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• W1980147218 abstract "Tumor necrosis factor-α receptor 1 and Fas recruit overlapping signaling pathways. To clarify the differences between tumor necrosis factor α (TNFα) and Fas pathways in hepatocyte apoptosis, primary mouse hepatocytes were treated with TNFα or an agonist anti-Fas antibody after infection with an adenovirus expressing an IκB superrepressor (Ad5IκB). Treatment with TNFα induced apoptosis in Ad5IκB-infected mouse hepatocytes, as we previously reported for rat hepatocytes. Ad5IκB plus anti-Fas antibody or actinomycin D plus anti-Fas antibody rapidly induced apoptosis, whereas anti-Fas antibody alone produced little cytotoxicity. The proteasome inhibitor (MG-132) and a dominant-negative mutant of nuclear factor-κB-inducing kinase also promoted TNFα- and Fas-mediated apoptosis. Expression of either crmA or a dominant-negative mutant of the Fas-associated death domain protein prevented TNFα- and Fas-mediated apoptosis. In addition, the caspase inhibitors, DEVD-cho and IETD-fmk, inhibited TNFα- and Fas-mediated apoptosis. In Ad5IκB-infected hepatocytes, caspases-3 and -8 were activated within 2 h after treatment with anti-Fas antibody or within 6 h after TNFα treatment. Confocal microscopy demonstrated onset of the mitochondrial permeability transition (MPT) and mitochondrial depolarization by 2–3 h after anti-Fas antibody treatment and 8–10 h after TNFα treatment, followed by cytochromec release. The combination of the MPT inhibitors, cyclosporin A, and trifluoperazine, protected Ad5IκB-infected hepatocytes from TNFα-mediated apoptosis. After anti-Fas antibody, cyclosporin A and trifluoperazine decreased cytochrome crelease but did not prevent caspase-3 activation and cell-death. In conclusion, nuclear factor-κB activation protects mouse hepatocytes against both TNFα- and Fas-mediated apoptosis. TNFα and Fas recruit similar but nonidentical, pathways signaling apoptosis. The MPT is obligatory for TNFα-induced apoptosis. In Fas-mediated apoptosis, the MPT accelerates the apoptogenic events but is not obligatory for them. Tumor necrosis factor-α receptor 1 and Fas recruit overlapping signaling pathways. To clarify the differences between tumor necrosis factor α (TNFα) and Fas pathways in hepatocyte apoptosis, primary mouse hepatocytes were treated with TNFα or an agonist anti-Fas antibody after infection with an adenovirus expressing an IκB superrepressor (Ad5IκB). Treatment with TNFα induced apoptosis in Ad5IκB-infected mouse hepatocytes, as we previously reported for rat hepatocytes. Ad5IκB plus anti-Fas antibody or actinomycin D plus anti-Fas antibody rapidly induced apoptosis, whereas anti-Fas antibody alone produced little cytotoxicity. The proteasome inhibitor (MG-132) and a dominant-negative mutant of nuclear factor-κB-inducing kinase also promoted TNFα- and Fas-mediated apoptosis. Expression of either crmA or a dominant-negative mutant of the Fas-associated death domain protein prevented TNFα- and Fas-mediated apoptosis. In addition, the caspase inhibitors, DEVD-cho and IETD-fmk, inhibited TNFα- and Fas-mediated apoptosis. In Ad5IκB-infected hepatocytes, caspases-3 and -8 were activated within 2 h after treatment with anti-Fas antibody or within 6 h after TNFα treatment. Confocal microscopy demonstrated onset of the mitochondrial permeability transition (MPT) and mitochondrial depolarization by 2–3 h after anti-Fas antibody treatment and 8–10 h after TNFα treatment, followed by cytochromec release. The combination of the MPT inhibitors, cyclosporin A, and trifluoperazine, protected Ad5IκB-infected hepatocytes from TNFα-mediated apoptosis. After anti-Fas antibody, cyclosporin A and trifluoperazine decreased