IKKi/IKKϵ Plays a Key Role in Integrating Signals Induced by Pro-inflammatory Stimuli
2003; Elsevier BV; Volume: 278; Issue: 29 Linguagem: Inglês
10.1074/jbc.m303001200
ISSN1083-351X
AutoresVladimir V. Kravchenko, John C. Mathison, Klaus Schwamborn, Frank Mercurio, Richard J. Ulevitch,
Tópico(s)Cytokine Signaling Pathways and Interactions
ResumoWe report that the product of the inducible gene encoding the kinase known as IKKi/IKKϵ (IKKi) is required for expression of a group of genes up-regulated by pro-inflammatory stimuli such as bacterial endotoxin (lipopolysaccharide (LPS)). Here, using murine embryonic fibroblasts obtained from mice bearing deletions in IKK2, p65, and IKKi genes, we provide evidence to support a link between signaling through the NF-κB and CCAAA/enhancer-binding protein (C/EBP) pathways. This link includes an NF-κB-dependent regulation of C/EBPβ and C/EBPδ gene transcription and IKKi-mediated activation of C/EBP. Disruption of the NF-κB pathway results in the blockade of the inducible up-regulation of C/EBPβ, C/EBPδ, and IKKi genes. Cells lacking IKKi are normal in activation of the canonical NF-κB pathway but fail to induce C/EBPδ activity and transcription of C/EBP and C/EBP-NF-κB target genes in response to LPS. In addition we show that, in response to LPS or tumor necrosis factor α, both β and δ subunits of C/EBP interact with IKKi promoter, suggesting a feedback mechanism in the regulation of IKKi-dependent cellular processes. These data are among the first to provide insights into the biological function of IKKi. We report that the product of the inducible gene encoding the kinase known as IKKi/IKKϵ (IKKi) is required for expression of a group of genes up-regulated by pro-inflammatory stimuli such as bacterial endotoxin (lipopolysaccharide (LPS)). Here, using murine embryonic fibroblasts obtained from mice bearing deletions in IKK2, p65, and IKKi genes, we provide evidence to support a link between signaling through the NF-κB and CCAAA/enhancer-binding protein (C/EBP) pathways. This link includes an NF-κB-dependent regulation of C/EBPβ and C/EBPδ gene transcription and IKKi-mediated activation of C/EBP. Disruption of the NF-κB pathway results in the blockade of the inducible up-regulation of C/EBPβ, C/EBPδ, and IKKi genes. Cells lacking IKKi are normal in activation of the canonical NF-κB pathway but fail to induce C/EBPδ activity and transcription of C/EBP and C/EBP-NF-κB target genes in response to LPS. In addition we show that, in response to LPS or tumor necrosis factor α, both β and δ subunits of C/EBP interact with IKKi promoter, suggesting a feedback mechanism in the regulation of IKKi-dependent cellular processes. These data are among the first to provide insights into the biological function of IKKi. Gene expression during innate and adaptive immune responses involves the combined effects of multiple transcription factors. Among these are members of the activating protein 1, NF-κB, signal transducers and activators of transcription, and CCAAA/enhancer-binding proteins (C/EBP) 1The abbreviations used are: C/EBP, CCAAA/enhancer-binding protein; RANTES, regulated on activation normal T cell expressed and secreted; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LPS, lipopolysaccharide; TNF, tumor necrosis factor; IL-1, interleukin-1; IKK, IκB kinase; MEF, murine embryonic fibroblast; imMEF, immortalized MEF; siRNA, small interfering RNA; EMSA, electrophoretic mobility shift assay; ChIP, chromatin immunoprecipitation; IRF-3, interferon regulatory factor-3; NEMO, NF-κB essential modulator. families (1Rincón M. Flavell R.A. Davis R.J. Oncogene. 