Abstract
Epidermal growth factor receptor-dependent CD95-tyrosine phosphorylation was recently identified as an early step in apoptosis induction via the CD95 system (Reinehr, R., Schliess, F., and Häussinger, D. (2003) FASEB J. 17, 731-733). The effect of peroxynitrite (ONOO(-)) on modulation of the hyperosmotic and CD95 ligand (CD95L)-induced CD95 activation process was studied. Pretreatment of hepatocytes with ONOO(-) inhibited CD95L- and hyperosmolarity-induced CD95 membrane trafficking and formation of the death-inducing signaling complex, but not epidermal growth factor receptor activation and its association with CD95. Under these conditions, however, no tyrosine phosphorylation of CD95 occurred; instead, CD95 was tyrosine-nitrated. When ONOO(-) was added after induction of CD95-tyrosine phosphorylation by CD95L or hyperosmolarity, tyrosine nitration of CD95 was largely prevented and death-inducing signaling complex formation occurred. CD95-tyrosine nitration abolished the hyperosmotic sensitization of hepatocytes toward CD95L-induced apoptosis. Additionally, in CD95-yellow fluorescent protein-transfected Huh7-hepatoma cells, ONOO(-) induced CD95 Tyr nitration and prevented CD95L-induced Tyr phosphorylation and apoptosis. Tyrosine-nitrated CD95 was also found in rat livers derived from an in vivo model of endotoxinemia. The data suggest that CD95-tyrosine nitration prevents CD95 activation by inhibiting CD95-tyrosine phosphorylation. Apparently, CD95-tyrosine phosphorylation and nitration are mutually exclusive. The data identify critical tyrosine residues of CD95 as another target of the anti-apoptotic action of NO.
Highlights
APO-1) system in response to hyperosmolarity, CD95 ligand, hydrophobic bile acids, or ethanol [1,2,3,4,5,6,7,8,9,10,11]
Peroxynitrite (ONOOϪ)-induced Protein-tyrosine Nitration— 24-h cultured and normoosmotically exposed rat hepatocytes were exposed for 15 min to a bolus of peroxynitrite (ONOOϪ; initial concentration 1–500 mol/liter), which has a half-life at pH 7.4 of ϳ2 s [24]
ONOOϪ was added for 15 min after the experimental period, i.e. after a 3-h incubation period with hyperosmolarity or CD95 ligand (CD95L), CD95Tyr nitration was strongly blunted (Fig. 1B), an overall formation of Tyr-nitrated proteins was still observed
Summary
Materials—The materials used were purchased as follows: collagenases from Roche Molecular Biochemicals (Mannheim, Germany); William’s E medium and taurolithocholate-3-sulfate from Sigma (Deisenhofen, Germany); penicillin/streptomycin from Biochrom (Berlin, Germany); fetal calf serum from Invitrogen (Gaithersburg, MD); Dulbecco’s modified Eagle’s medium/nut.mix F-12, LipofectAMINE 2000, and pTOPO-TA vector from Invitrogen (Karlsruhe, Germany); caspase-3 and -8 assays from R&D Systems (Wiesbaden, Germany); terminal deoxynucleotidyltransferase-mediated X-dUTP nick end labeling (TUNEL) assay from Roche Diagnostics (Mannheim, Germany). PSG without ONOOϪ addition was instituted for 15 min prior to the normoosmotic, hyperosmotic, or CD95L exposure. Increasing doses of ONOOϪ induced increasing amounts of total protein nitration as indicated by dot blot technique. ONOOϪ was instituted for 15 min either before or after the experimental periods, i.e. exposure to normoosmolarity (305 mosmol/liter), hyperosmolarity (405 mosmol/liter), or CD95L for the time periods indicated. For this purpose, medium was removed from the respective culture dishes and cells were washed twice with phosphate-buffered saline.
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