Abstract
23 min readThe IκB kinases (IKKs) lie downstream of the NF-κB-inducing kinase (NIK) and activate NF-κB by phosphorylation of IκBα. This leads to IκBα degradation and release of NF-κB. In U937 monocytic cells, interleukin (IL)-1β (1 ng/ml) and tumor necrosis factor (TNF)-α; 10 ng/ml) induced κB-dependent transcription equally. However, IKK activity was strongly induced by TNF-α but not by IL-1β. This was consistent with IκBα phosphorylation and degradation, yet TNF-α-induced NF-κB DNA binding was only 30–40% greater than for IL-1β. This was not explained by degradation of IκBβ, IκBε, or p105 nor nuclear translocation of NF-κB·IκBα complexes or degradation-independent release of NF-κB. Dominant negative (NIK) repressed TNF-α and IL-1β-induced κB-dependent transcription by ∼60% and ∼35%, respectively. These data reveal an imprecise relationship between IKK activation, IκBα degradation, and NF-κB DNA binding, suggesting the existence of additional mechanisms that regulate NF-κB activation. Finally, the lack of correlation between DNA binding and transcriptional activation plus the fact that PP1 and genistein both inhibited κB-dependent transcription without affecting DNA binding activity demonstrate the existence of regulatory steps downstream of NF-κB DNA binding. Therapeutically these data are important as inhibition of the NIK-IKK-IκBα cascade may not produce equivalent reductions in NF-κB-dependent gene expression. The IκB kinases (IKKs) lie downstream of the NF-κB-inducing kinase (NIK) and activate NF-κB by phosphorylation of IκBα. This leads to IκBα degradation and release of NF-κB. In U937 monocytic cells, interleukin (IL)-1β (1 ng/ml) and tumor necrosis factor (TNF)-α; 10 ng/ml) induced κB-dependent transcription equally. However, IKK activity was strongly induced by TNF-α but not by IL-1β. This was consistent with IκBα phosphorylation and degradation, yet TNF-α-induced NF-κB DNA binding was only 30–40% greater than for IL-1β. This was not explained by degradation of IκBβ, IκBε, or p105 nor nuclear translocation of NF-κB·IκBα complexes or degradation-independent release of NF-κB. Dominant negative (NIK) repressed TNF-α and IL-1β-induced κB-dependent transcription by ∼60% and ∼35%, respectively. These data reveal an imprecise relationship between IKK activation, IκBα degradation, and NF-κB DNA binding, suggesting the existence of additional mechanisms that regulate NF-κB activation. Finally, the lack of correlation between DNA binding and transcriptional activation plus the fact that PP1 and genistein both inhibited κB-dependent transcription without affecting DNA binding activity demonstrate the existence of regulatory steps downstream of NF-κB DNA binding. Therapeutically these data are important as inhibition of the NIK-IKK-IκBα cascade may not produce equivalent reductions in NF-κB-dependent gene expression. nuclear factor κB electromobility shift assay IκB kinase interleukin-1β NF-κB-inducing kinase tumor necrosis factor-α phenylmethylsulfonyl fluoride dithiothreitol glutathione S-transferase; polyacrylamide gel electrophoresis The acute phase transcription factor nuclear factor κB (NF-κB)1 is an inducible enhancer of many inflammatory genes including cytokines, chemokines, and adhesion molecules as well as enzymes such as inducible nitric-oxide synthase and cyclooxygenase-2 (reviewed in Refs. 1Barnes P.J. Karin M. N. Engl. J. Med. 1997; 336: 1066-1071Crossref PubMed Scopus (4338) Google Scholar and 2Siebenlist U. Franzoso G. Brown K. Annu. Rev. Cell Biol. 1994; 10: 405-455Crossref