Hyperosmotic stress induces axl activation and cleavage in cerebral endothelial cells

Due to the relative impermeability of the blood-brain barrier many drugs are unable to reach the CNS in therapeutically relevant concentration. One method to deliver drugs to the CNS is the osmotic opening of the blood-brain barrier using mannitol. Hyperosmotic mannitol induces a strong phosphorylation on tyrosine residues in a broad spectrum of proteins in cerebral endothelial cells, the principal components of the blood-brain barrier. Previously we have shown that among targets of tyrosine phosphorylation are ?-catenin, extracellular signal-regulated kinase 1/2 and the non-receptor tyrosine kinase Src. The aim of this study was to identify new signaling pathways activated by hypertonicity in cerebral endothelial cells. Using an antibody array and immunoprecipitation we identified the receptor tyrosine kinase Axl to become tyrosine phosphorylated in response to hyperosmotic mannitol. Besides activation, Axl was also cleaved in response to osmotic stress. Degradation of Axl proved to be metalloproteinase- and proteasome-dependent and resulted in 50-55 kDa C-terminal products which remained phosphorylated even after degradation. Specific knockdown of Axl increased the rate of apoptosis in hyperosmotic mannitol-treated cells, therefore we assume that activation of Axl may be a protective mechanism against hypertonicity-induced apoptosis. Our results identify Axl as an important element of osmotic stress-induced signaling

GAS6 Enhances Repair Following Cuprizone-Induced Demyelination

Growth arrest-specific protein 6 (gas6) activities are mediated through the Tyro3, Axl, and Mer family of receptor tyrosine kinases. Gas6 is expressed and secreted by a wide variety of cell types, including cells of the central nervous system (CNS). In this study, we tested the hypothesis that administration of recombinant human Gas6 (rhGas6) protein into the CNS improves recovery following cuprizone withdrawal. After a 4-week cuprizone diet, cuprizone was removed and PBS or rhGas6 (400 ng/ml, 4 µg/ml and 40 µg/ml) was delivered by osmotic mini-pump into the corpus callosum of C57Bl6 mice for 14 days. Nine of 11 (82%) PBS-treated mice had abundant lipid-associated debris in the corpus callosum by Oil-Red-O staining while only 4 of 19 (21%) mice treated with rhGas6 had low Oil-Red-O positive droplets. In rhGas6-treated mice, SMI32-positive axonal spheroids and APP-positive deposits were reduced in number relative to PBS-treated mice. Compared to PBS, rhGas6 enhanced remyelination as revealed by MBP immunostaining and electron microscopy. The rhGas6-treated mice had more oligodendrocytes expressing Olig1 in the cytoplasm, indicative of oligodendrocyte progenitor cell maturation. Relative to PBS-treated mice, rhGas6-treated mice had fewer activated microglia in the corpus callosum by Iba1 immunostaining. The data show that rhGas6 treatment resulted in more efficient repair following cuprizone-induced injury

Liability in Peacekeeping Missions: A Civil Cause of Action for the Mothers of Srebrenica Against the Dutch Government and the United Nations

Jasna Hasanbasic places The Mothers of Srebrenica\u27s lawsuits against the UN and The Netherlands in the context of liability and peacekeeping responsibilities. She argues that international law lacks the basic tort elements of adequate rules, rights, and remedies, and cannot provide legal redress to the women who lost husbands, brothers, and sons in the genocide at Srebrenica. Domestic law and courts are the answer. Given this assertion, will lawsuits against the UN and The Netherlands for grossly negligent peacekeeping encourage more effective planning and execution of future peacekeeping missions? Or will the UN withdraw from this international security function? Srebrenica provides a case study for potential similar legal claims, such as those that could arise from international involvement in Syria, in response to state-authorized attacks on civilians

Liability in Peacekeeping Missions: A Civil Cause of Action for the Mothers of Srebrenica Against the Dutch Government and the United Nations

Jasna Hasanbasic places The Mothers of Srebrenica\u27s lawsuits against the UN and The Netherlands in the context of liability and peacekeeping responsibilities. She argues that international law lacks the basic tort elements of adequate rules, rights, and remedies, and cannot provide legal redress to the women who lost husbands, brothers, and sons in the genocide at Srebrenica. Domestic law and courts are the answer. Given this assertion, will lawsuits against the UN and The Netherlands for grossly negligent peacekeeping encourage more effective planning and execution of future peacekeeping missions? Or will the UN withdraw from this international security function? Srebrenica provides a case study for potential similar legal claims, such as those that could arise from international involvement in Syria, in response to state-authorized attacks on civilians

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