2001; 20: 2490-2497Crossref PubMed Scopus (105) Google Scholar, 2Pomerantz J.L. Baltimore D. Mol. Cell. 2002; 10: 693-701Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar, 3Poli V. J. Biol. Chem. 1998; 273: 29279-29282Abstract Full Text Full Text PDF PubMed Scopus (562) Google Scholar). Prototypic activators of innate immunity such as bacterial endotoxin (lipopolysaccharide (LPS)) are known to regulate both the NF-κB and the C/EBP pathways (4Silverman N. Maniatis T. Genes Dev. 2001; 15: 2321-2342Crossref PubMed Scopus (777) Google Scholar, 5Akira S. Kishimoto T. Adv. Immunol. 1997; 65: 1-46Crossref PubMed Google Scholar). The C/EBP family is comprised of at least six proteins containing basic leucine zipper (bZIP) motifs (6Lekstrom-Himes J. Xanthopoulos K.G. J. Biol. Chem. 1998; 273: 28545-28548Abstract Full Text Full Text PDF PubMed Scopus (692) Google Scholar, 7McKnight S.L. Cell. 2001; 107: 259-261Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). The NF-κB family is made up of fewer members including p50, p52, p65/RelA, c-Rel, and RelB (8Ghosh S. May M.J. Kropp E.B. Annu. Rev. Immunol. 1998; 16: 225-260Crossref PubMed Scopus (4631) Google Scholar). These transcription factors regulate expression of distinct and overlapping subsets of genes encoding immune and pro-inflammatory modulators (3Poli V. J. Biol. Chem. 1998; 273: 29279-29282Abstract Full Text Full Text PDF PubMed Scopus (562) Google Scholar, 9Pahl H.L. Oncogene. 1999; 18: 6853-6866Crossref PubMed Scopus (3464) Google Scholar). Whether or not NF-κB and C/EBP family members influence each other is not well understood at this time. Current paradigms suggest that the rapidly and transiently activated NF-κB pathway is central in a primary wave of gene induction followed by a second wave of gene transcription some hours later mediated by other transcription factors including members of the C/EBP family (3Poli V. J. Biol. Chem. 1998; 273: 29279-29282Abstract Full Text Full Text PDF PubMed Scopus (562) Google Scholar, 5Akira S. Kishimoto T. Adv. Immunol. 1997; 65: 1-46Crossref PubMed Google Scholar). Despite participating in regulation of overlapping sets of genes, the NF-κB and the C/EBP pathways are activated by distinct intracellular signaling mechanisms. Activation of the canonical NF-κB pathway depends on stability of the inhibitor known as IκBα. It stabilizes NF-κB complexes so that after its degradation the remaining subunits translocate from the cytoplasm to the nucleus. NF-κB-dependent transcription of IκBα gene also provides a potent feedback mechanism maintaining the balance between cytoplasmic and nuclear localization of the NF-κB subunits (8Ghosh S. May M.J. Kropp E.B. Annu. Rev. Immunol. 1998; 16: 225-260Crossref PubMed Scopus (4631) Google Scholar). Treatment of cells with specific inducers, such as TNF, IL-1, or LPS, results in the phosphorylation of IκBα at two serines (Ser-32 and Ser-36). This is a signal for its rapid ubiquitin-dependent proteolysis and translocation of free NF-κB to the nucleus. IκBα phosphorylation is catalyzed by the IκB kinase (IKK), a complex composed of three subunits, IKKα/IKK1, IKKβ/IKK2, and NEMO/IKKγ/IKKAP1/FIP3. IKK1 and IKK2 are the catalytic subunits, whereas NEMO serves a non-enzymatic, regulatory function. Biochemical and genetic analyses demonstrate that IKK2 is essential for NF-κB activation in response to TNF, IL-1, and LPS, whereas IKK1 is not required for such responses (10Karin M. Ben-Neriah Y. Annu. Rev. Immunol. 2000; 18: 621-663Crossref PubMed Scopus (4106) Google Scholar). Targeted deletion of p65 or IKK2 gene affects TNF-induced transcription of the IκBα gene (11Beg A.A. Sha W.C. Bronson R.T. Ghosh S. Baltimore D. Nature. 1995; 376: 167-170Crossref PubMed Scopus (1640) Google Scholar, 12Li Q. Van Antwerp D. Mercurio F. Lee K.F. Verma I.M. Science. 1999; 284: 321-325Crossref PubMed Scopus (857) Google Scholar). Recently two additional IKK2-related kinases, IKKi/IKKϵ (13Shimada T. Kawai T. Takeda K. Matsumoto M. Inoue J. Tatsumi Y. Kanamaru A. Akira S. Int. Immunol. 