PubMed Scopus (2023) Google Scholar). Proinflammatory cytokines such as interleukin (IL)-1β and tumor necrosis factor (TNF)-α rapidly induce NF-κB DNA binding and κB-dependent transcription in most cell types (1Barnes P.J. Karin M. N. Engl. J. Med. 1997; 336: 1066-1071Crossref PubMed Scopus (4338) Google Scholar, 2Siebenlist U. Franzoso G. Brown K. Annu. Rev. Cell Biol. 1994; 10: 405-455Crossref PubMed Scopus (2023) Google Scholar). NF-κB DNA binding activity comprises homo- and, more usually, heterodimers of Rel proteins such as RelA (p65), RelB, c-Rel, NF-κB1 (p105/p50), and NF-κB2 (p100/p52) (2Siebenlist U. Franzoso G. Brown K. Annu. Rev. Cell Biol. 1994; 10: 405-455Crossref PubMed Scopus (2023) Google Scholar). NF-κB is held inactive in the cytoplasm by inhibitory IκB proteins including IκBα, IκBβ, IκBγ, and IκBε. Agonists such as TNF-α and IL-1β result in phosphorylation of IκBα at serines 32 and 36 (3Traenckner E.B. Pahl H.L. Henkel T. Schmidt K.N. Wilk S. Baeuerle P.A. EMBO J. 1995; 14: 2876-2883Crossref PubMed Scopus (937) Google Scholar, 4Brown K. Gerstberger S. Carlson L. Franzoso G. Siebenlist U. Science. 1995; 267: 1485-1488Crossref PubMed Scopus (1322) Google Scholar). These phosphorylation events lead to ubiquitination of IκBα followed by its rapid degradation by the 26 S proteasome (5Thanos D. Maniatis T. Cell. 1995; 80: 529-532Abstract Full Text PDF PubMed Scopus (1219) Google Scholar). This releases active NF-κB, typically p50/p65 heterodimers, which translocates to the nucleus and activates transcription via κB enhancer elements. Recently a high molecular mass, ≈700 kDa, complex has been described that contains kinase activities specific for serines 32 and 36 of IκBα (6DiDonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. Nature. 1997; 388: 548-554Crossref PubMed Scopus (1929) Google Scholar, 7Mercurio 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 (1861) Google Scholar, 8Zandi E. Rothwarf D.M. Delhase M. Hayakawa M. Karin M. Cell. 1997; 91: 243-252Abstract Full Text Full Text PDF PubMed Scopus (1609) Google Scholar). The two main IκBα kinase (IKK) activities in this complex, termed the IKK signalsome, have been cloned and are called IKKα and IKKβ. In addition, the upstream kinase, where the IL-1β and TNF-α signaling pathways converge before IKK activation, has been identified as a mitogen-activated protein kinase kinase kinase and named NF-κB-inducing kinase (NIK) (9Malinin N.L. Boldin M.P. Kovalenko A.V. Wallach D. Nature. 1997; 385: 540-544Crossref PubMed Scopus (1172) Google Scholar). As NIK interacts with and stimulates IKK activity and can phosphorylate IKKα on serine 176, it is likely that NIK lies immediately upstream of the IKKs in the NF-κB activation cascade (10Woronicz J.D. Gao X. Cao Z. Rothe M. Goeddel D.V. Science. 1997; 278: 866-869Crossref PubMed Scopus (1072) Google Scholar, 11Nakano H. Shindo M. Sakon S. Nishinaka S. Mihara M. Yagita H. Okumura K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3537-3542Crossref PubMed Scopus (474) Google Scholar, 12Ling L. Cao Z. Goeddel D.V. