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Differential requirement for c-Jun NH₂-terminal kinase in TNF- and Fas-mediated apoptosis in hepatocytes ¹

ROBERT F. SCHWABE^*,,2, HIROSHI UCHINAMI^*,, TING QIAN, BRYDON L. BENNETT, JOHN J. LEMASTERS and DAVID A. BRENNER^*,

^* Departments of Medicine,
Biochemistry and Biophysics, and
Cell and Developmental Biology, University of North Carolina, Chapel Hill, North Carolina, USA; and
Signal Research Division, Celgene Corporation, San Diego, California, USA

²Correspondence: E-mail: rfs2102{at}columbia.edu

SPECIFIC AIMS

JNK is strongly activated in TNF-mediated apoptosis in hepatocytes, but its effects on cell death are not known. This study analyzes how inhibition of JNK activity affects death receptor-mediated apoptosis and at what level JNK acts in death receptor-induced apoptosis in primary hepatocytes.

PRINCIPAL FINDINGS

1. SP600125 inhibits JNK in hepatocytes
SP600125 (20 µM) inhibited the phosphorylation of the JNK target c-Jun in TNF-treated primary rat hepatocytes. SP600125 only slightly diminished Akt activity and did not inhibit Erk phosphorylation or IB degradation.

2. TNF-induced JNK activation is prolonged in hepatocytes expressing an IB superrepressor
Prolonged JNK activation has been suggested to be essential to mediate proapoptotic effects of TNF in cells with inactivated NF-B pathways. Hepatocytes expressing IBsr displayed a sustained strong JNK activation lasting for 60 min after TNF whereas control hepatocytes only showed an activation up to the 15 min.

3. SP600125 inhibits TNF-induced apoptosis
Inhibition of JNK did not sensitize hepatocytes to TNF-mediated apoptosis, since hepatocytes which were incubated with SP600125 (20 µM) showed no signs of apoptosis after TNF whereas inhibition of NF-B by IBsr induced massive apoptosis after TNF. When IBsr-expressing hepatocytes were preincubated with SP600125 (20 µM), apoptosis was significantly inhibited between 8 h and 14 h after TNF (Fig. 1 A, B). Cell death was reduced by 60% (P<0.05) and caspase 3-like activity was reduced by of 67% (P<0.05) in SP600125 treated hepatocytes after 11 h (Fig. 1A, C ). DNA laddering (Fig. 1D ) and Annexin V staining (Fig. 1E ) were virtually absent in SP600125 treated hepatocytes after TNF. To exclude the possibility that SP600125 prevented cell death independent of its effect on JNK activity, SP600125 was tested in TRAF2dn expressing hepatocytes which show no JNK activation after TNF and should therefore not be protected by SP600125. As expected, SP600125 did not further reduce apoptosis in TRAF2dn-expressing hepatocytes.

View larger version (81K): [in this window] [in a new window]   Figure 1. SP600125 inhibits TNF-mediated apoptosis in AdIBsr sensitized hepatocytes. AdIBsr infected hepatocytes were pretreated with SP600125 (20 µM) or DMSO (0.1%) for 2 h followed by various lengths of TNF (30 ng/mL) treatment.A) Viability was determined by trypan blue exclusion as described in materials and methods. Shown is the mean of 3 independent experiments performed in duplicate. B) Caspase 3-like activity was measured by incubating cell extracts with DEVD-AFC and is expressed as AFC release (pmol) per µg protein extract after 2 h of incubation. Shown is one representative experiment performed in duplicate. C) Hepatocytes were infected with the indicated adenoviruses at an moi of 30 for 2 h. After pretreatment with SP600125 (20 µM) or DMSO for 2 h, hepatocytes were treated with TNF (30 ng/mL) for 8 h. Caspase 3-like activity was measured by AFC release assay is expressed as AFC release (pmol) per µg protein extract after 2 h of incubation. Each condition represents at least two independent experiments performed in duplicate. ANOVA analysis represents conditions that were performed at least three times. D) Hepatocytes were infected with AdIBsr at a moi of 30 for 2 h and pretreated with SP600125 (20 µM) or DMSO for 2 h. DNA was isolated after 8 h of TNF treatment and visualized on a 1.8% agarose gel. E) AdIBsr-infected hepatocytes were pretreated with SP600125 (20 µM) or DMSO for 2 h followed by treatment with TNF (30 ng/mL) for 8 h and stained with Annexin V and propidium iodide. Annexin V (green fluorescence) and propidium iodide (red fluorescence) are shown in the upper panel, the phase contrast image is shown in the lower panel.

