A Novel IκB Protein, IκB-ζ, Induced by Proinflammatory Stimuli, Negatively Regulates Nuclear Factor-κB in the Nuclei

Abstract

The transcription factor nuclear factor-κB (NF-κB) plays crucial roles in a wide variety of cellular functions and its activity is strictly regulated by cytosolic inhibitors known as IκBs. We here report a new member of the IκB protein family, IκB-ζ, harboring six ankyrin repeats at its carboxyl terminus. IκB-ζ mRNA is strongly induced after stimulation by lipopolysaccharide. The induction of IκB-ζ is also observed by stimulation with interleukin-1β but not by tumor necrosis factor-α. In contrast to cytosolic IκB-α, -β, and -ε, the induced IκB-ζ localizes in the nucleus via its amino-terminal region, which shows no homology with other proteins. Transiently expressed IκB-ζ inhibits the NF-κB activity without affecting the nuclear translocation of NF-κB upon stimulation. The expressed IκB-ζ preferentially associates with the NF-κB subunit p50 rather than p65 and recombinant IκB-ζ proteins inhibit the DNA binding of the p65/p50 heterodimer and the p50/p50 homodimer. Thus, IκB-ζ negatively regulates NF-κB activity in the nucleus, possibly in order to prevent excessive inflammation. Moreover, transfection of IκB-ζ renders cells more susceptible to apoptosis induced by tumor necrosis factor-α. The proapoptotic activity of IκB-ζ further suggests that it might be one of key regulators for inflammation and other biologically relevant processes. The transcription factor nuclear factor-κB (NF-κB) plays crucial roles in a wide variety of cellular functions and its activity is strictly regulated by cytosolic inhibitors known as IκBs. We here report a new member of the IκB protein family, IκB-ζ, harboring six ankyrin repeats at its carboxyl terminus. IκB-ζ mRNA is strongly induced after stimulation by lipopolysaccharide. The induction of IκB-ζ is also observed by stimulation with interleukin-1β but not by tumor necrosis factor-α. In contrast to cytosolic IκB-α, -β, and -ε, the induced IκB-ζ localizes in the nucleus via its amino-terminal region, which shows no homology with other proteins. Transiently expressed IκB-ζ inhibits the NF-κB activity without affecting the nuclear translocation of NF-κB upon stimulation. The expressed IκB-ζ preferentially associates with the NF-κB subunit p50 rather than p65 and recombinant IκB-ζ proteins inhibit the DNA binding of the p65/p50 heterodimer and the p50/p50 homodimer. Thus, IκB-ζ negatively regulates NF-κB activity in the nucleus, possibly in order to prevent excessive inflammation. Moreover, transfection of IκB-ζ renders cells more susceptible to apoptosis induced by tumor necrosis factor-α. The proapoptotic activity of IκB-ζ further suggests that it might be one of key regulators for inflammation and other biologically relevant processes. nuclear factor-κB lipopolysaccharide interleukin-1 tumor necrosis factor-α electrophoretic mobility shift assay IκB kinase toll-like receptor NF-κB-inducing kinase The nuclear factor-κB (NF-κB)1 is an evolutionarily conserved transcription factor that controls the expression of a large number of genes involved in a wide variety of biological functions, such as inflammation, stress and immune responses, embryonic development, and apoptosis (1Ghosh S. May M.J. Kopp E.B. Annu. Rev. Immunol. 1998; 16: 225-260Crossref PubMed Scopus (4550) Google Scholar, 2Hatada E.N. Krappmann D. Scheidereit C. Curr. Opin. Immunol. 2000; 12: 52-58Crossref PubMed Scopus (311) Google Scholar). The NF-κB activity is attributed to the homo- and heterodimers of Rel/NF-κB family proteins. In resting cells, NF-κB is present in the cytosol as an inactive complex with its inhibitor proteins, IκBs. Exposure of cells to various proinflammatory stimuli including microbial cell-wall components, viral products, cytokines, or cellular stresses induces proteasome-mediated degradation of IκB following its phosphorylation and ubiquitination. Thus liberated NF-κB translocates into the nucleus where it activates the transcription of target genes, such as cytokines/chemokines, cell adhesion molecules, the nitric-oxide synthase, or co-stimulatory molecules (1Ghosh S. May M.J. Kopp E.B. Annu. Rev. Immunol. 