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J. Biol. Chem., Vol. 275, Issue 47, 37187-37193, November 24, 2000
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From the Department of Experimental Pathology, Holland Laboratory,
American Red Cross, Rockville, Maryland 20855
Injury of endothelial cells induced by reactive
oxygen species plays an important role in the development of early
stages of vascular diseases such as hypertension and atherosclerosis. Exposure of human umbilical vein endothelial cells to hydrogen peroxide
(H2O2), a common form of reaction oxygen
species, triggers a series of intracellular events, including actin
cytoskeletal reorganization, cytoplasm shrinkage, membrane blebbing and
protein-tyrosine phosphorylation. The effect of
H2O2 on endothelial cells is dramatically enhanced when a survival pathway involving extracellular
signal-regulated kinase is blocked by PD098059. In contrast, the injury
of endothelial cells mediated by H2O2 is
inhibited by PP2, a selective specific inhibitor for protein-tyrosine
kinase Src. Cortactin, a filamentous actin (F-actin)-associated
protein, becomes phosphorylated at tyrosine residues upon stimulation
by H2O2 in a manner dependent on the activity
of Src. The level of tyrosine phosphorylation of cortactin is
correlated with the formation of membrane blebs. Overexpression of
wild-type cortactin tagged with green fluorescent protein in
endothelial cells via a retroviral vector substantiates the
H2O2-induced morphological changes, whereas
overexpression of a green fluorescent protein-cortactin mutant
deficient in tyrosine phosphorylation renders endothelial cells
resistant to H2O2. The functional role of
cortactin in H2O2-mediated shape changes was also evaluated in NIH 3T3 cells. Stable 3T3 transfectants expressing wild-type cortactin in the presence of either
H2O2/PD098059 or H2O2
alone at 200 µM exhibited a dramatic shape change
characterized by rounding up or aggregation. However, the similar
changes were not detected with cells overexpressing a cortactin mutant
deficient in tyrosine phosphorylation. These data demonstrate an
important role of the Src/cortactin-dependent actin
reorganization in the injury of endothelial cells mediated by reactive
oxygen species.
Tyrosine Phosphorylation of Cortactin Is Required for
H2O2-mediated Injury of Human Endothelial
Cells*
*
This work was supported in part by National Institutes of
Health Grant R01 HL52753-07, Department of Defense Grant
DAMD17-98-1-8278, and American Heart Association Established
Investigator Grant 0040135N (to X. Z.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Experimental
Pathology, Holland Laboratory, American Red Cross, 15601 Crabbs Branch
Way, Rockville, MD 20855. Tel.: 301-738-0568; Fax: 301-738-0879;
E-mail: zhanx@usa.redcross.org.
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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