4. SP600125 protects hepatocytes from TNFR1 mediated apoptosis
Mouse hepatocytes were treated with human TNF to selective stimulate TNFR1. Human TNF induced JNK activation in mouse hepatocytes and this activation was blocked by SP600125. SP600125 blocked human TNF-induced apoptosis in IBsr-expressing mouse hepatocytes. However, mouse TNF was more efficient in inducing apoptosis than human TNFa, indicating that the concurrent activation of TNFR1 and TNFR2 enhances cell death. This finding may be explained, in part, by the slighly more prolonged activation of JNK found in IBsr expressing mouse hepatocytes after mouse TNF.

5. Fas-induced apoptosis of hepatocytes is independent of JNK activation
Activation of Fas by the agonistic antibody Jo2 treatment failed to activate JNK. Treatment with CD95L overexpressing 3T3 cells (3T3CD95L) activated JNK, but to a much lesser degree than TNF, indicating that extensive receptor aggregation may be required. 3T3CD95L-induced c-Jun phosphorylation was decreased by SP600125 (20 µM). SP600125 did not affect cell death, caspase 3-like activity or DNA laddering after Jo2 plus actinomycin D (ActD) or after coculture with 3T3CD95L.

6. JNK exerts proapoptotic effects independently of transcription and c-Jun
Next we investigated whether the effects of SP600125 depended on transcription. In ActD-treated hepatocytes, SP600125 reduced TNF-induced cell death by 42% (P<0.05), caspase 3-like activity by 59% (P<0.05) and prevented DNA laddering. The mutated c-Jun TAM67 did not prevent apoptosis in IBsr-expressing hepatocytes and TAM67-expressing hepatocytes were rescued from TNF by SP600125, indicating the protective effects of SP600125 do not require c-Jun phosphorylation.

7. SP600125 inhibits the mitochondrial permeability transition, cytochrome c release and Bid cleavage after TNF
Mitochondrial permeability transition (MPT) regulates cytochrome c release and executioner caspase activation and is crucial for TNF-induced apoptosis in hepatocytes. Confocal microscopy of hepatocytes loaded with TMRM and calcein revealed the onset of the MPT, defined as loss of mitochondrial TMRM and disappearance of calcein voids 5 h after TNF in vehicle treated hepatocytes (Fig. 2 A). In SP600125 treated hepatocytes, the MPT was delayed as seen by the retention of TMRM and most of calcein voids even after 7 h (Fig. 2A ). S100 fractions showed a strong increase in cytosolic cytochrome c after 7 h of TNF in vehicle treated hepatocytes (Fig. 2B ) which was blunted in SP600125 treated hepatocytes. Bid degradation started 6 h after TNF (Fig. 2C ) in vehicle-treated hepatocytes, whereas high levels of full-length Bid were still present at 12 h in SP600125-treated hepatocytes.

View larger version (57K): [in this window] [in a new window]   Figure 2. JNK mediates apoptosis upstream of the mitochondria. IBsr expressing hepatocytes were pretreated with SP600125 (20 µM) or DMSO for 2h. A) Mitochondria were loaded with TMRM to monitor mitochondrial polarity and calcein to monitor mitochondrial permeability. Following TNF treatment (30 ng/mL), calcein and TMRM fluorescence were monitored for 7 h. B) For the detection of cytochrome c release, S-100 fraction were prepared from SP600125 (20 µM) or DMSO pretreated hepatocytes after 7 h of TNF and analyzed for cytochrome c release. C) Bid degradation was determined by Western blot analysis after various lengths of TNF treatment in SP600125 (20 µM) or DMSO-pretreated hepatocytes.

