title: Hyperosmotic stress induces Axl activation and cleavage in cerebral endothelial cells.
abstract: Because of the relative impermeability of the blood-brain barrier (BBB), 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 BBB 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 BBB. Previously, we have shown that among targets of tyrosine phosphorylation are beta-catenin, extracellular signal-regulated kinase 1/2 and the non-receptor tyrosine kinase Src. The aim of this study was to identify new signalling 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 signalling.
authors.affiliation: Institute of Biophysics, Biological Research Center, Szeged, Hungary.
authors.forename: Imola
authors.initials: I
authors.lastname: Wilhelm
authors.forename: Péter
authors.initials: P
authors.lastname: Nagyoszi
authors.forename: Attila E
authors.initials: AE
authors.lastname: Farkas
authors.forename: Pierre-Olivier
authors.initials: PO
authors.lastname: Couraud
authors.forename: Ignacio A
authors.initials: IA
authors.lastname: Romero
authors.forename: Babette
authors.initials: B
authors.lastname: Weksler
authors.forename: Csilla
authors.initials: C
authors.lastname: Fazakas
authors.forename: Ngo Thi Khue
authors.initials: NT
authors.lastname: Dung
authors.forename: Sándor
authors.initials: S
authors.lastname: Bottka
authors.forename: Hannelore
authors.initials: H
authors.lastname: Bauer
authors.forename: Hans-Christian
authors.initials: HC
authors.lastname: Bauer
authors.forename: István A
authors.initials: IA
authors.lastname: Krizbai
mesh_terms: D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D001812:Blood-Brain Barrier / Q000187:drug effects / Q000201:enzymology*; D002460:Cell Line; D002536:Cerebral Arteries / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D015536:Down-Regulation / Q000502:physiology; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D006801:Humans; D006982:Hypertonic Solutions / Q000494:pharmacology; D008353:Mannitol / Q000494:pharmacology; D045726:Metalloproteases / Q000378:metabolism; D015513:Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D009997:Osmotic Pressure / Q000187:drug effects*; D010766:Phosphorylation / Q000187:drug effects; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt / Q000187:drug effects / Q000378:metabolism; D034622:RNA Interference; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D000094963:Axl Receptor Tyrosine Kinase
chemical_list: D006982:Hypertonic Solutions; D015513:Oncogene Proteins; D011518:Proto-Oncogene Proteins; D008353:Mannitol; D020794:Receptor Protein-Tyrosine Kinases; D051057:Proto-Oncogene Proteins c-akt; D045726:Metalloproteases; D046988:Proteasome Endopeptidase Complex; D000094963:Axl Receptor Tyrosine Kinase; C000722289:AXL protein, human