Apoptosis-inducing Agents Cause Rapid Shedding of Tumor Necrosis Factor Receptor 1 (TNFR1)
1999; Elsevier BV; Volume: 274; Issue: 19 Linguagem: Inglês
10.1074/jbc.274.19.13643
ISSN1083-351X
AutoresLisa A. Madge, M. Rocı́o Sierra-Honigmann, Jordan S. Pober,
Tópico(s)Immune Response and Inflammation
ResumoSeveral chemical compounds not known to interact with tumor necrosis factor (TNF) signal transducing proteins inhibit TNF-mediated activation of vascular endothelial cells (EC). Four structurally diverse agents, arachidonyl trifluoromethylketone, staurosporine, sodium salicylate, and C6-ceramide, were studied. All four agents caused EC apoptosis at concentrations that inhibited TNF-induced IκBα degradation. However, evidence of apoptosis was not evident until after several (e.g. 3–12) hours of treatment, whereas 2 h of treatment was sufficient to inhibit TNF responses. IL-1-induced IκBα degradation was unaffected by these treatments. Inhibition of TNF signaling could not be prevented with either of the broad spectrum caspase inhibitors zVADfmk or yVADcmk. The inhibition of p38 kinase with SB203580 prevented the inhibition of TNF signaling by all agents except arachidonyl trifluoromethylketone. No changes in the levels or molecular weights of the adaptor proteins TRADD (TNF receptor-associated death domain), RIP (receptor-interacting protein), or TRAF2 (TNF receptor-associated factor-2) were caused by apoptogenic drugs. However, TNF receptor 1 (TNFR1) surface expression was significantly reduced by all four agents. Furthermore, TNF-dependent recruitment of TRADD to surface TNFR1 was also inhibited. These data suggest that several putative inhibitors of TNF signaling work by triggering apoptosis and that an early event coincident with the initiation of apoptosis, preceding evidence of injury, is loss of TNFR1. Consistent with this hypothesis, cotreatment of EC with the metalloproteinase inhibitor Tapi (TNF-α proteinase inhibitor) blocked the reduction in surface TNFR1 by apoptogenic drugs and prevented inhibition of TNF-induced IκBα degradation without blocking apoptosis. TNFR1 loss could be a mechanism to limit inflammation in response to apoptotic cell death. Several chemical compounds not known to interact with tumor necrosis factor (TNF) signal transducing proteins inhibit TNF-mediated activation of vascular endothelial cells (EC). Four structurally diverse agents, arachidonyl trifluoromethylketone, staurosporine, sodium salicylate, and C6-ceramide, were studied. All four agents caused EC apoptosis at concentrations that inhibited TNF-induced IκBα degradation. However, evidence of apoptosis was not evident until after several (e.g. 3–12) hours of treatment, whereas 2 h of treatment was sufficient to inhibit TNF responses. IL-1-induced IκBα degradation was unaffected by these treatments. Inhibition of TNF signaling could not be prevented with either of the broad spectrum caspase inhibitors zVADfmk or yVADcmk. The inhibition of p38 kinase with SB203580 prevented the inhibition of TNF signaling by all agents except arachidonyl trifluoromethylketone. No changes in the levels or molecular weights of the adaptor proteins TRADD (TNF receptor-associated death domain), RIP (receptor-interacting protein), or TRAF2 (TNF receptor-associated factor-2) were caused by apoptogenic drugs. However, TNF receptor 1 (TNFR1) surface expression was significantly reduced by all four agents. Furthermore, TNF-dependent recruitment of TRADD to surface TNFR1 was also inhibited. These data suggest that several putative inhibitors of TNF signaling work by triggering apoptosis and that an early event coincident with the initiation of apoptosis, preceding evidence of injury, is loss of TNFR1. Consistent with this hypothesis, cotreatment of EC with the metalloproteinase inhibitor Tapi (TNF-α proteinase inhibitor) blocked the reduction in surface TNFR1 by apoptogenic drugs and prevented inhibition of TNF-induced IκBα degradation without blocking apoptosis. TNFR1 loss could be a mechanism to limit inflammation in response to apoptotic cell