1999; 11: 1357-1362Crossref PubMed Scopus (312) Google Scholar, 14Peters R.T. Liao S.M. Maniatis T. Mol. Cell. 2000; 5: 513-522Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar) and TANK-binding kinase 1/NF-κB-activating kinase/T2K (15Pomerantz J.L. Baltimore D. EMBO J. 1999; 18: 6694-6704Crossref PubMed Google Scholar, 16Tojima Y. Fujimoto A. Delhase M. Chen Y. Hatakeyama S. Nakayama K. Kaneko Y. Nimura Y. Motoyama N. Ikeda K. Karin M. Nakanishi M. Nature. 2000; 404: 778-782Crossref PubMed Scopus (316) Google Scholar, 17Bonnard M. Mirtsos C. Suzuki S. Graham K. Huang J. Ng M. Itie A. Wakeham A. Shahinian A. Henzel W.J. Elia A.J. Shillinglaw W. Mak T.W. Cao Z. Yeh W.C. EMBO J. 2000; 19: 4976-4985Crossref PubMed Google Scholar) have been identified. Whether these latter proteins play a role as enzymatic or non-enzymatic regulatory factors is not fully understood (4Silverman N. Maniatis T. Genes Dev. 2001; 15: 2321-2342Crossref PubMed Scopus (777) Google Scholar). Regulatory mechanisms for the C/EBP pathways differ markedly from those of NF-κB and include transcriptional and/or post-translational mechanisms as well as protein-protein interactions via dimerization through leucine-zipper domains (6Lekstrom-Himes J. Xanthopoulos K.G. J. Biol. Chem. 1998; 273: 28545-28548Abstract Full Text Full Text PDF PubMed Scopus (692) Google Scholar, 7McKnight S.L. Cell. 2001; 107: 259-261Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 18Cao Z. Umek R.M. McKnight S.L. Genes Gev. 1991; 5: 1538-1552Crossref PubMed Scopus (1350) Google Scholar, 19Williams S.C. Cantwell C.A. Johnson P.F. Genes Dev. 1991; 5: 1553-1567Crossref PubMed Scopus (439) Google Scholar). Phosphorylation also regulates the C/EBP family by directing nuclear localization and transcription-activating potential (20Nakajima T. Kinoshita S. Sasagawa T. Sasaki K. Naruto M. Kishimoto T. Akira S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2207-2211Crossref PubMed Scopus (518) Google Scholar, 21Trautwein C. Caelles C. van der Geer P. Hunter T. Karin M. Chojkier M. Nature. 1993; 364: 544-547Crossref PubMed Scopus (293) Google Scholar, 22Chumakov A.M. Grillier I. Chumakova E. Chih D. Slater J. Koeffler H.P. Mol. Cell. Biol. 1997; 17: 1375-1386Crossref PubMed Google Scholar, 23Buck M. Zhang L. Halasz N.A. Hunter T. Chojkier M. EMBO J. 2001; 20: 6712-6723Crossref PubMed Scopus (85) Google Scholar). Some members of this family, specifically C/EBPβ and C/EBPδ, have been linked to gene expression in the acute phase response and during inflammation (24Akira S. Isshiki H. Sugita T. Tanabe O. Kinoshita S. Nishio Y. Nakajima T. Hirano T. Kishimoto T. EMBO J. 1990; 9: 1897-1906Crossref PubMed Scopus (1212) Google Scholar, 25Kinoshita S. Akira S. Kishimoto T. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1473-1476Crossref PubMed Scopus (262) Google Scholar, 26Juan T.S.-C. Wilson D.R. Wilde M.D. Darlington G.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2584-2588Crossref PubMed Scopus (126) Google Scholar). Furthermore, up-regulation of C/EBPβ and C/EBPδ gene expression occurs after exposure to pro-inflammatory stimuli such as TNF, IL-1, IL-6, or LPS (3Poli V. J. Biol. Chem. 1998; 273: 29279-29282Abstract Full Text Full Text PDF PubMed Scopus (562) Google Scholar, 5Akira S. Kishimoto T. Adv. Immunol. 1997; 65: 1-46Crossref PubMed Google Scholar). Here we have performed experiments to identify possible regulatory links between the NF-κB and C/EBP pathways, with an emphasis on events related to innate immune responses. We have used murine embryonic fibroblasts (MEFs) obtained from various strains of mice bearing targeted gene deletions in components of the NF-κB pathway. We show that fibroblasts isolated from p65–/– or IKK2–/–, but not from control, mice fail to induce C/EBPβ and C/EBPδ transcripts in response to cell stimulation with LPS. Furthermore, we observed that both p65 and IKK2 are required for induction of IKKi mRNA. In turn, in primary fibroblasts lacking the IKKi gene LPS induces C/EBPβ and C/EBPδ mRNAs and activates NF-κB, as observed in control