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3792-3797Crossref PubMed Scopus (454) Google Scholar). However, the degree to which these signaling events apply to different cell types and different inducing agents is presently unclear. In asthmatic individuals, elevated levels of activated NF-κB are found in sputum macrophage, suggesting that cells of the monocytic lineage may play a significant role in asthmatic inflammation (13Hart L.A. Krishnan V.L. Adcock I.M. Barnes P.J. Chung K.F. Am. J. Respir. Crit. Care Med. 1998; 158: 1585-1592Crossref PubMed Scopus (393) Google Scholar). Human monocytic U937 cells were therefore used to investigate the NIK-IKK-IκB signal transduction pathway in the activation of NF-κB and κB-dependent transcription by IL-1β and TNF-α. U937 cells (ECACC code 85011440) were cultured at 37 °C in a humidified atmosphere with 5% CO2 in RPMI 1640 medium (Sigma) supplemented with 10% (v/v) fetal calf serum (Sigma), 2 mml-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, and 2.8 μg/ml amphotericin B (complete medium) and were maintained between 2–9 × 105 cells/ml. Cells were stimulated in RPMI medium as above but supplemented with 2% fetal calf serum at 5 × 105cells/ml. IL-1β and TNF-α (R&D Systems, Abingdon, Oxon) were used at 1 ng/ml and 10 ng/ml, respectively. Where used, cycloheximide (10 μg/ml; Sigma) was added 5 min before stimulation,N-carbobenzyloxy-Leu-Leu-leucinal (MG-132) (10 μm; Sigma) was added 60 min before stimulation, and PP1 and genistein (Calbiochem) were added 30 min before stimulation at concentrations of 10 μm and 100 μm, respectively. Drugs were dissolved in dimethyl sulfoxide (Me2SO) and were diluted to final concentrations of less than 0.1% (v/v). At this level Me2SO had no effect on activation of NF-κB or κB-dependent transcription (data not shown). The NF-κB-dependent reporter, pGL3.6κB.BG.luc, contains two tandem repeats of the sequence 5′-GGG GAC TTT C CC TGG GGA CTT TCC CTGGGG ACT TTC CC-3′, which contains three copies of the decameric NF-κB binding site (underlined) upstream of a minimal β-globin promoter driving a luciferase gene as described previously (14Schwarzer N. Nost R. Seybold J. Parida S.K. Fuhrmann O. Krull M. Schmidt R. Newton R. Hippenstiel S. Domann E. Chakraborty T. Suttorp N. J. Immunol. 1998; 161: 3010-3018PubMed Google Scholar). The reporter, pGL3.6κBmut.luc, is as above except that the core NF-κB binding site is mutated to 5′-GCC ACT TTC C-3′ (mutated bases underlined). The NIK and dominant negative NIK expression vectors were gifts from David Wallach (9Malinin N.L. Boldin M.P. Kovalenko A.V. Wallach D. Nature. 1997; 385: 540-544Crossref PubMed Scopus (1172) Google Scholar). Aliquots of 10 × 106 cells in 250 μl of Hanks' balanced salt solution were electroporated with 10 μg of either pGL3.6κB.BG.luc or pGL3.6κB.BG.luc using a Gene Pulser II (Bio-Rad) set for 200 V, 950 microfarads. For overexpression studies, electroporation was performed using either 5 μg of pGL3.6κB.BG.luc plus 5 μg of pcDNA3 or NIK expression vector or using 2.5 μg of pGL3.6κB.BG.luc plus 10 μg of pcDNA3 or dominant negative NIK expression vector. Transfected cells were incubated in 10 ml of complete medium for 12 h before plating onto 2× 6-well plates in RPMI medium containing 2% fetal calf serum. Cells were harvested 6 h after stimulation and assayed for luciferase activity using a commercially available luciferase reporter gene assay (Promega, Southampton, UK). After normalization to protein concentration, data were expressed as fold activation. Extracts were prepared essentially according to Osborn et al. (15Osborn L. Kunkel S. Nabel G.J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2336-2340Crossref PubMed Scopus (1460) Google Scholar). Cells were washed twice with ice-cold Hanks' balanced salt solution before resuspension in 10 mm HEPES pH 7.9, 10 mm KCl, 1.5 mm MgCl2, 0.1% v/v Nonidet P40 (Nonidet P-40), 0.5 mm phenylmethylsulfonyl fluoride (PMSF), and 1 mm dithiothreitol (DTT) (Buffer