Reactive oxygen species activate focal adhesion kinase, paxillin and p130cas tyrosine phosphorylation in endothelial cells.
Reactive oxygen species (ROS), particularly hydroxyl radical (HO*), increase neutrophil adherence to hypoxanthine-xanthine oxidase (HX-XO)-treated human umbilical vein endothelial cells (HUVEC) in culture. This adherence is inhibited by the tyrosine kinase inhibitors genistein (30 microM) and herbimycin A (0.9 microM), suggesting the involvement of tyrosine kinase. Phosphorylation of several HUVEC proteins in the range of 120-130 and 70 kDa was found to depend on the XO concentration and stimulation time. This phosphorylation was inhibited by the antioxidants dimethylthiourea (DMTU, 0.75 to 7.5 mM) and pentoxifylline (Ptx, 0.1 mM), and by the iron chelators desferrioxamine (DF, 1 mM) and hydroxybenzyl ethylene diamine (HBED, 0.5 mM), suggesting the involvement of HO*. Three tyrosine-phosphorylated proteins, focal adhesion kinase (p125FAK), paxillin (PAX) and p130cas were isolated and characterized by immunoprecipitation and western blotting. Antioxidants and iron chelators reduced their phosphorylation. HUVEC treated with ROS for 15 min showed actin stress fiber formation. Cytochalasin D (5 microM) inhibited tyrosine phosphorylation and PMN-HUVEC adherence, showing the importance of cytoskeleton integrity in these two functions. In conclusion, HO*, which is involved in increased PMN-HUVEC adhesion, also increases tyrosine phosphorylation on three major cytoskeleton proteins which seem to play a role in this adhesion.
Gozin
A
A
INSERM U479 and Department of Hematology, CHU Xavier Bichat, Paris, France.
Franzini
E
E
Andrieu
V
V
Da Costa
L
L
Rollet-Labelle
E
E
Pasquier
C
C
D002448:Cell Adhesion; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D002478:Cells, Cultured; D050719:Crk-Associated Substrate Protein; D015638:Cytochalasin D / Q000494:pharmacology*; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D003599:Cytoskeleton / Q000378:metabolism; D004730:Endothelium, Vascular / Q000378:metabolism*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D006801:Humans; D017665:Hydroxyl Radical / Q000378:metabolism; D019271:Hypoxanthine / Q000378:metabolism; D007150:Immunohistochemistry; D007502:Iron Chelating Agents / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D009504:Neutrophils / Q000378:metabolism; D051419:Paxillin; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011506:Proteins; D017382:Reactive Oxygen Species / Q000378:metabolism*; D050717:Retinoblastoma-Like Protein p130; D014969:Xanthine Oxidase / Q000378:metabolism
C493841:BCAR1 protein, human; D015815:Cell Adhesion Molecules; D050719:Crk-Associated Substrate Protein; D003598:Cytoskeletal Proteins; D004791:Enzyme Inhibitors; D007502:Iron Chelating Agents; C496061:PXN protein, human; D051419:Paxillin; D010750:Phosphoproteins; D011506:Proteins; D017382:Reactive Oxygen Species; D050717:Retinoblastoma-Like Protein p130; D019000:Phosphotyrosine; D015638:Cytochalasin D; D019271:Hypoxanthine; D017665:Hydroxyl Radical; D014969:Xanthine Oxidase; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human