cells, but importantly, fail to induce C/EBPδ-specific DNA binding activity. Furthermore, we report that LPS treatment of IKKi–/– MEFs reveals deficits in expressions of genes associated with immune and pro-inflammatory responses. In totality, these data support the contention that there is a link between the NF-κB and C/EBP pathways and that IKKi may be a key element in integration of signals from both pathways during inflammatory and immune responses. Cells, Antibodies, and Reagents—RelA/p65-, IKK2-, IKKi-, or Egr1-deficient MEFs and the corresponding immortalized MEFs (imMEFs) were generated and maintained as described (11Beg A.A. Sha W.C. Bronson R.T. Ghosh S. Baltimore D. Nature. 1995; 376: 167-170Crossref PubMed Scopus (1640) Google Scholar, 12Li Q. Van Antwerp D. Mercurio F. Lee K.F. Verma I.M. Science. 1999; 284: 321-325Crossref PubMed Scopus (857) Google Scholar, 27Takeda K. Takeuchi O. Tsujimura T. Itami S. Adachi O. Kawai T. Sanjo H. Yoshikawa K. Terada N. Akira S. Science. 1999; 284: 313-316Crossref PubMed Scopus (538) Google Scholar, 28Yan S.F. Fujita T. Lu J. Okada K. Shan Zou Y. Mackman N. Pinsky D.J. Stern D.M. Nat. Med. 2000; 6: 1355-1361Crossref PubMed Scopus (402) Google Scholar). Human umbilical vein endothelial cells were purchased from Clonetics Corp. and maintained in EGM media (Cambrex, Gaithersburg, MD). The IKKi, IKK2, IKK1, NEMO, p65, p50, c-Rel, C/EBPβ, and C/EBPδ antibodies were purchased from Santa Cruz. LPS (Escherichia coli 0111:B4) was purchased from List Biological Laboratories. Total RNA was prepared by using TRIzol reagent (Invitrogen). The NF-κB, Oct-1, and C/EBP gel shift oligonucleotides were from Santa Cruz. RNA oligonucleotides were purchased from Dharmacon Research. Double-stranded small interfering RNAs (siRNAs) (IKKi, 5′-GUGAAGGUCUUCAACACUACC-3′× 5′-UAGUGUUGAAGACCUUCACAG-3′; firefly luciferase as a nonspecific control, 5′-CGUACGCGGAAUACUUCGAAA-3′× 5′-UCGAAGUAUUCCGCGUACGUG-3′) were prepared and used for transfection of human umbilical vein endothelial cells (∼5 × 106 cells per transfection) by using electroporation performed as described (29Gitlin L. Karelsky S. Andino R. Nature. 2002; 418: 430-434Crossref PubMed Scopus (520) Google Scholar). Assays—Samples of total RNA (10 μg) were analyzed by Northern blot as described (13Shimada T. Kawai T. Takeda K. Matsumoto M. Inoue J. Tatsumi Y. Kanamaru A. Akira S. Int. Immunol. 1999; 11: 1357-1362Crossref PubMed Scopus (312) Google Scholar). A blot was hybridized with specific antisense oligonucleotide labeled by T4 polynucleotide kinase using [γ-32P]ATP. Nuclear extracts were prepared and used for electrophoretic mobility shift assay (EMSA) as described (30Kravchenko V.V. Pan Z. Han J. Herbert J.-M. 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Chromatin immunoprecipitation (ChIP) were performed as described (31Ivanov V.N. Bhoumik A. Krasilnikov M. Raz R. Owen-Schaub L.B. Levy D. Horvath C.M. Ronai Z. Mol. Cell. 2001; 7: 517-528Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar). The antibodies specific for C/EBPβ, C/EBPδ, or p65 were used for the ChIP assay. The levels of IKKi or IκBα promoter DNA were determined by PCR using oligonucleotides from the 5′-untranslated region of IKKi gene (5′-TCTGTAAAGCAATGAGCAAG-3′; 5′-AGGAAGCTGACACAGTGTGG-3′) or IκBα gene (5′-AGGGAAAGAAGGGTTCTTGC-3′; 5′-CTGACTGTTGTGGGCTCG-3′). Metabolic Labeling—106 cells were plated per 60-mm dish. On the second day, the cells were washed 3 times with phosphate-free Dulbecco's modified Eagle's medium containing 5% of dialyzed fetal bovine serum and then incubated in the same medium containing 400 mCi/ml [32P]H3PO4 for 4 h. The last 2 h some cells were incubated with 100 ng/ml LPS. The cells were then washed three times with cold phosphate-buffered saline and used for preparation of nuclear extract according to EMSA protocol (see above). The nuclear extracts were diluted by the addition of 10 