cytochrome crelease but did not prevent caspase-3 activation and cell-death. In conclusion, nuclear factor-κB activation protects mouse hepatocytes against both TNFα- and Fas-mediated apoptosis. TNFα and Fas recruit similar but nonidentical, pathways signaling apoptosis. The MPT is obligatory for TNFα-induced apoptosis. In Fas-mediated apoptosis, the MPT accelerates the apoptogenic events but is not obligatory for them. tumor necrosis factor tumor necrosis factor-a receptor Fas-associated death domain protein tumor necrosis factor-α receptor-associated death domain protein tumor necrosis factor-α receptor-associated factor nuclear factor κB NF-κB-inducing kinase IκB kinase adenovirus expressing IκBα superrepressor (532A, 536A) mitochondrial permeability transition cyclosporin A hormonally defined medium hemagglutinin terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling tetramethylrhodamine methyl ester actinomycin D multiplicity of infection trifluoperazine amino-4-trifluoromethyl coumarin Apoptosis, a morphologically and biochemically distinct form of cell death, is an important physiologic process in both normal development and in pathological processes. Two death factors, Fas ligand and tumor necrosis factor-α (TNFα),1 bind to their receptors and induce apoptosis, killing the cells within hours (1.Nagata S. Cell. 1997; 88: 355-365Abstract Full Text Full Text PDF PubMed Scopus (4557) Google Scholar). Apoptosis controlled by such death receptor pairs can cause tissue destruction (2.Ogasawara J. Watanabe-Fukunaga R. Adachi M. Matsuzawa A. Kasugai T. Kitamura Y. Itoh N. Suda T. Nagata S. Nature. 1993; 364: 806-809Crossref PubMed Scopus (1814) Google Scholar). Hepatocyte apoptosis, mainly induced by death domain receptor ligands such as Fas ligand and TNFα, is implicated in several experimental and human liver diseases including viral hepatitis, alcoholic hepatitis, acute liver failure, ischemia/reperfusion injury, diseases of the bile ducts, graft-versus-host disease, and hepatocellular carcinoma (3.Galle P.R. Krammer P.H. Semin. Liver Dis. 1998; 18: 141-151Crossref PubMed Scopus (123) Google Scholar). The TNF receptor family includes Fas, the receptor for Fas ligand, and the two TNFα receptors (TNFR) (1.Nagata S. Cell. 1997; 88: 355-365Abstract Full Text Full Text PDF PubMed Scopus (4557) Google Scholar). Upon binding to Fas ligand, Fas forms a complex with the associated protein, Fas-associated death domain protein (FADD), which directly binds and activates caspase-8. Recent studies showed that FLICE-associated huge protein interacts with FADD and caspase-8 (4.Imai Y. Kimura T. Murakami A. Yajima N. Sakamaki K. Yonehara S. Nature. 1999; 398: 777-785Crossref PubMed Scopus (204) Google Scholar). FLICE-associated huge protein may control apoptosis at the level of caspase activation. TNFR1 interacts with the adaptor protein TNFR-associated death domain protein (TRADD) that recruits FADD, which again directly activates caspase-8. TNFα also induces other signaling pathways via TRADD including the protein kinase receptor interacting protein and TNF receptor-associated factor 2 (TRAF2). TNFα activates the mitogen-activated protein kinase kinase kinase, NF-κB-inducing kinase (NIK), via either protein kinase receptor interacting protein or TRAF2 (5.Malinin N.L. Boldin M.P. Kovalenko A.V. Wallach D. Nature. 1997; 385: 540-544Crossref PubMed Scopus (1164) Google Scholar, 6.Natoli G. Costanzo A. Moretti F. Fulco M. Balsano C. Levrero M. J. Biol. Chem. 1997; 272: 26079-26082Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). NIK in turn phosphorylates and activates the IκB kinase (IKK) complex (7.Woronicz J.D. Gao X. Cao Z. Rothe M. Goeddel D.V. Science. 