Background: Gas6 has been shown to interact with Axl in endothelial cells and to induce several signaling pathways involved in cell survival and proliferation. However, the interaction of Gas6/Axl with lipid raft/caveolin-1 in endothelial cells and its role in thrombosis are unknown.
Objectives: We tested whether Axl and/or caveolin-1 is involved in Gas6-induced Akt, ERK1/2, and c-Src activation leading to altered tissue factor expression in endothelial cells.
Methods: Gas6-treated endothelial cells were transfected with small interfering RNA (siRNA) for Axl, caveolin-1, c-Src, and Akt or treated with pharmacological inhibitors of c-Src and ERK1/2. Sucrose gradient centrifugation and confocal microscopy were used to study lipid raft/caveolin-1-enriched fractions. Akt, ERK1/2, p38, and c-Src activation was analyzed by Western blot analysis. Tissue factor expression was assessed by real-time quantitative polymerase chain reaction and immunofluorescence.
Results and conclusion: Gas6 induced Axl and c-Src localization into lipid raft/caveolin-1-enriched fractions. Gas6 increased the phosphorylation of Akt, ERK1/2, and c-Src but not p38. Using siRNA, we demonstrated that Axl is required for Akt, ERK1/2, and c-Src activation after Gas6 stimulation. siRNA for caveolin-1 blocked Gas6-induced phosphorylation of Akt, ERK1/2, and c-Src. c-Src downregulation inhibited Gas6-induced Akt but not ERK1/2 phosphorylation. Finally, Gas6 increased tissue factor mRNA and protein expression in endothelial cells. Tissue factor expression was blocked by siRNA for Axl, caveolin-1, or Akt as well as c-Src inhibition. These data demonstrate that the signaling pathway Gas6/Axl/caveolin-1/c-Src/Akt is required for tissue factor expression in endothelial cells, providing mechanistic insight into how Gas6 exerts its prothrombotic role in the vasculature.
Keywords: axl receptor tyrosine kinase; c-src kinase; caveolin-1; growth arrest-specific protein 6; tissue factor.
© 2013 International Society on Thrombosis and Haemostasis.