A). After a 2-min incubation on ice, nuclei were separated by centrifugation at 1000 g for 10 min. Supernatants (cytoplasmic extracts) were retained. For electrophoretic mobility shift assay (EMSA), nuclei were resuspended in 20 mm HEPES, pH 7.9, 420 mm NaCl, 1.5 mm MgCl2, 0.2 mm EDTA, 25% v/v glycerol, 0.5 mm PMSF, and 1 mm DTT (Buffer B) and incubated on ice for 60 min with vigorous mixing. Nuclear debris was removed by centrifugation, and supernatants were diluted 4-fold in 20 mm HEPES, pH 7.9, 50 mm KCl, 0.2 mm EDTA, 0.5 mm PMSF, and 1 mm DTT (Buffer C). For Western blot analysis, nuclear pellets were rinsed twice in Hanks' balanced salt solution and then resuspended in 10 mm Tris-HCl, pH 7.5, 150 mmNaCl, 1.5 mm MgCl2, 0.65% Nonidet P-40, 0.5 mm PMSF, and 1 mm DTT (lysis buffer) and subjected to one freeze/thaw cycle. Nuclear proteins, 5 μg, were used in binding reactions as described previously (16Newton R. Adcock I.M. Barnes P.J. Biochem. Biophys. Res. Commun. 1996; 218: 518-523Crossref PubMed Scopus (82) Google Scholar). Consensus NF-κB probe (Promega) containing the decameric NF-κB site (underlined) was 5′-AGT TGA GGG GAC TTT CCC AGG-3′ (sense stand). Specificity was determined by the prior addition of a 100-fold excess of unlabeled competitor consensus oligonucleotide. For supershift analysis, nuclear extracts were incubated on ice for 90 min with antisera raised to various Rel proteins (Santa Cruz Biotechnology, Santa Cruz, CA) at 0.4 μg/ml before the addition of radiolabeled oligonucleotide. Reactions were separated on 6% nondenaturing acrylamide gels in 0.25 × Tris-buffered EDTA. Gels were dried, and protein-DNA complexes were visualized by autoradiography. Cytoplasmic or nuclear extract, 20 μg, was added to an equal volume of 125 mm Tris-HCl, pH 6.8, 1% w/v SDS, 10% v/v glycerol, 0.1% w/v bromphenol blue, 2% v/v 2-mercaptoethanol (2× SDS loading buffer) and boiled for 5 min. Samples were separated by 10% SDS-PAGE and transferred to hybond-ECL membranes (Amersham Pharmacia Biotech). Membranes were probed with rabbit anti-sera to human IκBα (C-21), IκBβ (C-20), IκBγ (5177C), IκBε (M-121), or p65 (C-20) antibody (Santa Cruz) diluted 1:4000, 1:1000, 1:500, 1:500, or 1:2000, respectively. The p50/p105 antibody (Upstate Biotechnology, Lake Placid, NY) was used at 1 μg/μl. For detecting the serine 32-phosphorylated IκBα, rabbit anti-human IκBα antibody (number 9240) (New England Biolabs, Hitchin, Herts) was used at 1:1000 dilution. After washing, membranes were incubated with horseradish peroxidase-linked anti-rabbit immunoglobulin (DAKO Ltd, High Wickham, Buckinghamshire) and detected by enhanced chemiluminescence (ECL) (Amersham Pharmacia Biotech). Cytoplasmic extracts, 100 μg, prepared in Buffer A supplemented with 25 μg/ml aprotinin, 10 μg/ml leupeptin, 0.5 mm Na3VO4, 1 mm sodium pyrophosphate, 50 mm NaF, and 5 μg/ml N-tosyl-l-phenylalanine chloromethyl ketone (TPCK) were precleared by the addition of 1.0 μg of normal rabbit IgG (Santa Cruz) together with 20 μl of protein A-Agarose (Santa Cruz) for 1 h at 4 °C. After centrifugation, supernatants were incubated with 5 μl of p65 antibody-agarose conjugate (SC-109AC) (Santa Cruz) for 2 h at 4 °C, and immunoprecipitates were collected by centrifugation. After washing 4 times with buffer A supplemented with inhibitors and 0.05% Tween 20, immunocomplexes were resuspended in 40 μl of 1× SDS loading buffer and boiled for 5 min before Western blot analysis. IKK signalsomes were immunoprecipitated from cytoplasmic extracts, 200 μg, using 1 μg of IKKα antibody (H744) (Santa Cruz) and kinase assays performed as described previously (7Mercurio F. Zhu H. Murray B.W. Shevchenko A. 