death. The biological effects elicited by cytokines such as TNF 1The abbreviation used is: TNF, tumor necrosis factor; AP-1, activator protein-1; ATK, arachidonyl trifluoromethylketone; EC, endothelial cell(s); FACS, fluorescence-activated cell sorter; mAb, monoclonal antibody; ICAM-1, intercellular adhesion molecule-1; IL-1, interleukin-1; M199, medium 199; NFκB, nuclear factor κB; IκBα, inhibitor of NFκBα; PARP, poly(ADP-ribose) polymerase; PBS, phosphate-buffered saline; RIP, receptor-interacting protein; stauro, staurosporine; Tapi, TNF-α proteinase inhibitor; TNFR1 and TNFR2, TNF receptor 1 and 2, respectively; TRADD, TNF receptor 1-associated death domain protein; TRAF, TNF receptor-associated factor; VCAM-1, vascular cell adhesion molecule-1. and IL-1, which include inflammation and tissue injury, are initiated by ligand-induced formation of distinct multiprotein receptor complexes. Activation of the TNF signal transduction cascade is initiated by the interaction of TNF with two distinct surface receptors, TNFR1 (55 kDa) and TNFR2 (75 kDa) (reviewed in Ref. 1Tartaglia L.A. Goeddel D.V. Immunol. Today. 1992; 13: 151-153Abstract Full Text PDF PubMed Scopus (1001) Google Scholar). Although both TNFR1 and TNFR2 may mediate the activation of independent downstream signaling pathways (2Engelmann H. Holtmann H. Brakebusch C. Avni Y.S. Sarov I. Nophar Y. Hadas E. Leitner O. Wallach D. J. Biol. Chem. 1990; 265: 14497-14504Abstract Full Text PDF PubMed Google Scholar, 3Tartaglia L.A. Weber R.F. Figari I.S. Reynolds C. Palladino Jr., M.A. Goeddel D.V. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9292-9296Crossref PubMed Scopus (770) Google Scholar, 4Haridas V. Darnay B.G. Natarajan K. Heller R. Aggarwal B.B. J. 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Within the assembled type 1 IL-1 receptor complex, IL-1 receptor-associated kinase becomes autophosphorylated (25Cao Z. Henzel W.J. Gao X. Science. 1996; 271: 1128-1131Crossref PubMed Scopus (777) Google Scholar) and subsequently leaves the complex to associate with cytosolic TRAF6 (26Cao Z. Xiong J. Takeuchi M. Kurama T. Goeddel D.V. Nature. 1996; 383: 443-446Crossref PubMed Scopus (1123) Google Scholar). The IL-1 receptor-associated kinase·TRAF6 complex, like the TRADD·RIP·TRAF2 complex, lies upstream of the activation of NFκB-inducing kinase, creating a point of convergence with the TNF signaling pathway. Vascular endothelial cells (EC) are a major target for the pro-inflammatory actions of TNF and IL-1. One of the major proinflammatory responses in EC initiated by the TNF and IL-1 signal transduction cascades described above is the expression of the leukocyte adhesion molecules E-selectin, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1). E-selectin is a surface glycoprotein that mediates the initial tethering and rolling of neutrophils. The cytokine-dependent transcription of this adhesion molecule has an absolute requirement for the activation of NFκB (27Read M.A. Whitley M.Z. Williams A.J. Collins T. J. Exp. Med. 1994; 179: 503-512Crossref PubMed Scopus (381) Google Scholar) and can be significantly enhanced by the co-binding of ATF2/c-Jun heterodimers (a form of AP-1) to the E-selectin enhanceosome (28De Luca L.G. Johnson D.R. Whitley M.Z. Collins T. Pober J.S. J. Biol. Chem. 1994; 269: 19193-19196Abstract Full Text PDF PubMed Google Scholar). Inhibition of the activation of NFκB or AP-1 in response to TNF or IL-1 is therefore a potential target for novel anti-inflammatory reagents. A wide variety of of diverse pharmacological agents including sodium salicylate, arachidonic acid, short chain ceramide (C6-ceramide), and staurosporine (stauro) have been reported to inhibit TNF-mediated activation of NFκB, expression of E-selectin, or both in EC (29Lane T.A. Lamkin G.E. Wancewicz E.V. Biochem. Biophys. Res. Commun. 1990; 172: 1273-1281Crossref PubMed Scopus (86) Google Scholar, 30Stuhlmeier K.M. Kao J.J. Bach F.H. J. Biol. Chem. 1997; 272: 24679-24683Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 31Slowik M.R. De Luca L.G. Min W. Pober J.S. Circ. Res. 1996; 79: 736-747Crossref PubMed Scopus (44) Google Scholar, 32Pierce J.W. Read M.A. Ding H. Luscinskas F.W. Collins T. J. Immunol. 