1997; 278: 866-869Crossref PubMed Scopus (1068) Google Scholar, 8.Cohen L. Henzel W.J. Baeuerle P.A. Nature. 1998; 395: 292-296Crossref PubMed Scopus (269) Google Scholar, 9.Zhao Q. Lee F.S. J. Biol. Chem. 1999; 274: 8355-8358Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar). IKKs phosphorylate IκB, targeting it for NF-κB activation (10.DiDonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. Nature. 1997; 388: 548-554Crossref PubMed Scopus (1911) Google Scholar, 11.Mercurio F. Zhu H. Murray B.W. Shevchenko A. Bennett B.L. Li J. Young D.B. Barbosa M. Mann M. Manning A. Rao A. Science. 1997; 278: 860-866Crossref PubMed Scopus (1852) Google Scholar, 12.Zandi E. Rothwarf D.M. Delhase M. Hayakawa M. Karin M. Cell. 1997; 91: 243-252Abstract Full Text Full Text PDF PubMed Scopus (1584) Google Scholar). Recent studies indicate that NF-κB activation by TNFα protects cells from TNF cytotoxicity (13.Van Antwerp D.J. Martin S.J. Kafri T. Green D.R. Verma I.M. Science. 1996; 274: 787-789Crossref PubMed Scopus (2447) Google Scholar, 14.Liu Z.G. Hsu H. Goeddel D.V. Karin M. Cell. 1996; 87: 565-576Abstract Full Text Full Text PDF PubMed Scopus (1783) Google Scholar, 15.Wang C.Y. Mayo M.W. Baldwin Jr., A.S. Science. 1996; 274: 784-787Crossref PubMed Scopus (2511) Google Scholar). TNFα binding to the TNF receptor potentially both initiates apoptosis and activates NF-κB, which suppresses apoptosis by induction of NF-κB-responsive genes, including TRAF1, TRAF2, and the inhibitor of apoptosis proteins (16.Wang C.Y. Mayo M.W. Korneluk R.G. Goeddel D.V. Baldwin Jr., A.S. Science. 1998; 281: 1680-1683Crossref PubMed Scopus (2577) Google Scholar). The expression of the IκB superrepressor by an IκBα (S32A, S36A)-expressing adenovirus (Ad5IκB), which blocks NF-κB activation, sensitizes primary rat hepatocytes to TNFα-mediated apoptosis (17.Bradham C.A. Qian T. Streetz K. Trautwein C. Brenner D.A. Lemasters J.J. Mol. Cell. Biol. 1998; 18: 6353-6364Crossref PubMed Scopus (367) Google Scholar). Furthermore, TNFα-mediated cytotoxicity is enhanced by the addition of inhibitors of protein or RNA synthesis (cyclohexamide and actinomycin D) (18.Leist M. Gantner F. Jilg S. Wendel A. J. Immunol. 1995; 154: 1307-1316PubMed Google Scholar). Fas induces NF-κB binding activity in certain, but not all, cell types. Fas can stimulate the DNA binding activity of NF-κB in a variety of tumor cells irrespective of their sensitivity or resistance to Fas-mediated cytotoxicity (19.Ponton A. Clement M.V. Stamenkovic I. J. Biol. Chem. 1996; 271: 8991-8995Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Another report showed that the activation of NF-κB can induce target gene expression that rescues TNFα- but not Fas-mediated apoptosis in T24 cell lines (13.Van Antwerp D.J. Martin S.J. Kafri T. Green D.R. Verma I.M. Science. 1996; 274: 787-789Crossref PubMed Scopus (2447) Google Scholar). However, whether NF-κB is activated in Fas-mediated apoptosis in nontumor cells, such as hepatocytes, is not clear. Anti-Fas antibody injection into mice induces severe liver failure with apoptosis of hepatocytes (2.Ogasawara J. Watanabe-Fukunaga R. Adachi M. Matsuzawa A. Kasugai T. Kitamura Y. Itoh N. Suda T. Nagata S. Nature. 1993; 364: 806-809Crossref PubMed Scopus (1814) Google Scholar). However, anti-Fas antibody alone induces apoptosis in less than 20% of the cultured hepatocytes in vitro, whereas all cells were killed by anti-Fas antibody in the presence of actinomycin D or cycloheximide (20.Ni R. Tomita Y. Matsuda K. Ichihara A. Ishimura K. Ogasawara J. Nagata S. Exp. Cell Res. 1994; 215: 332-337Crossref PubMed Scopus (183) Google Scholar, 21.Rouquet N. Carlier K. Briand P. Wiels J. Joulin V. Biochem. Biophys. Res. Commun. 