The role of Gas6-Axl in vascular biology /

Gas6, the product of a growth arrest specific gene, is a member of the vitamin K-dependent family of gamma-carboxylated proteins and the ligand for the tyrosine kinase receptor Axl. Gas6-Axl interactions are important in many biological functions such as cell survival, mitogensis and regulation of thrombosis. We show that gas6 plays an important role in endothelial cell survival and that its post-translational modification (gamma-carboxylation) is necessary for its biologic activity. Using flow cytometry, we first demonstrate that gas6 can prevent apoptosis induced by serum starvation of primary cultures of human umbilical vein endothelial cells (HUVECs). This effect is mediated through activation of the phosphatidylinositol 3-kinase (PI3 kinase) and Akt pathway, known anti-apoptosic regulators. In addition, in the presence of the PI3 kinase inhibitor, wortmannin, gas6 is unable to induce Akt phosphorylation during serum-stress resulting in endothelial cell apoptosis. Similarly, dominant negative Akt constructs prevent gas6-induced endothelial cell survival, underscoring the importance of Akt activation in gas6-mediated survival.Several downstream regulators of this survival pathway were also identified in HUVECs, namely, NF-kappaB as well as the antiapoptotic and proapoptotic proteins Bcl-2 and caspase 3, respectively. Luciferase assay experiments indicate that gas6 induces a rapid increase in NF-kappaB transcriptional activity. We also show that NF-kappaB is phosphorylated early after gas6 treatment as evidenced by Western Blot analysis. As well, the level of Bcl-2 protein increased, supporting the notion that the Bcl-2 antiapoptotic pathway is stimulated, and levels of caspase 3 activation, a know proapoptotic regulator, are significantly reduced with gas6 treatment. These initial results indicate that gas6-Axl interactions are an important mediator of endothelial cell survival during serum stress through activation of classical antiapoptotic pathway, namely, Akt phosphorylation, NF-kappaB activation, increased Bcl-2 protein expression, and a reduction in caspase 3 activation.A most intriguing feature of gas6 as a mediator of cell survival is its unusual posttranslational modification, gamma-carboxylation. Thus, understanding the role of Gla domain of gas6 in gas6-Axl interaction is of fundamental importance since gamma-carboxylation may influence the anti-apoptotic property of gas6-Axl interaction. Here for the first time we show that Gla domain of gas6 is indispensable for its anti-apoptotic function. Initially, we show that carboxylated gas6 binds to phosphatidylserine-containing phospholipid membranes in an analogous manner to other gamma-carboxylated proteins whereas decarboxylated gas6 does not. Further, the gamma-carboxylation inhibitor, warfarin, abrogates gas6-mediated protection of NIH3T3 fibroblasts from serum starvation-induced apoptosis. A similar effect is observed on endothelium where only carboxylated but not decarboxylated gas6 protects endothelial cells from serum starvation-induced apoptosis. In addition, we also demonstrate that proper gamma-carboxylation of gas6 affects activation of its downstream targets, Axl and Akt. These results thus clearly indicate that this post-translational modification imparts a significant role in function of gas6.Therefore, we propose that gas6 plays important role in endothelial cell survival and that its post-translational modification, gamma-carboxylation, is crucial for this function