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Hyperosmolarity and CD95L trigger CD95/EGF receptor association and tyrosine phosphorylation of CD95 as prerequisites for CD95 membrane trafficking and DISC formation ¹

Clinic for Gastroenterology, Hepatology and Infectiology, Heinrich-Heine-University Düsseldorf, Germany

²Correspondence: Universitätsklinikum Düsseldorf, Klinik für Gastroenterologie, Hepatologie und Infektiologie, Moorenstrasse 5, D-40225 Düsseldorf, Germany. E-mail: haeussin{at}uni-duesseldorf.de

SPECIFIC AIMS

The study was performed to gain insight into the molecular mechanisms underlying hyperosmotic- or CD95 ligand (CD95L) -induced trafficking of CD95 receptor (CD95) to the plasma membrane and induction of the death-inducing signaling complex (DISC) in isolated rat hepatocytes, thereby addressing the important issue of apoptosis initiation.

PRINCIPAL FINDINGS

1. Hyperosmotic exposure or addition of CD95 ligand generate oxidative stress, activation of c-Jun-amino-terminal kinase (JNK), and the epidermal growth factor receptor (EGF-R)
Hyperosmotic (405 mosmol/L) exposure of 24 h cultured rat hepatocytes induces almost instantaneously an oxidative stress response, as evidenced by DCFDA (carboxy-H2-dichlorodihydrofluorescein) fluorescence, an antioxidant- and genistein-sensitive tyrosine phosphorylation of the EGF-R within 1 min and an antioxidant-sensitive JNK activation after 5 min. Already small osmolarity increases (by 40 mosmol/L) were sufficient to trigger EGF-R-tyrosine phosphorylation. Likewise, addition of CD95L produced antioxidant-sensitive activation of JNK and the EGF-R (Fig. 1 ).

View larger version (48K): [in this window] [in a new window]   Figure 1. Activation of JNK by CD95L and inhibitor sensitivity of CD95L-induced EGF-R-tyrosine phosphorylation, EGF-R/CD95 association, CD95-tyrosine phosphorylation, and DISC formation in normo-osmotically exposed rat hepatocytes. 24 h cultured rat hepatocytes were exposed to CD95L (100 ng/mL), thereby maintaining normo-osmolarity (305 mosmol/L). When indicated, cells were preincubated with the antioxidant N-acetylcysteine (NAC, 30 mmol/L), the specific EGF-R-tyrosine kinase inhibitor AG1478 (5 µmol/L), the unspecific tyrosine kinase inhibitor genistein (100 µmol/L), the JNK inhibitor L-JNKI-1 (5 µmol/L), or the PKC inhibitor Gö6850 (10 µmol/L) for 30 min before CD95L (100 ng/mL) addition. Hyperosmotic medium (405 mosmol/L) served as a positive control. Representative blots from at least 3 independent experiments are given. A) Phospho-JNK-1/-2 ( 46/54 kDa) as a marker of JNK activation was detected by Western blot. CD95L leads within 5 min to JNK activation vs. control, which is strongly blunted in the presence of N-acetylcysteine (30 mmol/L; added 15 min before CD95L addition). B) EGF-R-tyrosine phosphorylation, EGF-R/CD95 association, CD95-tyrosine phosphorylation, and DISC formation were detected by Western blot. Total CD95 amount ( 48 kDa) from each sample served as a loading control and was detected in each experiment (not shown). 1: EGF-R-tyrosine phosphorylation (P-EGF-R), a marker for EGF-R activation, was detected by Western blot 10 min after CD95L addition or hyperosmolarity. CD95L-induced EGF-R phosphorylation is sensitive to NAC and genistein but not to AG1478, L-JNKI-1, or Gö6850. 2: EGF-R/CD95 association (EGF-R/CD95) was detected by CD95 immunoprecipitation and subsequent EGF-R Western blot 60 min after CD95L addition or hyperosmolarity. CD95L-induced EGF-R/CD95 association is sensitive to NAC and L-JNKI-1 but not to AG1478, genistein, and Gö6850. 3: CD95-tyrosine phosphorylation (CD95-P-Tyr) was detected by CD95 immunoprecipitation and subsequent phospho-tyrosine Western blot 60 min after CD95L addition or hyperosmolarity. CD95L-induced CD95-tyrosine phosphorylation was sensitive to NAC, AG1478, genistein, and L-JNKI-1, whereas Gö6850 was ineffective. 4: DISC formation (caspase 8/CD95 and FADD/CD95) was detected using CD95 immunoprecipitation and FADD and caspase 8 Western blot 3 h after CD95L addition or hyperosmolarity. CD95L-induced FADD/caspase 8 association with CD95 is sensitive to NAC, AG1478, genistein, and L-JNKI-1; Gö6850 is ineffective.

2. EGF-R associates with CD95 and triggers CD95-tyrosine phosphorylation
Coimmunoprecipitation studies revealed an association of EGF-R with CD95 within 30 min of hyperosmotic exposure and subsequent tyrosine phosphorylation of CD95 (Fig. 1) . Inhibition of JNK by a JNK inhibitory peptide or of protein kinase C (PKC) by Gö6850 had no effect on EGF-R phosphorylation, but abolished CD95/EGF-R association and prevented CD95-tyrosine phosphorylation, indicating the requirement of JNK- and PKC signals for hyperosmotic CD95/EGF-R association. In line with this, prevention of the hyperosmotic JNK signal by N-acetylcysteine (NAC) also inhibited EGF-R-CD95 association and CD95-tyrosine phosphorylation. Specific inhibition of EGF-R-tyrosine kinase activity by AG1478 prevented CD95-tyrosine phosphorylation and DISC formation but had no effect on hyperosmolarity-induced EGF-R phosphorylation and EGF-R association with CD95. Likewise, genistein, which inhibited hyperosmotic EGF-R activation but not EGF-R/CD95 association, prevented CD95-tyrosine phosphorylation. These findings suggest that EGF-R-tyrosine kinase activity is required for tyrosine phosphorylation of CD95. Similar findings were obtained upon addition of CD95L to hepatocytes (Fig. 1) , except that PKC inhibition by Gö6850 was unable to prevent CD95/EGF-R association and CD95-tyrosine phosphorylation. EGF-R activation also occurred in response to EGF, but failed to trigger EGF-R/CD95 association and CD95-tyrosine phosphorylation and activation.

