Gas6‐induced tissue factor expression in endothelial cells is mediated through caveolin‐1–enriched microdomains

Summary 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. Objectiv...

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Published in:	Journal of thrombosis and haemostasis Vol. 12; no. 3; pp. 395 - 408
Main Authors:	Laurance, S., Aghourian, M. N., Jiva Lila, Z., Lemarié, C. A., Blostein, M. D.
Format:	Journal Article
Language:	English
Published:	England Elsevier Limited 01-03-2014
Subjects:	Axl Receptor Tyrosine Kinase Caveolin 1 - metabolism caveolin‐1 Cell Proliferation Cell Survival CSK Tyrosine-Protein Kinase c‐src kinase Endothelial Cells - cytology growth arrest–specific protein 6 Human Umbilical Vein Endothelial Cells Humans Intercellular Signaling Peptides and Proteins - metabolism Kinases Lipids Medical research Membrane Microdomains - chemistry Phosphorylation Proto-Oncogene Proteins - metabolism Proto-Oncogene Proteins c-akt - metabolism Receptor Protein-Tyrosine Kinases - metabolism Signal Transduction src-Family Kinases - metabolism Thromboplastin - metabolism Thrombosis - metabolism tissue factor caveolin-1 c-src kinase growth arrest-specific protein 6 tissue factor axl receptor tyrosine kinase
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Summary:	Summary 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.
Bibliography:	Manuscript handled by: W. Ruf Final decision: P. H. Reitsma, 1 December 2013 ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	1538-7933 1538-7836 1538-7836
DOI:	10.1111/jth.12481