volumes of standard radioimmune precipitation assay buffer, and C/EBPδ or p65 proteins were recovered by immunoprecipitation with specific antibodies as indicated in Fig. 5C. The immunoprecipitates were analyzed by SDS-PAGE and autoradiography. We first sought an experimental system to investigate relationships between the C/EBP and NF-κB pathways. The promoter region of C3 gene contains C/EBP sites (32Wilson D.R. Juan T.S. Wilde M.D. Fey G.H. Darlington G.J. Mol. Cell. Biol. 1990; 10: 6181-6191Crossref PubMed Scopus (66) Google Scholar); it is known that C3 expression is regulated by C/EBPδ (26Juan T.S.-C. Wilson D.R. Wilde M.D. Darlington G.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2584-2588Crossref PubMed Scopus (126) Google Scholar). Despite the fact that there are no identifiable NF-κB sites in the C3 promoter, transcription of the C3 gene is induced by NF-κB activators including LPS (9Pahl H.L. Oncogene. 1999; 18: 6853-6866Crossref PubMed Scopus (3464) Google Scholar, 33Rus H.G. Kim L.M. Niculesen F.I. Shin M.L. J. Immunol. 1992; 148: 928-933PubMed Google Scholar). Thus, we reasoned that measurements of LPS induction of C3 mRNA would be an appropriate marker for initial studies to investigate possible relationships between the NF-κB and C/EBP pathways. Here we have used MEFs from mice bearing targeted deletions of genes encoding IKK2, p65, and IKKi. We first examined induction of C3 mRNA in LPS-treated MEFs derived from IKK2–/– and control (IKK2+/+) embryos; the induction of C3 mRNA was observed in control cells but not in the IKK2-deficient cells (Fig. 1A). In contrast, LPS-mediated induction of c-jun mRNA was nearly identical in both types of MEFs. Moreover, as shown here and as previously noted, IKK2–/– MEFs showed LPS-induced IκBα mRNA and NF-κB DNA binding activity that was partially reduced when IKK2–/– and IKK2+/+ cells were compared (Ref. 12Li Q. Van Antwerp D. Mercurio F. Lee K.F. Verma I.M. Science. 1999; 284: 321-325Crossref PubMed Scopus (857) Google Scholar; Fig. 1, A and B). Similar findings were noted in experiments with spontaneously imMEFs derived from IKK2–/– and control cells (Fig. 1C). Thus, the absence of IKK2 revealed a deficiency in LPS-induced C3 induction that is not likely to result from loss of LPS responsiveness. To further probe the role of the NF-κB pathway we also examined the effects of p65 deficiency on LPS induction of C3 and IκBα mRNA. LPS-mediated induction of both IκBα and C3 mRNAs was completely abolished in p65–/– imMEFs (Fig. 1D). These data suggest that regulation of C3 gene expression requires an intact NF-κB pathway. However, we hypothesize that the role of NF-κB involves indirect mechanisms and requires additional gene expression under the control of NF-κB. In fact it has been shown that protein synthesis is required for LPS-induced expression of C3 mRNA (34Cardinaux J.-R. Allaman I. Magistretti P.J. Glia. 2000; 29: 91-97Crossref PubMed Scopus (159) Google Scholar). The observation that LPS induces IKKi mRNA (13Shimada T. Kawai T. Takeda K. Matsumoto M. Inoue J. Tatsumi Y. Kanamaru A. Akira S. Int. Immunol. 1999; 11: 1357-1362Crossref PubMed Scopus (312) Google Scholar) prompted us to evaluate the effects of IKKi deletion on C3 gene expression. Thus, we next measured LPS-induced C3, IκBα, and IKKi mRNA in IKKi–/– and control MEFs as well as in IKK2–/– cells (Fig. 2A). As shown above, LPS-mediated induction of IκBα mRNA was reduced in cells lacking IKK2, whereas IKKi deficiency had no effect on LPS-induced expression of IκBα mRNA. In contrast, the induction of C3 mRNA was abolished in the IKKi–/– cells as well as in the IKK2–/– cells. Furthermore, we failed to observe induction of IKKi mRNA in the IKK2–/– cells. Nearly identical results were also obtained in similar experiments with LPS-stimulated IKKi–/– and IKKi+/+ imMEFs derived from the corresponding primary isolates of MEFs (Fig. 2B). As an additional