1998; 16: 225-260Crossref PubMed Scopus (4550) Google Scholar, 2Hatada E.N. Krappmann D. Scheidereit C. Curr. Opin. Immunol. 2000; 12: 52-58Crossref PubMed Scopus (311) Google Scholar). Although the activation of NF-κB is essential for the initiation of inflammation to eliminate pathogens, its activity must be tightly regulated: too strong or prolonged activation of NF-κB leads to overproduction of cytokines, which culminates in the induction of fever or life-threatening shock, and thus is seriously detrimental to the host. In order to regulate the activity, NF-κB activates genes, such as IκB-α (3Brown K. Park S. Kanno T. Franzoso G. Siebenlist U. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2532-2536Crossref PubMed Scopus (574) Google Scholar, 4Sun S.-C. Ganchi P.A. Ballard D.W. Greene W.C. Science. 1993; 259: 1912-1915Crossref PubMed Scopus (947) Google Scholar, 5de Martin R. Vanhove B. Cheng Q. Hofer E. Csizmadia V. Winkler H. Bach F.H. EMBO J. 1993; 12: 2773-2779Crossref PubMed Scopus (290) Google Scholar, 6Scott M.L. Fujita T. Liou H.-C. Nolan G.P. Baltimore D. Genes Dev. 1993; 7: 1226-1276Crossref Scopus (296) Google Scholar) and A20 (7Opipari A.W. Boguski M.S. Dixit V.M. J. Biol. Chem. 1990; 265: 14705-14708Abstract Full Text PDF PubMed Google Scholar, 8Jaattela M. Mouritzen H. Elling F. Bastholm L. J. Immunol. 1996; 156: 1166-1173PubMed Google Scholar), whose products inhibit the activity or activation of NF-κB itself, thus comprising negative-feedback loop. Lipopolysaccharide (endotoxin or LPS), a major cell wall component of Gram-negative bacteria, is one of the strongest stimulators to activate NF-κB for monocytes and macrophages, which play critical roles in innate immunity (9Ulevitch R.J. Immunol. Res. 2000; 21: 49-54Crossref PubMed Scopus (71) Google Scholar). The cellular activation triggered by LPS and some of the other microbial cell-wall components has recently been shown to be mediated by members of the toll-like receptor (TLR) family (10Medzhitov R. Preston-Hurlburt P. Janeway Jr., C.A. Nature. 1997; 388: 394-397Crossref PubMed Scopus (4344) Google Scholar, 11Rock F.L. Hardiman G. Timans J.C. Kastelein R.A. Bazan J.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 588-593Crossref PubMed Scopus (1430) Google Scholar, 12Aderem A. Ulevitch R.J. Nature. 2000; 406: 782-787Crossref PubMed Scopus (2589) Google Scholar, 13Akira S. Biochem. Soc. Trans. 2000; 28: 551-556Crossref PubMed Scopus (97) Google Scholar). TLRs share a cytoplasmic domain (TIR domain) with homology to that of the interleukin (IL)-1 receptor (IL-1R) or the IL-1 receptor accessory protein (IL-1RAcP), and it is suggested that the intracellular pathways for the LPS and IL-1 signaling are shared. In this study, to better understand the mechanisms regulating inflammation or NF-κB activity, we screened for genes that are induced by proinflammatory stimuli on these cells. During the screening, we identified a protein with partial similarity with the IκB protein family. The protein, induced by the proinflammatory stimuli, negatively regulates the NF-κB activity in the nucleus, whereas typical IκB proteins, represented by IκB-α, -β, and -ε, are constitutively expressed and present in the cytosol to prevent nuclear translocation of NF-κB. The distinct characteristics of this protein from the cytosolic IκB proteins would expand our understanding of mechanisms for the regulation of NF-κB and inflammatory reactions. Peritoneal macrophages were collected by peritoneal lavage with Hank's balanced salt solution (Life Technologies) at 4 or 5 days after intraperitoneal injection of 2 ml of 3% sterile thioglycollate (Difco) into 8–10-week-old mice