8. JNK overexpression induces apoptosis through a mitochondrial pathway
Overexpression of JNK1 activated the JNK pathway as apparent by an increase in c-Jun phosphorylation. JNK1-overexpressing hepatocytes displayed a moderate increase in cell death (10% vs. 4% in the GFP control), increased caspase 3-like activity and DNA laddering. Caspase 3-like activity and DNA laddering were prevented by the MPT inhibitors cyclosporine A and trifluoperazine indicating that JNK acts upstream of the MPT.

CONCLUSIONS AND SIGNIFICANCE

The functions of JNK in apoptosis remain controversial. Although recent studies have correlated JNK activity with apoptotic cell death in many cell types, JNK may also mediate anti-apoptotic signals depending on cell type, stimulus, and the concurrent activation of other signaling pathways. Our study proposes that JNK acts as a proapoptotic signaling molecule in TNF-induced apoptosis of primary hepatocytes as seen 1) by the protective effects of SP600125 and 2) the ability of JNK overexpression to induce apoptosis. JNK has been shown to mediate apoptosis through a transcriptionally independent pathway in UV-mediated apoptosis. In our study, the proapoptotic effect of JNK in TNF-mediated apoptosis did not depend on transcription, since SP600125 protected ActD treated hepatocytes to a similar degree as IBsr expressing hepatocytes. In TNF-mediated apoptosis in hepatocytes, JNK appears to have targets besides c-Jun since hepatocytes transduced with the mutated c-Jun TAM67 were not protected from TNF. In contrast to TNF-induced apoptosis, SP600125 did not affect Fas-mediated apoptosis induced by either the agonistic antibody Jo2 in combination with ActD or coculture with 3T3CD95L. These results are consistent with studies in other cell types showing that JNK activation and ASK1 are not required for Fas-mediated apoptosis. Despite its potent effects during the first 14h of TNF treatment, SP600125 did not completely rescue hepatocytes from apoptosis. It appears that JNK acts in concert with other TNF-induced proapoptotic factors to enhance TNF-mediated apoptosis. Consistent with this hypothesis is the finding that isolated JNK activation in JNK1 overexpressing hepatocytes induced apoptosis in a much smaller percentage of hepatocytes than TNF. SP600125 delayed the occurrence of the MPT, decreased mitochondrial cytochrome c release and prevented Bid degradation in TNF-treated hepatocytes indicating that JNK interacts with factors upstream of the mitochondria. This hypothesis is further confirmed by the fact that overexpression of JNK1 induced an activation of the mitochondrial death pathway in hepatocytes. JNK is able to phosphorylate Bcl-2 family members which are important regulators of the mitochondrial death pathway. Consistent with previous studies, we did not detect Bcl-2 expression in hepatocytes, nor did we observe Bcl-xl phosphorylation after TNF. It is likely that JNK has other targets in TNF-mediated hepatocyte apoptosis such as the Bax family members Bim and Bmf which recently have been shown to be phosphorylated by JNK.

TNF is a mediator of hepatocellular damage in reperfusion injury and alcoholic liver injury. Based on our results, small molecule JNK inhibitors may represent a novel pharmacological option for the treatment of reperfusion injury, alcoholic hepatitis and other types of TNF-mediated liver injury. Data from our laboratory indeed show a strong protective effect of small molecule JNK inhibitors in reperfusion injury of the liver with significant improvement in mortality. Further studies have to define the relative role of JNK1 and JNK2 in TNF-induced apoptosis in hepatocytes and targets of JNK.

View larger version (70K): [in this window] [in a new window]   Figure 3. Schematic diagram.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0771fje; doi: 10.1096/fj.03-0771fje

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