1996; 156: 3961-3969PubMed Google Scholar). However, none of these agents has been shown to interact with any of the molecules identified to date in the TNF signaling pathway. During the course of our investigation of the signaling role of arachidonic acid, we noted that concentrations of this lipid or its structural analogues, such as the cytosolic phospholipase A2inhibitors arachidonyl trifluoromethylketone (ATK) and methylarachidonyl fluorophosphonate, inhibited E-selectin induction but only at concentrations that induced apoptosis. However, these agents were able to block the activation of NFκB before morphological evidence of apoptosis became detectable. These observations led us to hypothesize that coincident with the induction of apoptosis is a common mechanism by which diverse pharmacological compounds can selectively inhibit the EC response to TNF. Here we present evidence in support of this hypothesis and identify the loss of TNFR1 surface expression as the mechanism by which apoptogenic drugs selectively inhibit TNF responses. In accordance with an approved protocol by the Yale University Human Investigations Committee, human umbilical vein EC were isolated and cultured as described previously on gelatin (J. T. Baker Inc.)-coated tissue culture plastic (Falcon, Lincoln Park, NJ) in medium 199 (M199) supplemented with 20% fetal calf serum, 200 μm l-glutamine (all from Life Technologies Inc.), 50 μg/ml EC growth factor (Collaborative Biomedical Products, Bedford, MA), 100 μg/ml porcine heparin (Sigma), 100 units/ml penicillin, and 100 μg/ml streptomycin (Life Technologies Inc.). All experiments were performed using EC at passage 3 or 4. Recombinant human TNF was a gift from Biogen (Cambridge, MA). Recombinant human IL-1β was purchased from R & D Systems Inc. (Minneapolis, MN). zVADfmk was purchased from Enzyme Systems Products (Dublin, CA). SB203580, C6-ceramide, stauro, and yVADcmk were purchased from Calbiochem. R32W TNF mutein protein was a gift from W. Fiers (State University of Ghent, Belgium). ATK was purchased from Cayman Chemical Co. (Ann Arbor, MI), and a fresh vial was used each day. A tert-butyl derivative of Tapi was a gift of the Immunex Corp. (Seattle, WA). All other reagents were purchased from Bio-Rad or Sigma. Rabbit anti-human IκBα antibody was purchased from Santa Cruz Biotechology (Santa Cruz, CA). Mouse mAb to human TRADD was purchased from Transduction Laboratories (Lexington, KY). Mouse mAb to TNFR1 was a gift from D. Goeddel (Tularik Inc., South San Francisco, CA). Mouse mAb to TNFR2 was purchased from Genzyme Diagnostics (Cambridge, MA). Rabbit anti-human TRAF2 and RIP antibodies were also a gift from D. Goeddel. Mouse mAb to poly(ADP-ribose) polymerase (PARP) was a gift from G. Poirier, CHUL Research Center (Quebec, Canada). Horseradish peroxidase-conjugated secondary antibodies for Western blotting were purchased from Jackson ImmunoResearch (West Grove, PA). For FACS analysis, fluorescein isothiocyanate-conjugated anti-mouse IgG (H + L) F(ab′)2 was purchased from Roche Molecular Biochemicals. For experimental manipulation, EC were plated on human plasma fibronectin-coated multiwell chamber slides (Falcon). After treatment, cells were fixed by the addition of an equal volume of paraformaldehyde (1% final) in PBS for 30 min at room temperature. The slide was subsequently washed twice in PBS, permeabilized for 30 s in PBS containing 0.1% Triton X-100, washed a further four times in PBS, and mounted in Gel Mount (Biomedia Corp., Foster City, CA) containing the nuclear stain 4′,6-diamidino-2-phenylindole·HCl (Dapi, ∼0.0001%, Molecular Probes, Eugene, OR). Specimens were examined by immunofluorescence microscopy using a Nikon diaphot microscope with a 360-nm filter. EC plated on gelatin-coated 24- or 96-well plates were treated as described. After experimental manipulation, medium was removed, and cells were washed twice