Abstract
Abstract 3308
Gas6 is the ligand for the TAM family of receptors, which are composed of three members namely Tyro3, Axl and Mer. These receptors belong to the large family of type I transmembrane receptor tyrosine kinases. Our laboratory has identified important intracellular signaling pathways important in gas6-Axl mediated protection of endothelial cells from apoptosis. However, as both gas6 and Axl null mice are protected from lethal thromboembolism, we sought to explore novel gas6-Axl intracellular signaling pathways regulating thrombosis. Caveolae have been shown to play a crucial role in the activation of signaling cascades following ligand binding to receptor tyrosine kinases. Caveolae are formed from lipid rafts by polymerization of caveolins. Caveolin-1 is the most abundant protein found in caveolae. Caveolin-1-enriched microdomains are well known to play a role as a docking platform for receptor tyrosine kinases and intracellular adaptor signaling proteins. Axl association with these highly specialized domains of the plasma membrane has not been previously elucidated. In the present study, we investigated the role of caveolin-1-enriched microdomains in gas6/Axl signaling in endothelial cells. First, we demonstrated that gas6-induced Akt and Erk1/2 phosphorylation required the presence of a functional Axl receptor as shown by Axl siRNA knockdown experiments in human umbilical vein endothelial cells. Then, caveolin-1 fractions, enriched by a detergent-free cell lysis followed by sucrose gradient ultra centrifugation, were studied by western blot analysis. After 5 and 10 min of gas6 treatment, Axl colocalized with caveolin-1 suggesting Axl recruitment into caveolin-1-enriched cell fractions. We found that c-Src, a signaling molecule known to behave as a transient docking platform in lipid rafts, also moved in caveolin-1-enriched cell fractions after gas6 stimulation. Caveolin-1 siRNA abolished gas6-induced Akt, Erk1/2 and c-Src phosphorylation suggesting that caveolin-1 enriched fractions are required for gas6-Axl signaling. Interestingly, we have shown that gas6-induced Akt phosphorylation required c-Src activation using c-Src siRNA and the pharmacological inhibitor (PP2). However, gas6-induced Erk1/2 phosphorylation was independent of c-Src. Finally, we found that gas6 increased tissue factor expression through the Axl-c-Src-Akt signaling cascade. Taken together, our results demonstrate that caveolin-1-enriched domains are required for gas6-Axl signaling and lead to the upregulation of tissue factor expression by gas6 in endothelial cells. These results highlight new insights of gas6-Axl signaling and function in endothelial cells.
Disclosures:
No relevant conflicts of interest to declare.
Convallatoxin, the primary cardiac glycoside in lily of the valley (
Convallaria majalis
), induces tissue factor expression in endothelial cells