1996; 229: 27-35Crossref PubMed Scopus (44) Google Scholar). These results suggest that cultured mouse hepatocytes may express protective proteins against apoptosis. Furthermore, Fas-mediated apoptosis was delayed in hepatocytes during liver regeneration in mice (22.Takehara T. Hayashi N. Mita E. Kanto T. Tatsumi T. Sasaki Y. Kasahara A. Hori M. Hepatology. 1998; 27: 1643-1651Crossref PubMed Scopus (54) Google Scholar). This suggests that TNFα may act as one of the protective factors against Fas-mediated hepatocyte apoptosis, because initiation of liver regeneration requires TNFα. Perhaps TNFα-induced activation of NF-κB protects hepatocytes from Fas-mediated apoptosis. Mitochondria play a key role in the regulation of apoptosis (23.Green D.R. Reed J.C. Science. 1998; 281: 1309-1312Crossref PubMed Google Scholar, 24.Susin S.A. Zamzami N. Kroemer G. Biochim. Biophys. Acta. 1998; 1366: 151-165Crossref PubMed Scopus (758) Google Scholar, 25.Reed J.C. Jurgensmeier J.M. Matsuyama S. Biochim. Biophys. Acta. 1998; 1366: 127-137Crossref PubMed Scopus (351) Google Scholar, 26.Cai J. Yang J. Jones D.P. Biochim. Biophys. Acta. 1998; 1366: 139-149Crossref PubMed Scopus (637) Google Scholar). Opening of the mitochondrial permeability transition (MPT) pore, which is regulated by members of the Bcl-2 family, causes the release of soluble proteins, such as cytochrome c and apoptosis-inducing factor, from the intermembrane space. Inhibitors of MPT pore opening, including cyclosporin A (CsA), block apoptosis in some systems (27.Zamzami N. Marchetti P. Castedo M. Hirsch T. Susin S.A. Masse B. Kroemer G. FEBS Lett. 1996; 384: 53-57Crossref PubMed Scopus (388) Google Scholar, 28.Zamzami N. Susin S.A. Marchetti P. Hirsch T. Gomez-Monterrey I. Castedo M. Kroemer G. J. Exp. Med. 1996; 183: 1533-1544Crossref PubMed Scopus (1267) Google Scholar). The MPT is an essential component in the signaling pathways in TNFα-mediated cytotoxicity in the L929 line of mouse fibroblast (29.Pastorino J.G. Simbula G. Yamamoto K. Glascott Jr., P.A. Rothman R.J. Farber J.L. J. Biol. Chem. 1996; 271: 29792-29798Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar) and TNFα-induced apoptosis in rat hepatocytes (17.Bradham C.A. Qian T. Streetz K. Trautwein C. Brenner D.A. Lemasters J.J. Mol. Cell. Biol. 1998; 18: 6353-6364Crossref PubMed Scopus (367) Google Scholar). Anti-apoptotic Bcl-2 family proteins reside in mitochondria and can prevent the MPT. Bcl-xL/Bcl-2 prevented the release of cytochromec, yet other aspects of mitochondrial dysfunction still transpired and cells died (30.Gross A. Yin X.M. Wang K. Wei M.C. Jockel J. Milliman C. Erdjument-Bromage H. Tempst P. Korsmeyer S.J. J. Biol. Chem. 1999; 274: 1156-1163Abstract Full Text Full Text PDF PubMed Scopus (931) Google Scholar), suggesting that the release of cytochrome c may not be required for cell death. Some studies using nonhepatic cells demonstrate that the translocation of cytochrome c from mitochondria to cytosol does not require a mitochondrial transmembrane depolarization (31.Bossy-Wetzel E. Newmeyer D.D. Green D.R. EMBO J. 1998; 17: 37-49Crossref PubMed Scopus (1107) Google Scholar, 32.Jurgensmeier J.M. Xie Z. Deveraux Q. Ellerby L. Bredesen D. Reed J.