CD95-tyrosine nitration inhibits hyperosmotic and CD95 ligand-induced CD95 activation in rat hepatocytes

Epidermal growth factor receptor (EGFR)-dependent CD95-tyrosine phosphorylation was recently identified as an early step in apoptosis induction via the CD95 system (Reinehr et al., FASEB J 2003). The effect of peroxynitrite (ONOO ---- ) on modulation of the hyperosmotic and CD95L-induced CD95 activation process was studied. Pretreatment of hepatocytes with ONOO ---- inhibited CD95 ligand (CD95L)- and hyperosmolarity-induced CD95 membrane trafficking and formation of the death-inducing signaling complex (DISC), but not EGFR 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 DISC formation occurred. CD95-tyrosine nitration abolished the hyperosmotic sensitization of hepatocytes towards CD95L-induced apoptosis. Also in CD95-YFP-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 antiapoptotic action of NO.


Introduction
Apoptosis plays an important role in the pathogenesis of liver injury with activation of the CD95 (also known as Fas/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] In hepatocytes, CD95 activation is a complex process, which involves rapid activation of the EGFR, its JNK-dependent association with CD95 and subsequent tyrosine phosphorylation of CD95 by the EGFR-tyrosine kinase activity. 4,6 CD95tyrosine phosphorylation then triggers CD95 membrane trafficking, DISC formation and in the case of hydrophobic bile acids and CD95L execution of apoptosis. 4,6 CD95 activation and DISC formation are also triggered by hyperosmotic cell shrinkage, however, despite this hepatocytes do not undergo spontaneous apoptosis, but are sensitized towards CD95Linduced apoptosis. 1 The critical role of CD95-tyrosine phosphorylation for apoptosis induction/sensitization is underlined by the findings that inhibitors of EGFR activation or its tyrosine kinase activity abolish CD95 activation, membrane trafficking and apoptosis. 4,6 Reinehr et al. Thereafter phosphate-saline-glucose buffer (PSG) 23 containing 100 mmol/L KH 2 PO 4 , 10 mmol/L NaCl and 5 mmol/L glucose (pH 7.4) was installed for 15 min. If indicated, ONOO − − − − was added as a bolus at the respective concentration to the PSG buffer, followed instantly by tilting of the culture dish. 23  incubation with horseradish peroxidase-coupled anti-mouse, anti-sheep or anti-rabbit IgG antibody (all diluted 1:10000) at room temperature for 2 hours respectively, the blots were washed extensively and developed using enhanced chemiluminescent detection (Amersham Pharmacia Biotech, Upsala, Sweden). Blots were exposed to Kodak X-OMAT AR-5 film (Eastman Kodak Co., Rochester, NY, USA).

CD95-membrane trafficking
For determination of membrane surface trafficking of CD95 in primary rat heaptocytes, cells were cultured for 24 hours on collagen-coated glass coverslips (∅ 30 mm) in 6-well culture plates (Falcon, Heidelberg, Germany) and subsequently exposed to hyperosmotic medium or CD95L at the given concentrations. Permeabilized and non-permeabilized cells were stained as published recently 1 using a polyclonal rabbit anti-CD95 antibody (dilution 1:500 in PBS) and a secondary anti-rabbit Cy3-conjugated antibody. Cells were visualized using an Axioskop (Zeiss, Oberkochen, Germany) and pictures were taken with a 3CCD-Camera (Intas, Göttingen, Germany). Receptor membrane trafficking was defined as the appearance of fluorescent spotting on the surface of the non-permeabilized cells compared to the non-permeabilized control cells. 1,4,6 For each condition, a blinded observer scored at least 100 cells per independent experiment from at least three different cell preparations for CD95membrane trafficking.
For determination of membrane surface trafficking of CD95-YFP in Huh7, cells were transfected as described above and plated on glass bottom dishes (Mattek, Ashland, MA, USA). Confocal pictures were taken using the LSM 510 META (Zeiss, Oberkochen, Germany). YFP was excited with 514 nm. 24 h after transfection CD95-YFP was detected in Reinehr et al. JBC -M3:11997-Rev 11 living cells. At least 100 cells from three independent experiments were counted in a humidified 5% CO 2 -atmosphere at 37°C.

Detection of apoptosis
Caspase 3 and 8 activation was determined by in vitro caspase assays from cytosol of rat hepatocytes treated with hyperosmotic medium or CD95L for the indicated time periods. The colorimetric assay (R&D Systems, Wiesbaden, Germany) and the Bio-Rad Lowry-protein assay (Bio-Rad Labs., Hercules, CA, USA) were used as published recently. 1 Data were corrected for total protein amount and are expressed as relative increase compared to the control level (normoosmotical medium without CD95L set to 1).
Terminal deoxynucleotidyl transferase-mediated X-dUTP nick-end labelling of FITCconjugated deoxyuridine triphosphate (TUNEL) technique was performed as described recently. 1 The number of apoptotic cells was determined by an independent examinator by counting the percentage of fluorescein-positive cells. At least 100 cells from three independent experiments from three different cell preparations were counted for each condition. Cells were visualised on an Axioskop (Zeiss, Oberkochen, Germany).