in PBS. The remaining cells were fixed and stained by the addition of 70% ethanol containing 100 μg/ml Hoescht 33258 reagent (Molecular Probes) for 30 min at room temperature. Cells were again washed twice with PBS before fluorescence was recorded (λex = 360 nm, λem = 460 nm) using a fluorescence plate reader (Perspective Biosystems Inc., Framingham, MA). For analysis of the characteristic cleavage of PARP during apoptosis, EC seeded in six-well plates were treated for 3 h with serum-free M199 containing ATK (0–50 μm) in the presence and absence of zVADfmk (40 μm). Both floating and attached cells were harvested and lysed in TNT solution (50 mm Tris-HCl, pH 6.8, 150 mm NaCl, and 1% Triton X-100) containing Pefablock (1 mm), aprotinin (10 μg/ml), pepstatin (1 μg/ml), leupeptin (10 μg/ml), NaF (10 mm), Na3VO4 (1 mm), and β-glycerophosphate (1 mm) for 20 min on ice. For all other immunoblots, each well was washed twice in ice-cold PBS and lysed by the addition of 100 μl of TNT as described above. DNA in cell lysates was sheared by passing the lysates through a 0.5-cc syringe fitted with a 28.5-gauge needle two or three times. For each sample, an equal amount of protein (20 μg) was size-fractionated by SDS-polyacrylamide gel electrophoresis (33Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar) and then transferred to a polyvinylidene difluoride membrane (Immobilon P, Millipore, Milford, MA) and immunoblotted. Detection of the immunogen by enhanced chemiluminesence was performed according to the manufacturer's instructions (Pierce). Immunoprecipitation was performed by the lysis of confluent human umbilical vein EC seeded on three 10-cm dishes (approximately 107 cells/sample) in a total volume of 3 ml of TNT. The lysates were centrifuged at 735 × g for 10 min at 4 °C before being precleared by incubation with 25 μl of a 1:1 slurry of Gamma Bind-Sepharose (Amersham Pharmacia Biotech) for 2–3 h at 4 °C on a rocking platform. After centrifugation at 14,000 rpm for 10 s, the cleared lysates were transferred to another tube and incubated with mAb to TNFR1 (1 μl/sample) overnight at 4 °C on a rocking platform before the addition of 50 μl of the 1:1 Gamma Bind-Sepharose slurry to each lysate. Incubation was continued at 4 °C for a further 4–6 h. Immune complexes, collected by centrifugation at 16,000 × g for 3 s, were washed once in ice-cold TNT and three to four times in ice-cold PBS and solubilized by the addition of 25 μl of sample buffer (33Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar). The entire sample was subjected to SDS-polyacrylamide gel electrophoresis and Western blot analysis. Indirect immunofluorescence was used to quantify the surface amount of TNFR1 and TNFR2 after treatment of EC with ATK, C6-ceramide, sodium salicylate, and stauro. EC were treated as described, harvested by collagenase (Life Technologies Inc.) digestion, and washed in PBS containing 1% fetal calf serum before incubation for 30 min at 4 °C with saturating amounts of each antibody or nonbinding control mAb (K16/16) as described previously (34Slowik M.R. De Luca L.G. Fiers W. Pober J.S. Am. J. Pathol. 1993; 143: 1724-1730PubMed Google Scholar). Cells were washed three times before the addition of fluorescein isothiocyanate-conjugated anti-mouse secondary Ab and incubation for a further 30 min at 4 °C. Labeled cells were washed an additional three times, fixed in paraformaldehyde (2%), and analyzed by FACS (FACSort, Becton Dickinson, San Jose, CA). EC on gelatin-coated six-well plates were treated for 2 h with serum-free M199 ± ATK (50 μm) or sodium salicylate (20 mm). After incubation the culture medium was harvested, and the amount of soluble TNFR1 in the medium was determined using a soluble TNFR1 enzyme-linked immunosorbent assay kit (Immunotech, Miami, FL). Reports from the literature and our own observations indicate that the cytosolic phospholipase A2inhibitor ATK, the kinase inhibitor stauro, the anti-inflammatory agent sodium salicylate, and the cell-permeant ceramide analogue, C6-ceramide, are all able to inhibit TNF-induced adhesion molecule expression (29Lane T.A. Lamkin G.E. Wancewicz E.V. Biochem. Biophys. Res. Commun. 