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4997-5002Crossref PubMed Scopus (1373) Google Scholar, 33.Green D.R. Cell. 1998; 94: 695-698Abstract Full Text Full Text PDF PubMed Scopus (1102) Google Scholar), whereas others show that mitochondrial depolarization accompanies cytochromec release (34.Heiskanen K.M. Bhat M.B. Wang H.W. Ma J. Nieminen A.L. J. Biol. Chem. 1999; 274: 5654-5658Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar). Thus, mitochondrial involvement and the role of cytochrome c and MPT in apoptosis are still controversial. The purpose of this study was to elucidate the differences between Fas and TNFα pathways in hepatocyte apoptosis and the roles of NF-κB activation and MPT in Fas-mediated apoptosis. The results show that NF-κB activation has a protective role in not only TNFα- but also Fas-mediated apoptosis. Furthermore, we show that Fas agonistic antibody induces the MPT, which accelerates apoptosis, but is not essential for it. About 8-week-old C57Bl6 male mice were anesthetized with ketamine/acepromazine malate administered by intraperitoneal injection. Hepatocytes were then isolated by a retrograde, nonrecirculating in situ collagenase perfusion of livers cannulating through the inferior vena cava by a procedure modified from Moldeus et al. (35.Moldeus P. Hogberg J. Orrenius S. Methods Enzymol. 1978; 52: 60-71Crossref PubMed Scopus (1268) Google Scholar). Livers were first perfused in situ with an oxygenated 0.5 mm EGTA containing calcium-free salt solution (8 ml/min, 37 °C for 5 min), followed by perfusion with solution containing 0.04% collagenase D (Roche Molecular Biochemicals) for 10 min. The liver was then gently minced on a Petri dish and filtered with polyamide mesh (I 003 Y NITEX 3–60/45, TETKO Inc., NY). Hepatocytes were washed two times and centrifuged at 50 × g for 2 min. Cell viability was consistently >90% as determined by trypan blue exclusion. Hepatocyte cultures contained less than 1% Kupffer cells and the stellate cells as determined by fluorescein isothiocyanate-labeled latex beads (1 μm, Polysciences, Warrington, PA) and autofluorescence, respectively. 5 × 105 cells were plated on 6-well plates coated with mouse collagen type I in Waymouth's medium containing 10% fetal bovine serum, 0.1 μm insulin, and 0.1 μmdexamethazone. 1.5 × 106, 2.5 × 106, or 8 × 106 cells were plated on a 60-, 100-, or 150-mm dish, respectively. After 2 h, the culture was washed with phosphate-buffered saline and changed to hormonally defined medium (HDM) containing 0.1 μm insulin, 2 mml-glutamine, 5 μg/ml transferrin, 3 μm selenium, and 10 nm free fatty acids in RPMI basal medium. Cells were infected with recombinant adenoviruses in HDM containing 30 plaque-forming units/cell for 2 h at 37 °C and then changed to HDM containing recombinant murine TNFα (R&D Systems, Minneapolis, MN), Jo-2 (Pharmingen, San Diego, CA), or other treatments. All animals received humane care in compliance with the guidelines of the University of North Carolina. The adenovirus 5 variants Ad5IκB, Ad5LacZ, Ad5ΔFADD and Ad5crmA, expressing HA-IκBα (S32A, S36A), β-galactosidase, a truncated form of FADD, and crmA, respectively, have been described elsewhere (17.Bradham C.A. Qian T. Streetz K. Trautwein C. Brenner D.A. Lemasters J.J. Mol. Cell. Biol. 1998; 18: 6353-6364Crossref PubMed Scopus (367) Google Scholar, 36.Iimuro Y. Nishiura T. Hellerbrand C. Behrns K.E. Schoonhoven R. Grisham J.W. Brenner D.A. J. Clin. Invest. 1998; 101: 802-811Crossref PubMed Scopus (423) Google Scholar). The Ad5 vector expressing ΔNIK (Ad5Δ NIK) was constructed by cre-lox recombination as described (37.Anton M. Graham F.L. J. Virol. 1995; 69: 4600-4606Crossref PubMed Google Scholar). An insert from pCDNA-HA2101 (deletion of amino acids 1–623, a gift from Dr. G. Natoli) (6.Natoli G. Costanzo A. Moretti F. Fulco M. Balsano C. Levrero M. J. Biol. Chem. 1997; 272: 26079-26082Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar) was subcloned into the shuttle vector pAdlox using standard techniques, and the construct was confirmed by restriction digests. Expression of the ΔNIK construct was confirmed with a luciferase reporter gene assay in monkey kidney fibroblasts (COS-7, ATCC-CRL-1651, American Type Culture Collections) as described previously (36.Iimuro Y. Nishiura T. Hellerbrand C. Behrns K.E. Schoonhoven R. Grisham J.W. Brenner D.A. J. Clin. Invest. 