and maintained in RPMI 1640 medium containing 10% heat-inactivated fetal calf serum supplemented with 100 units/ml penicillin and 100 µg/ml streptomycin. The other cells were cultured in Dulbecco's modified Eagle's medium with the heat-inactivated fetal calf serum, penicillin, and streptomycin. LPS from Escherichia coli0111:B4 was purchased from List Biological Laboratories, Inc. Tumor necrosis factor (TNF)-α and IL-1β were from Genzyme Corp. Cambridge, MA. Subtractive hybridization was performed using polymerase chain reaction-select cDNA subtraction kit (CLONTECH Laboratories, Inc.) with cDNAs from LPS-treated (1 µg/ml, 1 h) and untreated RAW264.7 cells, according to the manufacturer's instructions. A partial cDNA fragment obtained by the subtractive hybridization was used as a probe for Northern blotting to confirm the induction by LPS and for the screening of a cDNA library constructed from LPS-stimulated RAW264.7 cells. The 5′ non-coding region was isolated by 5′-rapid amplification of cDNA ends to confirm the initiation codon. The expression plasmids for transfection were constructed by subcloning the DNA fragments obtained by polymerase chain reaction into pcDNA3 (Invitrogen Corp.), pCI (Promega Corp.), or pEGFP-N1 (CLONTECH Laboratories, Inc.) with or without NH2-terminal Myc- or Flag-tag. pRK-IKK-β-Flag (14Woronicz J.D. Gao X. Cao Z. Rothe M. Goeddel D.V. Science. 1997; 278: 866-869Crossref PubMed Scopus (1060) Google Scholar) and pcDNA3-Flag-NIK (15Malinin N.L. Boldin M.P. Kovalenko A.V. Wallach D. Nature. 1997; 385: 540-544Crossref PubMed Scopus (1157) Google Scholar) were described previously. The entire coding region or the COOH-terminal region (amino acids 315–629) of IκB-ζ was subcloned into pMALg (provided by Dr. T. Ito) or pGEX-2T (Amersham Pharmacia Biotech AB), respectively, to prepare recombinant proteins. RAW264.7 or HEK293 cells were transfected with the indicated expression plasmid together with pELAM1-Luc (16Irie T. Muta T. Takeshige K. FEBS Lett. 2000; 467: 160-164Crossref PubMed Scopus (161) Google Scholar) and pRL-TK (Promega Corp.) by the FuGENE™ 6 method (Roche Molecular Biochemicals) or the calcium-phosphate method (17Chen C. Okayama H. Mol. Cell. Biol. 1987; 7: 2745-2752Crossref PubMed Scopus (4799) Google Scholar), respectively. Two days after transfection, the cells were stimulated as indicated at 37 °C for 6 h. Luciferase activities were measured by using the Dual-luciferase Reporter System (Promega Corp.). EMSA was performed essentially as described in Ref. 18Yamamoto H. Hanada K. Nishijima M. Biochem. J. 1997; 325: 223-228Crossref PubMed Scopus (35) Google Scholar with annealed oligonucleotides (5′-TTAACAGAGGGGACTTTCCGAG-3′ and 5′-GGCTCGGAAAGTCCCCTCTGTTAA-3′) as a probe. HeLa and COS-7 cells grown on a coverslip were transfected with the indicated expression vector with FuGENE™ 6. Twenty-four hours after transfection, the cells were fixed with 4% paraformaldehyde in phosphate-buffered saline at room temperature for 1 h. The cells were permeablized with 0.1% Triton X-100 and then incubated with anti-p65 (Santa Cruz Biotechnology), anti-Myc (9E10, Roche Molecular Biochemicals), or anti-Flag (M2, Sigma) in phosphate-buffered saline containing 2% fetal calf serum. After washing, the cells were further incubated with Alexa 488-conjugated goat anti-mouse IgG F(ab′)2 fragment (Molecular Probes) and/or Cy™ 3-labeled goat anti-rabbit IgG (Amersham Pharmacia BiotechAB). Cells were stained with 4′,6-diamidino-2-phenylindole before analyses under a fluorescent microscopy. HEK293T cells were transfected with the indicated expression plasmids by the calcium phosphate method (17Chen C. Okayama H. Mol. Cell. Biol. 1987; 7: 2745-2752Crossref PubMed Scopus (4799) Google Scholar). Cells were lysed with phosphate-buffered saline containing 1% Nonidet P-40, 0.1% sodium deoxycholate, 0.01% SDS, and 10 µg/ml aprotinin. Immunoprecipitate with anti-Flag (M2) or anti-Myc (9E10) antibody was resolved by 10% SDS-polyacrylamide gel electrophoresis and probed with the indicated antibody. Cells were transfected with the indicated plasmid with LipofectAMINE™ 2000 (Life Technologies). Twenty-four hours after transfection, cells were treated with 50 ng/ml TNF-α for 24 h and stained with annexin V-biotin (BD Pharmingen), followed by streptavidin-TRIcolor (Caltag Laboratories) and analyzed by flow cytometry. To identify genes that were up-regulated after LPS stimulation in macrophages, subtractive hybridization was performed with cDNAs from LPS-treated and untreated murine macrophage-like cell line RAW264.7. Among the clones specifically induced by LPS treatment of the cells, one clone was identified to encode a protein containing the ankyrin repeats as found in IκBs. The protein was named IκB-ζ and further analyzed, since it exhibited similarity to known IκB family proteins both in structure and function but had distinct characteristics (see below). No or mRNA for IκB-ζ was in the resting cells, but it was strongly induced by LPS treatment in RAW264.7 cells A induction was observed with peritoneal macrophages 1 The induction of IκB-ζ at 1 h after the stimulation. The of the mRNA then but expression was 24 and h after the stimulation 1 as ng/ml LPS IκB-ζ mRNA and the induction was by a that the biological of LPS 1 In order to the for the the expression of IκB-ζ was with cells stimulated with proinflammatory cytokines, tumor necrosis factor (TNF)-α and mRNA for IκB-ζ was also induced by IL-1β treatment in cells, with a to that observed in the LPS-stimulated cells 1 In its induction by TNF-α was 1 To the cells were by these stimuli, the expression of IκB-α was in as a for a typical induced by NF-κB (3Brown K. Park S. Kanno T. Franzoso G. Siebenlist U. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2532-2536Crossref PubMed Scopus (574) Google Scholar, 4Sun S.-C. Ganchi P.A. Ballard D.W. Greene W.C. Science. 1993; 259: 1912-1915Crossref PubMed Scopus (947) Google Scholar, 5de Martin R. Vanhove B. Cheng Q. Hofer E. Csizmadia V. Winkler H. Bach F.H. EMBO J. 1993; 12: 2773-2779Crossref PubMed Scopus (290) Google Scholar, 6Scott M.L. Fujita T. Liou H.-C. Nolan G.P. Baltimore D. Genes Dev. 1993; 7: 1226-1276Crossref Scopus (296) Google Scholar). The IκB-α mRNA was strongly induced by that these stimuli induced cells to activate NF-κB. To the functions of IκB-ζ, cDNAs for IκB-ζ were isolated from a cDNA library constructed from LPS-stimulated RAW264.7 cells. The of the partial cDNA clones an for acids of IκB-ζ the in the cDNA was by 5′-rapid amplification of cDNA IκB-ζ a COOH-terminal region with 6 which was to that of 2 the other the NH2-terminal region not homology to proteins in the expression of IκB-ζ in the was by Northern a mRNA for IκB-ζ was in the and but was in the and 2 A strong with a was in the but its During the of the following a protein to IκB-ζ was as H. K. K. M. M. FEBS Lett. 2000; PubMed Scopus Google Scholar). of the IκB proteins thus identified repeats of the ankyrin which with the homology domain of the Rel/NF-κB proteins to nuclear and DNA To the of IκB-ζ in the activation of we transfected plasmid into cells and measured NF-κB activities in to proinflammatory LPS stimulation of RAW264.7 cells induced strong activation of as measured with an of IκB-ζ in a of the NF-κB activity in the stimulated cells To identify the for the we constructed of IκB-ζ and The COOH-terminal acids 315–629) of IκB-ζ containing the region the NF-κB The NH2-terminal region acids without the but activity A LPS stimulation of RAW264.7 cells to strong induction of IκB-ζ 1 we the of expression of IκB-ζ on the NF-κB activities upon LPS by the induction with for We transfected a for or into RAW264.7 cells and stimulated with LPS In contrast to the of the NF-κB activities were by the mRNA for IκB-ζ, possibly by of induced IκB-ζ the the had on NF-κB Thus, LPS stimulation of the cells, to activation of induces IκB-ζ which is for the NF-κB In to NF-κB activity, transfected IκB-ζ the and NF-κB activities and NF-κB is by degradation of cytosolic IκB-α or -β, which is induced by phosphorylation by (14Woronicz J.D. Gao X. Cao Z. Rothe M. Goeddel D.V. Science. 