3. Tyrosine-phosphorylated CD95 is targeted to the plasma membrane and undergoes DISC formation
After 1 h of hyperosmotic exposure, CD95 is targeted from the cellular interior to the plasma membrane and recruits Fas-associated death domain (FADD) and caspase 8 (DISC formation). Subfractionation studies revealed a strong enrichment of tyrosine-phosphorylated CD95 in the plasma membrane, but not in the cytosol. All maneuvers preventing CD95-tyrosine phosphorylation—i.e., JNK or PKC inhibition, NAC, genistein, and AG1478—inhibited hyperosmotic-induced CD95 membrane trafficking (Fig. 2 ) and DISC formation. Similar findings were obtained in response to CD95L (Fig. 1) , except that PKC inhibition was ineffective in preventing CD95 membrane trafficking and DISC formation.

View larger version (37K): [in this window] [in a new window]   Figure 2. Effect of hyperosmolarity and CD95L on CD95 immunolocalization in permeabilized and nonpermeabilized rat hepatocytes. 24 h cultured hepatocytes were exposed for 3 h to either normo- (305 mosmol/L, A, B), hyperosmotic medium (405 mosmol/L, C–F), or CD95L (100 ng/mL, G, H), then immunostained for CD95. Representative samples from at least 3 independent experiments are shown. A, B) Whereas nonpermeabilized normo-osmotically exposed hepatocytes (A) show virtually no CD95 immunostaining, permeabilized cells (B) show strong staining for CD95, indicating the intracellular localization of CD95 in normo-osmotically exposed hepatocytes. C–F) Hyperosmotic exposure leads to the appearance of CD95 staining in nonpermeabilized hepatocytes (C), indicating hyperosmotic CD95 membrane trafficking. This effect of hyperosmolarity is sensitive to a 30 min preincubation with the antioxidant NAC (30 mmol/L, D), the specific EGF-R-tyrosine kinase inhibitor AG1478 (5 µmol/L, E), or the unspecific tyrosine kinase inhibitor genistein (100 µmol/L, F). Inserts show phase contrast recordings, indicating that the presence of cells in case of CD95 was not detectable by surface immunocytochemistry (A, D–F). G + H) In normo-osmotic incubations, CD95L leads to a CD95 membrane staining in nonpermeabilized cells (G), which was sensitive to a 30 min preincubation with genistein (100 µmol/L, H; insert shows phase contrast recording).

CONCLUSIONS AND SIGNIFICANCE

CD95L is a potent inducer of hepatocyte apoptosis, and hyperosmotic hepatocyte shrinkage activates caspase 8 and sensitizes hepatocytes toward apoptosis. As shown in the present study, CD95 membrane trafficking and DISC formation as initial steps in programmed cell death in response to CD95L addition or hyperosmotic exposure involve a complex regulation and interplay between death and growth factor receptors. The data suggest that moderate hyperosmolarity triggers oxidative stress, resulting in EGF-R and JNK activation. This is followed by a JNK- and PKC-dependent EGF-R/CD95 association and tyrosine phosphorylation of CD95, probably through EGF-R-tyrosine kinase activity. Tyrosine phosphorylation of CD95 may provide a signal for CD95 trafficking to the plasma membrane and FADD/caspase 8 recruitment. Apparently, JNK- and PKC-induced signals are the triggers for EGF-R/CD95 association, whereas EGF-R activation is required to induce tyrosine phosphorylation of CD95 as a prerequisite for CD95 membrane targeting and DISC formation. Similar mechanisms account for the CD95L-induced CD95 activation, membrane trafficking, and DISC formation (Fig. 3 ); in contrast to hyperosmotic CD95 activation, however, PKC apparently is not involved. The hyperosmotic and CD95L-induced DISC formation shows an interesting interaction between a growth factor receptor and a death receptor in the regulation of cell function and provides novel aspects on the regulation of cell death and proliferation in general. Recent evidence suggests that death receptors can couple to both cell proliferation and cell death; recently, sequestration of CD95 by the HGF receptor Met has been proposed to act as a mechanism for cell survival by preventing activation of the death receptor. The present study adds another aspect on the interesting topic, namely, that CD95 activation requires the interaction with a growth factor receptor, i.e., EGF-R. The data further suggest an involvement of reactive oxygen species and EGF-R activation in sensing hyperosmotic stress by rat hepatocytes.

View larger version (19K): [in this window] [in a new window]   Figure 3. Schematic diagram. Activation of CD95 by hyperosmolarity and CD95L in hepatocytes. The scheme summarizes events in response to hyperosmolarity or CD95L addition. Both stimuli generate oxidative stress, which triggers EGF-R-tyrosine phosphorylation and JNK activation. Both signals may converge together with a PKC signal in order to allow association of EGF-R with CD95, which apparently is a substrate for EGF-R-tyrosine kinase activity. After CD95-tyrosine phosphorylation, CD95 membrane trafficking and DISC formation (FADD and caspase 8/CD95 association) are induced. *PKC has a modulatory role only in hyperosmotic (but not CD95L) -induced CD95 activation (see text).

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0915fje; to cite this article, use FASEB J. (February 5, 2003) 10.1096/fj.02-0915fje

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