1998; 101: 802-811Crossref PubMed Scopus (423) Google Scholar) and Western blotting using a mouse anti-HA monoclonal antibody (Babco, Berkeley, CA). Briefly, When COS cells reached subconfluence on 6-well culture plates, the cells were transfected with 3 μg of DNA and 1 μg of (κB)3-Luc, a reporter plasmid containing three copies of the NF-κB binding site (38.Westwick J.K. Bielawska A.E. Dbaibo G. Hannun Y.A. Brenner D.A. J. Biol. Chem. 1995; 270: 22689-22692Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar), using LipofectAMINE (Life Technologies, Inc.). Twenty-four h after transfection, medium was replaced with Dulbecco's modified Eagle's medium containing 10% fetal bovine serum with or without 20 ng/ml of TNFα. After a 5-h incubation, cellular extracts were prepared using enhanced luciferase assay reagents (Analytical Luminescence, San Diego, CA). Some cells were infected with Ad5ΔNIK 24 h after transfection of (κB)3-Luc, were stimulated, and were harvested as described the above. For quantitation of cell viability (presented as mean ± S.E.), cells were infected and treated as described above. After 17–20 h of TNFα or Jo-2 treatment, cell viability was determined by exclusion of trypan blue. Viable cells were counted in three different 200× power fields, and the percentage of treated viable cells to untreated viable cells was determined as a percentage of control viability. For propidium iodide nuclear staining, cells were fixed in 3:1 methanol/acetic acid, stained with 10 μg/ml popidium iodide, and viewed with an Olympus fluorescence microscope using a rhodamine filter set. Hepatocyte cell death was confirmed as apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) (Roche Molecular Biochemicals). TUNEL staining was performed according to the manufacture's instructions. Positive (apoptotic) cells were counted in three different 200× power fields. To assess DNA ladder formation, 2 × 106 cells were digested overnight at 37 °C in 0.5 mg/ml proteinase K, 0.5% sarcosyl in phosphate-buffered saline, treated with 10 μg of RNase for 1 h at 37 °C, gently extracted with phenol and chloroform, and analyzed on 2% agarose gels. Amino-4-trifluoromethyl courmarin (AFC) release assays for caspase-3 and -8 activities were performed using the FluorAce kit (Bio-Rad) according to the manufacturer's instructions. Briefly, whole cell lysates were combined with 25 μm z-DEVD-AFC or IETD-AFC (Enzyme and Systems Products, Livermore, CA) and were incubated 2 h at 37 °C. The change in fluorescence (excitation at 370 nm and emission at 490 nm) was monitored at 1-h intervals, converted to picomoles of AFC released by using a standard curve, and normalized for protein concentration. Nuclear protein extracts were prepared from primary mouse hepatocytes as described previously (17.Bradham C.A. Qian T. Streetz K. Trautwein C. Brenner D.A. Lemasters J.J. Mol. Cell. Biol. 1998; 18: 6353-6364Crossref PubMed Scopus (367) Google Scholar, 36.Iimuro Y. Nishiura T. Hellerbrand C. Behrns K.E. Schoonhoven R. Grisham J.W. Brenner D.A. J. Clin. Invest. 1998; 101: 802-811Crossref PubMed Scopus (423) Google Scholar). Protein-DNA binding reactions were carried out for 20 min on ice using 5 μg of nuclear extract and 32P-labeled DNA probes for the NF-κB consensus binding site (39.Bradham C.A. Stachlewitz R.F. Gao W. Qian T. Jayadev S. Jenkins G. Hannun Y. Lemasters J.J. Thurman R.G. Brenner D.A. Hepatology. 