specificity control, we also examined the LPS responses in MEFs derived from Egr-1-deficient embryos (28Yan S.F. Fujita T. Lu J. Okada K. Shan Zou Y. Mackman N. Pinsky D.J. Stern D.M. Nat. Med. 2000; 6: 1355-1361Crossref PubMed Scopus (402) Google Scholar). The LPS-mediated induction of C3 or IKKi mRNA was practically identical in Egr1–/– and Egr1+/+ cells (Fig. 2C), emphasizing the specificity of the effects observed here with IKK2–/–, p65–/–, and IKKi–/– MEFs. Thus, IKKi appears to be a key molecule for transcriptional induction of C3 gene in response to LPS, and its expression requires an intact NF-κB pathway. To further define how IKKi participates in gene regulation we performed the following experiments. Extracts from IKK2–/–, IKK2+/+, IKKi–/– (as negative control), or IKKi+/+ (as positive control) were immunoprecipitated to enrich the samples for IKKi, and the resultant immunoprecipitates were electrophoresed and then subjected to Western blot analysis (Fig. 3A). As was expected, expression of IKKi protein was not detected in IKKi–/– cells. In contrast, IKKi+/+ or IKK2+/+ cells showed detectable IKKi protein expression that was increased after LPS addition. Compared with IKK2+/+ or IKKi+/+ cells, the basal level of IKKi protein was significantly reduced in IKK2-deficient cells. Importantly, LPS addition failed to up-regulate IKKi expression in this cell type. These data together with Northern blot studies support the contention that IKK2 is required for the inducible expression of IKKi mRNA and protein. These data prompted us to address the question of how IKKi regulates LPS-induced C3 gene expression. Specifically. we asked whether the failure to induce C3 results from the absence of IKKi protein or whether essential signaling including expression and/or activation of IKK2 is also affected by IKKi deficiency. To address these issues, we used Western blot analysis to compare the levels of IKK2 protein in extracts from IKKi–/–, IKKi+/+, IKK2–/– (as negative control), or IKK2+/+ (as positive control) cells. The results showed that IKKi–/–, IKKi+/+, and IKK2+/+ cells express nearly identical levels of IKK2 protein (Fig. 3B). Expression of IKK1 and NEMO subunits of the IKK complex was also unchanged in IKKi–/– cells (Fig. 3B). Thus, the absence of IKKi does not reduce the protein expression of other key members of the IKK complex. Furthermore, it is unlikely that IKKi is required for activation of IKK complex because LPS treatment up-regulated the kinase activity of the IKK similarly in IKKi–/– and IKKi+/+ cells (Fig. 3C). Thus, the absence of IKKi does not alter signaling that is directly related to NF-κB activation. The same extracts were subjected in parallel to an immunoprecipitation/kinase assay for IKKi. In addition LPS treatment did not alter the kinase activity of IKKi (Fig. 3C), in keeping with observations of others that pro-inflammatory mediators do not alter IKKi kinase activity but, rather, up-regulate its expression (13Shimada T. Kawai T. Takeda K. Matsumoto M. Inoue J. Tatsumi Y. Kanamaru A. Akira S. Int. Immunol. 1999; 11: 1357-1362Crossref PubMed Scopus (312) Google Scholar). To investigate whether IKKi deficiency alters the rate and extent of LPS-induced expression of a variety of genes associated with innate immune and inflammatory responses we measured LPS-induced expression of TNF, IL-1, IL-6, IP-10, RANTES, and COX-2 mRNA. LPS treatment increased mRNA expression for each of these genes in IKKi+/+ cells (Fig. 4A); we also showed that TNF, IL-1, and IL-6 protein levels also increased (Fig. 4, B and C). In contrast IKKi deficiency resulted in a marked reduction of LPS-induced mRNA expression for each of this group of genes (Fig. 4A). Parallel decreases in protein expression for TNF, IL-1, and IL-6 were also noted (Figs. 4, B and C). The fact that both cell lines (IKKi–/– and IKKi+/+
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