1990; 172: 1273-1281Crossref PubMed Scopus (86) Google Scholar, 31Slowik M.R. De Luca L.G. Min W. Pober J.S. Circ. Res. 1996; 79: 736-747Crossref PubMed Scopus (44) Google Scholar, 32Pierce J.W. Read M.A. Ding H. Luscinskas F.W. Collins T. J. Immunol. 1996; 156: 3961-3969PubMed Google Scholar). This inhibition of E-selectin expression is paralleled by an inhibition of TNF-induced degradation of inhibitory protein IκBα. Optimal inhibitory concentrations, determined in preliminary experiments, are 50 μm ATK, 100 nm stauro, 20 mm sodium salicylate, and 50 μm C6-ceramide. We have found that these concentrations of all of these agents cause EC to undergo apoptosis; suboptimal inhibitory concentrations were less apoptogenic (Fig. 1 and data not shown). This correlation between inhibition of TNF responses and induction of apoptosis may have been missed in prior studies (29Lane T.A. Lamkin G.E. Wancewicz E.V. Biochem. Biophys. Res. Commun. 1990; 172: 1273-1281Crossref PubMed Scopus (86) Google Scholar, 31Slowik M.R. De Luca L.G. Min W. Pober J.S. Circ. Res. 1996; 79: 736-747Crossref PubMed Scopus (44) Google Scholar, 32Pierce J.W. Read M.A. Ding H. Luscinskas F.W. Collins T. J. Immunol. 1996; 156: 3961-3969PubMed Google Scholar) because biochemical and morphological evidence of apoptosis, such as PARP cleavage or nuclear condensation, typically did not become apparent for several (3Tartaglia L.A. Weber R.F. Figari I.S. Reynolds C. Palladino Jr., M.A. Goeddel D.V. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9292-9296Crossref PubMed Scopus (770) Google Scholar, 4Haridas V. Darnay B.G. Natarajan K. Heller R. Aggarwal B.B. J. Immunol. 1998; 160: 3152-3162PubMed Google Scholar, 5Tartaglia L.A. Pennica D. Goeddel D.V. J. Biol. Chem. 1993; 268: 18542-18548Abstract Full Text PDF PubMed Google Scholar, 6Pinckard J.K. Sheehan K.C. Schreiber R.D. J. Biol. Chem. 1997; 272: 10784-10789Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 7Hsu H. Xiong J. Goeddel D.V. Cell. 1995; 81: 495-504Abstract Full Text PDF PubMed Scopus (1749) Google Scholar, 8Hsu H. Shu H.B. Pan M.G. Goeddel D.V. Cell. 1996; 84: 299-308Abstract Full Text Full Text PDF PubMed Scopus (1738) Google Scholar, 9Hsu H. Huang J. Shu H.B. Baichwal V. Goeddel D.V. Immunity. 1996; 4: 387-396Abstract Full Text Full Text PDF PubMed Scopus (983) Google Scholar, 10Lee S.Y. Reichlin A. Santana A. Sokol K.A. Nussenzweig M.C. Choi Y. Immunity. 1997; 7: 703-713Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar, 11Yeh W.C. Shahinian A. Speiser D. Kraunus J. Billia F. Wakeham A. de la Pompa J.L. Ferrick D. Hum B. Iscove N. Ohashi P. Rothe M. Goeddel D.V. Mak T.W. Immunity. 1997; 7: 715-725Abstract Full Text Full Text PDF PubMed Scopus (712) Google Scholar, 12Kelliher M.A. Grimm S. Ishida Y. Kuo F. Stanger B.Z. Leder P. Immunity. 1998; 8: 297-303Abstract Full Text Full Text PDF PubMed Scopus (926) Google Scholar) hours, the precise timing depending on the drug used (data not shown). More importantly, no evidence of apoptosis was ever detectable in the first 2 h of treatment, by which time inhibition of TNF-mediated activation was already evident. Cytokine-induced E-selectin expression is absolutely dependent on the activation of NFκB (27Read M.A. Whitley M.Z. Williams A.J. Collins T. J. Exp. Med. 1994; 179: 503-512Crossref PubMed Scopus (381) Google Scholar). Therefore we examined the effect of 2 h of pretreatment of each apoptosis-inducing reagent on TNF- and IL-1-dependent degradation of IκBα, a critical step in the NFκB pathway. As shown in Fig. 2,A and B, TNF-induced degradation of IκBα is significantly reduced following 2 h of pretreatment with each apoptogenic drug. Surprisingly, no inhibition of IκBα degradation induced by IL-1 is detectable in replicate cultures. The observed effect of apoptogenic agents on TNF-dependent degradation of IκBα is not simply a delay in the kinetics of the degradation of this protein, because a complete time course