1997; 25: 1128-1135Crossref PubMed Scopus (188) Google Scholar). Complexes were separated by electrophoresis on nondenaturing 5% acrylamide gels and assayed by autoradiography and PhosphorImager analysis (Molecular Dynamics, Sunnyvale, CA). For supershift assays, 8 μg of antibody against p65 or p50 subunit of the NF-κB complex (Santa Cruz Biotechnology, Santa Cruz, CA) were added to the reaction mixture, and the incubation time was extended for an additional 30 min. The preparation of cytosolic S100-fractions and Western blot analysis was performed as described previously (17.Bradham C.A. Qian T. Streetz K. Trautwein C. Brenner D.A. Lemasters J.J. Mol. Cell. Biol. 1998; 18: 6353-6364Crossref PubMed Scopus (367) Google Scholar). Briefly, S-100 fractions were prepared from 8 × 106 hepatocytes by differential centrifugation in buffer containing 250 mm sucrose. Lysates containing 25 μg of protein was separated by electrophoresis on 15% acrylamide SDS gels and transferred into nitocellulose membranes (Schleicher & Schuell). Equal loading was confirmed by Ponceau S staining. Cytochromec was detected using primary monoclonal anti-cytochromec antibody (Pharmingen, San Diego, CA) and secondary anti-mouse horseradish peroxidase-conjugated antibody (Santa Cruz Biotechnology). Proteins were detected with ECL detection reagents (Amersham Pharmacia Biotech). Cell loading and confocal microscopy were carried out as described previously (40.Qian T. Nieminen A.L. Herman B. Lemasters J.J. Am. J. Physiol. 1997; 273: C1783-C1792Crossref PubMed Google Scholar). Briefly, 1–2 × 106 hepatocytes plated on collagen-coated 40-mm-diameter glass coverslips were infected with Ad5IκB in HDM supplemented with 50 mm HEPES (pH 7.0) to stabilize pH during the confocal measurements. The cells were loaded with 250 nmtetramethylrhodamine methyl ester (TMRM, Molecular Probes, Eugene, OR) and 1 μm calcein-acetoxymethyl ester (Molecular Probes) in Krebs Ringer-Hepes buffer for 15 min at 37 °C. The coverslips were mounted on a Nikon microscope (Nikon, Melville, NY) in HDM-HEPES containing 100 nm TMRM, and the temperature was maintained at 37 °C. The first image (time point 0) was then recorded. Subsequently, TNFα or Jo-2 was added to the medium, and images were collected at given time-points. Calcein and TMRM fluorescence were excited with an argon laser through a double dichroic reflector at 488 and 568 nm, respectively. TMRM was imaged through a 590-nm-long path emission filter using a Bio-Rad MRC-600 confocal system (Bio-Rad). Calcein fluorescence was collected through a 515–560-nm band path emission filter. A numerical aperture 1.4, 60× objective lens was used, and pinholes were set to 4 in both channels. Laser attenuation and power were set at 0.3% and low, respectively. Hepatocytes are resistant to TNFα-mediated apoptosis unless they are also treated with an inhibitor of protein synthesis (i.e. cycloheximide), RNA synthesis (i.e. actinomycin D) (18.Leist M. Gantner F. Jilg S. Wendel A. J. Immunol. 1995; 154: 1307-1316PubMed Google Scholar, 41.Leist M. Gantner F. Kunstle G. Bohlinger I. Tiegs G. Bluethmann H. Wendel A. Mol. Med. 1996; 2: 109-124Crossref PubMed Google Scholar), or NF-κB activity (i.e. an IκB superrepressor) (17.Bradham C.A. Qian T. Streetz K. Trautwein C. Brenner D.A. Lemasters J.J. Mol. Cell. Biol. 1998; 18: 6353-6364Crossref PubMed Scopus (367) Google Scholar). To extend these studies into Fas-mediated apoptosis, we switched to primary cultures of adult mouse hepatocytes, because rat hepatocytes only express low levels of Fas (42.Fladmark K.E. Gjertsen B.T. Doskeland S.O. Vintermyr O.K. Biochem. Biophys. Res. Commun. 