1997; 278: 866-869Crossref PubMed Scopus (1060) Google Scholar). In order to the signaling of the IκB-ζ of we NF-κB by or the of which is a known for (15Malinin N.L. Boldin M.P. Kovalenko A.V. Wallach D. Nature. 1997; 385: 540-544Crossref PubMed Scopus (1157) Google Scholar), and the of the of of IκB-ζ both and activation of NF-κB and the activity of transfected p65/p50 of the NF-κB was by IκB-ζ The and also had this that IκB-ζ inhibits NF-κB at the than The activity of might be to expression of this with the IκB-ζ or not The to its on the nuclear translocation of NF-κB upon stimulation. HeLa cells transfected with IκB-α or IκB-ζ were treated with and then nuclear translocation of the p65 subunit of NF-κB was p65 in the cytosol in cells 4 and was into the nucleus upon TNF-α stimulation cells in of IκB-α in of the nuclear translocation of p65 by the stimulation 4 the transfected cells shown by with the In contrast to the of IκB-α, IκB-ζ not the translocation of p65 4 We found that IκB-ζ was in the nucleus, whereas IκB-α the cells 4 The were observed in COS-7 cells 4 Thus, IκB-ζ inhibits the NF-κB in the nucleus rather than affecting its nuclear We further the of the of IκB-ζ 4 the NH2-terminal 4 nuclear to that of IκB-ζ the COOH-terminal was the entire cells, that the nuclear is present in the NH2-terminal the were also with protein proteins not which had activities We also found that the cytosolic the nuclear translocation of NF-κB upon stimulation not which might the activity of this A To further the mechanisms the of NF-κB by IκB-ζ, we the of IκB-ζ with the NF-κB IκB-α or IκB-ζ was with p65 or p50 in HEK293T cells. The cell were with anti-Flag followed by with anti-Myc antibody. IκB-ζ was found to with but its with p65 was was observed IκB-α and both p65 and and not IκB-ζ a for p50 to be since the ankyrin region of which is to that of IκB-ζ, been to preferentially with p50 rather than p65 M. Scheidereit C. Nature. PubMed Scopus Google Scholar). The nuclear and its with the p50 subunit that IκB-ζ might the DNA binding activity of NF-κB. In the of recombinant IκB-ζ on the DNA binding activity of NF-κB was by NF-κB probe was incubated with nuclear from RAW264.7 cells treated with LPS The was with the probe and by anti-p65 or that it represented the p65/p50 heterodimer not The of a recombinant protein protein with the IκB-ζ the of the 6 protein with the COOH-terminal ankyrin repeats exhibited the activity, whereas no was observed with protein or we the p65/p50 heterodimer and the p50 by both p65 and p50 or p50 into HEK293 cells. The nuclear from the cells transfected with p65 and p50 6 of which was to be the p65/p50 heterodimer and the p50/p50 by analyses not The recombinant IκB-ζ protein the of both 6 The COOH-terminal also exhibited the activity and the of the p50 was than that of the p65/p50 heterodimer 6 Thus, IκB-ζ inhibits the DNA binding of p65/p50 heterodimers as as the p50 its with the p50 The activity of IκB-ζ on which as an factor M. PubMed Scopus Google Scholar, Baltimore D. Science. 1996; PubMed Scopus Google Scholar, Jr., Science. 1996; PubMed Scopus Google Scholar, Martin T. Science. 