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To check loading and confirm the presence of IKKs, remaining immunoprecipitates were subject to SDS-PAGE and immunoblotted using 1:500 dilutions of IKKα and IKKβ (H740) antibodies (Santa Cruz). Cells were incubated in phosphate-free RPMI media for 2 h before the addition of [32P]orthophosphate (Amersham Pharmacia Biotech) at 0.2 mCi/ml and incubated for a further 4 h. Cells were then treated as indicated before harvesting in 200 μl of 1× phosphate-buffered saline, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS (radioimmune precipitation buffer) supplemented with 0.5 mmPMSF, 2 mm Na3VO4, 10 μg/ml leupeptin, 25 μg/ml aprotinin, and 50 mm NaF on ice for 15 min. Immunoprecipitation of p65 was performed as above except that radioimmune precipitation buffer was used in place of Buffer A. Samples were run on 10% SDS-PAGE before autoradiography or Western blotting. Transient transfection of the NF-κB-dependent reporter, pGL3.6κB.BG.luc, into U937 cells showed that IL-1β (1 ng/ml) and TNF-α (10 ng/ml) activate κB-dependent transcription to similar extents (8.3 ± 1.9-fold for IL-1β and 8.5 ± 1.6-fold for TNF-α) (Fig.1 A). These responses were κB-specific, as a reporter with mutated κB recognition sites, pGL3.6κB.mut.BG.luc, produced 10–100-fold lower luciferase expression, which was unaffected by TNF-α or IL-1β treatment (data not shown). EMSA revealed that both IL-1β and TNF-α strongly induced NF-κB DNA binding (Fig. 1 B). However, the TNF-α response occurred with faster kinetics than the IL-1β response and generally showed the highest overall activation (Fig. 1 B). Thus TNF-α-induced NF-κB was near maximal by 15 min, whereas IL-1β-induced NF-κB was not maximal until 30 min post-stimulation. Supershift analysis was performed to identify the Rel proteins involved in these responses (Fig. 1 C). In IL-1β and TNF-α treated extracts, anti-p50 antisera and anti-p65 antisera resulted in reduced mobility of DNA binding complexes. The NF-κB complex was unaffected by anti-c-Rel, RelB, and p52 antisera. These data indicate that in U937 cells, IL-1β- and TNF-α-inducible NF-κB DNA binding complexes are made up of both p50 and p65 proteins and not c-Rel, RelB, or p52. The fact that heterogeneous bands were observed suggests that both homo- and heterodimers of p50 and p65 may be present. Western blot analysis was performed on cytoplasmic extracts to examine the effect of IL-1β and TNF-α on IκBα degradation. Cells treated with vehicle showed no change in IκBα protein for the duration of the experiment (Fig. 2 A, left panel). After IL-1β treatment, little or no change in IκBα levels was observed during the first 15 min post-stimulation, and by 30 min, a maximum of only 40% loss of IκBα was observed (Fig.2 A, middle panel). By 1 h, IκBα protein levels had returned to above resting levels, which is consistent with NF-κB-dependent activation of the IκBαgene (17Le Bail O. Schmidt Ullrich R. Israel A. EMBO J. 1993; 12: 5043-5049Crossref PubMed Scopus (295) Google Scholar, 18Ito C.Y. Kazantsev A.G. Baldwin Jr., A.S. Nucleic Acids Res. 1994; 22: 3787-3792Crossref PubMed Scopus (209) Google Scholar). In marked contrast, TNF-α caused a rapid loss of IκBα within 15 min of stimulation, and again resynthesis was observed 1 h post-stimulation (Fig. 2 A, right panel). These data agree with the fact that NF-κB DNA binding was induced more