2272
• Main Authors: Bertin, F. R., Lemarié, C. A., Robins, R. S., Blostein, M. D.
• Format: Article
• Language: English
• Subjects: Animals; Bone Marrow Cells - metabolism; Cell Adhesion; Cell adhesion & migration; Cells, Cultured; endothelial cells; Endothelial Cells - drug effects; Endothelial Cells - metabolism; Forkhead Box Protein O1; Forkhead Transcription Factors - genetics; Forkhead Transcription Factors - metabolism; FoxO1 protein, mouse; Gene Expression Regulation; Genotype; growth arrest‐specific protein 6; Intercellular Signaling Peptides and Proteins - deficiency; Intercellular Signaling Peptides and Proteins - genetics; Intercellular Signaling Peptides and Proteins - metabolism; Male; Mice, Inbred C57BL; Mice, Knockout; Mutation; Phenotype; Phosphorylation; Protein Kinase Inhibitors - pharmacology; Proto-Oncogene Proteins c-akt - antagonists & inhibitors; Proto-Oncogene Proteins c-akt - metabolism; RNA Interference; Signal Transduction; Thrombin - pharmacology; thromboembolism; Time Factors; Transfection; Vascular Cell Adhesion Molecule-1 - genetics; Vascular Cell Adhesion Molecule-1 - metabolism; VCAM‐1; Venous Thromboembolism - genetics; Venous Thromboembolism - metabolism
• ISSN: 1538-7933; 1538-7836; 1538-7836
• DOI: 10.1111/jth.13156

Summary 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.