1996; PubMed Scopus Google Scholar), suggests that IκB-ζ might also be involved in the regulation of apoptosis upon inflammatory stimuli or in other relevant processes. To this we the of IκB-ζ expression on HeLa cells transfected with an a of IκB-α, or IκB-ζ were stimulated with TNF-α for 24 h and were under a as the cells the IκB-α, the cells exhibited of cells with more we stained cells with and analyzed by flow cytometry. transfected cells were analyzed after TNF-α the cells exhibited of annexin cells the other the cells analyzed under the of cells. The that expression of IκB-ζ was to that of the IκB-α The IκB-ζ expression not apoptosis without the stimulation. Thus, IκB-ζ possibly by the NF-κB We described in this a protein to the IκB family. No or expression of IκB-ζ is observed in macrophages and it is induced by a of proinflammatory stimuli, such as LPS and which activate containing the and respectively. Although both the and signaling activate NF-κB and protein TNF-α induces induction of The induction of IκB-ζ was by and that NF-κB is involved in the induction of T. and K. of the NF-κB subunit p65 in of IκB-α but induction of IκB-ζ mRNA was not The that IκB-ζ is induced by a from that of IκB-α, with NF-κB activation for the A transcription that is essential for the induction of IκB-ζ to be specifically by the signaling the cytoplasmic of TLRs and of IκB-ζ the NF-κB activities induced by LPS and IL-1 as as TNF-α regulation of NF-κB would be to prevent inflammatory which to or other detrimental The of for IκB-ζ in of NF-κB activity upon stimulation with LPS of IκB-ζ in the regulation of inflammatory reactions. IκB-ζ expression was induced with as as ng/ml LPS and was for at 2 days and it play a in the of LPS in cells. LPS and IL-1β stimulation is at in by IκB-ζ, since both stimuli activate IκB-ζ induction J. Immunol. 2000; PubMed Scopus Google Scholar). The activities of IκB-ζ or than of IκB-α or -β, but this might be to the that IκB-ζ or was not as expressed as the other proteins not IκB-ζ might be to degradation in the cells via its NH2-terminal region it not to the for phosphorylation by for the ubiquitination. In contrast to IκB-α, -β, and -ε, of which are in the the induced IκB-ζ localizes in the nucleus the NH2-terminal region where it negatively regulates the DNA binding of NF-κB via with the p50 subunit and are with the that IκB-ζ not the translocation of NF-κB into the H. K. K. M. M. FEBS Lett. 2000; PubMed Scopus Google Scholar) isolated the proteins, and the of which is to The protein to was also more recently by as an protein H. A. K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The induction by LPS stimulation and the nuclear of IκB-ζ described in this is with also in cells that transfection of the cDNA in the of which is also regulated by NF-κB. The be attributed to assay or cell IκB-ζ might also be involved in some activation as in the of which has been shown to as a of transcription V. Franzoso G. V. Park S. Kanno T. K. Siebenlist U. Cell. 1993; Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). IκB-ζ exhibited the proapoptotic activity upon TNF-α stimulation which as a receptor for microbial products to activate has been to apoptosis upon the stimulation S. B. J.D. P. A. Science. PubMed Scopus Google Scholar, P. A. EMBO J. 2000; PubMed Scopus Google Scholar). also a IκB-ζ expression is induced by the activation of and would regulate NF-κB activity and The proapoptotic activity of IκB-ζ was to that of IκB-α its activity on NF-κB was The that IκB-ζ other activities to apoptosis than NF-κB. IκB-ζ was induced by both LPS and IL-1β but not by TNF-α The of the IκB-ζ induction that these proinflammatory cytokines, IL-1β and activities that would in distinct The of the induction of IκB-ζ was by the induction of IκB-α, an the induction of IκB-α was for in cells, where IκB-ζ induction was whereas the induction was in or LPS-treated cells 1 of are inflammation, and a cell is to a of The of the cellular responses, such as activation of NF-κB and induction of IκB-ζ, the of the cells, including NF-κB regulates not inflammation but also wide of biologically IκB-ζ might play critical roles in such and analyses for the IκB-ζ induction and its function would more on the regulation of inflammatory the of inflammatory and other biological regulated by NF-κB. We D. V. Goeddel D. Wallach of and T. for the expression plasmids for and and respectively. We are to T. for of IκB-α plasmids and for