of TNF treatment from 5 to 60 min revealed that the degradation of IκBα is inhibited at all times examined (data not shown). These observations led us to hypothesize that an early common event in the biochemical program of apoptosis selectively inhibits TNF (but not IL-1) signal transduction. We next examined whether specific effector molecules implicated in the death pathway could inhibit TNF signal transduction. Activation of effector caspases is a common late step in apoptosis (35Cohen G.M. Biochem. J. 1997; 326: 1-16Crossref PubMed Scopus (4146) Google Scholar). To determine whether the activation of caspases participated in the inhibition of TNF-mediated NFκB activation by pretreatment with inducers of apoptosis, we employed the broad spectrum peptide-based caspase inhibitor zVADfmk. This caspase inhibitor (40 μm) was unable to prevent inhibition of TNF-mediated IκBα degradation when administered during pretreatment with ATK (50 μm) over a 2-h period (Fig. 3A) even though it was able to inhibit the characteristic caspase-dependent cleavage of PARP from a 116- to a 85-kDa fragment over a 3-h incubation with ATK (Fig. 3B). Similar results using zVADfmk were obtained with the other three initiators of apoptosis as well as with another broad spectrum caspase inhibitor, yVADcmk (40 μm) (data not shown). We conclude that activation of zVADfmk- and yVADcmk-sensitive caspases do not participate in the inhibition of TNF-mediated NFκB activation. Vilcek and colleagues (36Schwenger P. Bellosta P. Vietor I. Basilico C. Skolnik E.Y. Vilcek J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2869-2873Crossref PubMed Scopus (256) Google Scholar, 37Schwenger P. Alpert D. Skolnik E.Y. Vilcek J. Mol. Cell. Biol. 1998; 18: 78-84Crossref PubMed Google Scholar) have reported that the p38 kinase inhibitor SB203580 can prevent both sodium salicylate-induced apoptosis and inhibition of TNF signaling in fibroblasts. To determine whether p38 kinase played a role in the inhibition of TNF-dependent activation of NFκB in EC, we employed the same inhibitor. In accord with the fibroblast results, blocking of p38 kinases does relieve the inhibition of TNF-induced IκBα degradation in EC following pretreatment with sodium salicylate (Fig. 4). However, in contrast to the previous study, SB203580 was unable to prevent sodium salicylate-induced apoptosis of EC. SB203580 was similarly effective in preventing the inhibition of TNF-dependent IκBα degradation resulting from pretreatment with C6-ceramide and stauro. Despite a corresponding degree of inhibition of TNF responses by ATK compared with sodium salicylate, SB203580 was ineffective in preventing inhibition of TNF signaling by ATK. Both ATK and sodium salicylate treatment resulted in activation of p38 MAPK measured by Western blotting of the active form of the kinase with a phospho-p38-specific antibody. However, the activation of p38 in response to sodium salicylate was detected after only a 15-min treatment with the reagent and decreased over time, whereas the activation of p38 in respect to ATK was not increased until after 2 h of treatment with the reagent. At this time point the activation of p38 was slightly stronger than the activation initially observed with sodium salicylate. The inability of SB203580 to prevent ATK-dependent inhibition of TNF signaling may therefore result from incomplete blocking of p38 activation. Alternatively, these data may suggest a primary role of p38 kinase for responses to sodium salicylate but a secondary role for responses to ATK. The differential effects of apoptosis inducers on TNF- and IL-1-dependent degradation of IκBα suggest that the point of inhibition caused by the signaling cascade leading to apoptosis must be upstream of the convergence of TNF and IL-1 signal transduction cascades. In EC, TNF signaling begins with ligand binding to TNFR2, which passes the TNF trimer to TNFR1. Previous studies in EC have established that the R32W TNF mutein protein directly and exclusively signals thr
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