rapidly by TNF-α than by IL-1β. However, by 30 min, NF-κB DNA binding was essentially similar with both stimuli, yet loss of IκBα by TNF-α stimulation was almost total, whereas IL-1β resulted in a mere 40% reduction. Furthermore, by 15 min, IL-1β resulted in only a 15% loss of IκBα yet caused over 60% relative DNA binding activity. One explanation for these discrepancies could be that IL-1β-dependent induction of IκBα resynthesis occurred more rapidly such that total disappearance of IκBα was prevented. This possibility was addressed by stimulation in the presence of cycloheximide to prevent new protein synthesis (Fig.2 B). In control cells, cycloheximide had little effect on IκBα protein levels (Fig. 2 B, left panel). After IL-1β treatment in the presence of cycloheximide, loss of IκBα protein was less than 50% that of control at 30 min. This contrasts with the complete loss of IκBα within 15 min of TNF-α treatment in the presence of cycloheximide. The above data raise the possibility that IκBα-dependent release of NF-κB may not fully account for the induction NF-κB DNA binding activity observed after IL-1β treatment in U937 cells. We therefore focused on the potential role of other IκB proteins in activation of NF-κB. At 60 min, IκBβ showed a maximal loss of 30–40% with IL-1β and more than 50% loss after TNF-α treatment (Fig. 2 C). The kinetics of TNF-α-induced loss of IκBβ were delayed with respect to loss of IκBα and the repression of IκBβ protein levels, although modest, were more prolonged. The effects of IL-1β on loss of IκBβ were less pronounced than for TNF-α and only observed around 1–2 h post-stimulation. Consequently, IκBβ does not appear to contribute to levels of activated NF-κB observed before 1 h after IL-1β treatment. Again, relatively minor decreases were observed for IκBε after IL-1β and TNF-α treatments, and in each case, the kinetics were similar, suggesting that IκBε does not play a major role in release of active NF-κB (Fig. 2 D). In addition, degradation of the p50 precursor, p105, via the ubiquitin pathway may release active p50/p65 heterodimers (19Palombella V.J. Rando O.J. Goldberg A.L. Maniatis T. Cell. 1994; 78: 773-785Abstract Full Text PDF PubMed Scopus (1930) Google Scholar), whereas in some cells types, including lymphoid cells, the C-terminal part of p105 can be independently transcribed to produce IκBγ (20Inoue J. Kerr L.D. Kakizuka A. Verma I.M. Cell. 1992; 68: 1109-1120Abstract Full Text PDF PubMed Scopus (207) Google Scholar, 21Liou H.C. Nolan G.P. Ghosh S. Fujita T. Baltimore D. EMBO J. 1992; 11: 3003-3009Crossref PubMed Scopus (139) Google Scholar). Western blot analysis with an antibody for IκBγ, which also reacts with p105, revealed only p105 in U937 cells. Loss of p105 after treatment with IL-1β or TNF-α occurred with similar kinetics and in each case was no more than 50% (Fig. 2 E). In addition, a p50 antibody that cross-reacts with p105 also detected p105 and gave similar results to the IκBγ antibody (data not shown). Furthermore and consistent with the supershift data, immunoblot analysis failed to detect either p52 or precursor protein p100 (data not shown). These data suggest that IκBβ, IκBε, or p105 may not be the major sources of active NF-κB observed on EMSA after IL-1β or TNF-α stimulation in U937 cells. In Jurkat T-cells, pervanadate causes release of NF-κB without degradation of IκBα, and this event involves tyrosine phosphorylation rather than serine phosphorylation of IκBα (22Imber
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