Statistics
Results from at least three independent experiments are expressed as means ± SEM (standard error of the mean). n refers to the number of independent experiments. Results were analyzed using the Student's t-test: p<0.05 was considered statistically significant. Reinehr   suggest that phosphorylation and nitration of CD95-tyrosine residues are apparently mutually exclusive and that Tyr-nitrated CD95 can no longer be Tyr-phosphorylated by hyperosmolarity or CD95L. Likewise, after hyperosmotically-or CD95L-induced CD95 Tyr- CD95-Tyr-nitration also abolished CD95 membrane trafficking in response to hyperosmolarity and CD95L ( Table 1). The immunolocalization of CD95 was studied in 24 h cultured, normoosmotically (305 mosmol/L) exposed rat hepatocytes. Non-permeabilized cells were used in order to detect CD95 in the plasma membrane, whereas CD95 immunostaining of permeabilized cells reflects CD95 at the cell surface and the cellular interior. As shown in Fig. 4A, no significant CD95 immunostaining was found at the cell surface in non-permeabilized hepatocytes, whereas staining in the cellular interior was intense, when permeabilized hepatocytes were used (Fig. 4B). This indicates that CD95 is located inside the cell with little or no CD95 at the cell surface under normoosmotic conditions. 1 When, however, hepatocytes were exposed to hyperosmotic medium ( 5B). In addition, CD95L (50 ng/mL) induced a significant apoptosis only in Huh7 transfected with CD95-YFP, but not in cells, which were transfected with YFP only ( Table 2).  (Table 2). These data indicate that CD95-Tyr-nitration protects against CD95L-induced apoptosis.

Peroxynitrite (ONOO − ) inhibits hyperosmolarity-induced caspase 8-and 3-activation and sensitization towards CD95L-induced apoptosis in rat hepatocytes
Hyperosmolarity (405 mosmol/L) was recently shown to activate caspases 8 and 3 and to sensitize rat hepatocytes towards CD95L-induced apoptosis. 1  induced apoptosis, as it was also shown above for Huh7 cells. As shown recently, 1 hyperosmotic preconditioning had by itself had no effect on hepatocyte apoptosis and the number of TUNEL-positive cells (1.1 ± 0.2 %) did not significantly differ from normoosmotic controls (0.9 ± 0.2 %) ( Table 3). However, a 3 h-hyperosmotic priming period sensitized the hepatocytes towards CD95L (50 ng/ml)-induced apoptosis, as reported previously. 1 Upon exposure to CD95L about 96 % of the cells were TUNEL-positive after hyperosmotic priming, compared to only 30.2 ± 1.4 % with normoosmotic priming conditions. As further shown in Table 3 − was added for 15 min after 3 h of exposure towards hyperosmolarity, no effect on the hyperosmotic sensitization towards CD95L-induced apoptosis was observed. These data suggest that CD95-Tyr-nitration abolishes the hyperosmotic sensitization towards CD95Linduced apoptosis.

LPS treatment induces CD95-Tyr-nitration in vivo
In vivo treatment of rats with LPS (4 mg / kg body weight i.p.) is known to induce iNOS 29 and results within 24 h in an increased myeloperoxidase (MPO) expression in the liver (Fig.   8B). MPO was recently suggested to mediate the production of nitrating oxidants in vivo. 30 In vivo LPS treatment also induced tyrosine nitration of distinct proteins with a molecular mass of 35-75 kDa (Fig. 8A,C), however, the pattern of Tyr-nitrated proteins in response to LPS was apparently more specific than that obtained after ONOO − − − − treatment of hepatocytes (Fig.   1A). In CD95 immunoprecipitates from livers of LPS-treated animals strong Tyr-nitration of CD95 was detectable (Fig. 8D). These findings suggest that CD95 Tyr-nitration may also occur in vivo. Reinehr  were shown to be poor substrates for tyrosine phosphorylation reactions 36,37 and evidence for an impairment of signal transduction cascades that depend on reversible tyrosine phosphorylation events by 3-nitration has been given recently. 19,20,38 In line with this and as shown in the present study, CD95-Tyr-nitration blocks CD95 signaling towards apoptosis, which may be relevant for the known antiapoptotic effect of NO. [16][17][18]

CD95-Tyr-nitration and inhibition of apoptosis
Induction of hepatocyte apoptosis by hydrophobic bile acids, CD95L or hyperosmolarity involves a complex mechanism, which leads to an oxidative stress response, a subsequent antioxidant-sensitive JNK and EGFR activation, being followed by EGFR/CD95 association                  Table 3 Priming