1997; 232: 20-25Crossref PubMed Scopus (58) Google Scholar). TNFα alone had no cytotoxicity in primary mouse hepatocytes, but actinomycin D (ActD) plus TNFα caused massive cell death (Fig. 1, 16.2 ± 0.4%, % control viability at 17 h after treatment). Also, treatment of Ad5IκB-infected mouse hepatocytes expressing the IκB superrepressor with TNFα induced cell death (12.0 ± 1.1%). Cells expressing IκB superrepressor but not treated with TNFα did not lose viability. Furthermore, Ad5LacZ had a minimal effect on cell viability after TNFα treatment (82.0 ± 2.5%), compared with noninfected hepatocytes after TNFα. To study the role of NF-κB activation on Fas-mediated apoptosis, we treated cells with anti-Fas agonist-like antibody Jo2. Ad5IκB plus Jo2 (12.0 ± 1.2%) or ActD plus Jo2 (8.7 ± 0.9%) rapidly induced massive cell death, whereas Jo2 alone had low cytotoxicity (78.0 ± 11.5%). Furthermore, Jo2 did not induce significant cell death in Ad5LacZ-infected hepatocytes (75.0 ± 1.1%). The cytotoxic effects of Jo2 were dose-dependent in mouse hepatocytes expressing the IκB superrepressor (data not shown). The Ad5IκB-infected hepatocytes treated with Jo2 displayed nuclear condensation and fragmentation by propidium iodide staining, characteristic of apoptosis (Fig.2 A, lower panel), whereas uninfected cells displayed normal nuclear morphology after Jo2 treatment (Fig. 2 A, upper panel). To confirm hepatocyte death as apoptosis, TUNEL assay was performed. Although TUNEL positive cells were minimal after TNFα or Jo2 treatment, significant positive hepatocytes were observed after TNFα or Jo2 treatment in ActD-sensitized or Ad5IκB-infected hepatocytes (Fig.2 B). These results were consistent with cytotoxicity determined by the trypan blue extraction test. Furthermore, apoptosis was confirmed by the detection of fragmented chromosome DNA in infected cells after exposure to TNFα or Jo2 (Fig. 2 C). However, no DNA fragmentation was observed in the uninfected cells after Jo2 treatment. We also documented the role of the IκB/NFκB system in TNFα- and Fas-mediated apoptosis with a proteasome inhibitor, because proteasome inhibitors block IκBα degradation and reduce NF-κB activation (43.Jobin C. Hellerbrand C. Licato L.L. Brenner D.A. Sartor R.B. Gut. 1998; 42: 779-787Crossref PubMed Scopus (108) Google Scholar). MG-132, a potent and specific proteasome inhibitor (Fig. 1, MG-132 alone, 92.0 ± 4.6%), promotes TNFα- and Fas-mediated apoptosis (9.0 ± 3.6% and 0%, respectively). To examine whether Jo2 directly activates NF-κB, NF-κB DNA binding activity was assessed by electrophoretic mobility shift assay using an NF-κB binding site as probe. TNFα treatment for 30 min induced an increase in NF-κB DNA binding activity (2.2-fold increase,p < 0.001, versus untreated hepatocytes) (Fig. 3, A and B). Jo2 treatment also induced NF-κB binding activity (1.5-fold increase,p < 0.005, versus untreated hepatocytes), although to a less extent than TNFα. This activation was observed even at 15 min after Jo2 treatment with the peak at 30 min after Jo2 (data not shown). The NF-κB complex activated by Jo2 treatment of mouse hepatocytes was composed of p50-p65 dimers, as determined by supershifts (Fig. 3 A). These results show that TNFα and Jo2 activate NF-κB in mouse hepatocytes and blocking NF-κB sensitizes mouse hepatocytes to TNFα- and Fas-mediated apoptosis. NIK has been identified as a TRAF2-interacting protein that signals for NF-κB activation (5.Malinin N.L. Boldin M.P. Kovalenko A.V. Wallach D. Nature. 1997; 385: 540-544Crossref PubMed Scopus (1164) Google Scholar). Adenovirus (Ad5ΔNIK)-mediated overexpression of the C-terminal NIK fragment (NIKΔ2101) impaired the induction of NF-κB by TNFα in a reporter gene assay in COS cells (Fig. 3 C). HA-tagged ΔNIK was expressed in primary mouse hepatocytes by infection of Ad5ΔNIK at 10, 30, and 50 m.o.i. (Fig.3 D). Dominant-negative expression of NIK sensitized mouse hepatocytes to TNFα- and Fas-mediated cell de" @default.
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