Lin Feng Chen

Smads are signal transducers for members of the transforming growth factor-β (TGF-β) superfamily. Upon ligand stimulation, receptor-regulated Smads (R-Smads) are phosphorylated by serine/threonine kinase receptors, form complexes with common-partner Smad, and translocate into the nucleus, where they regulate the transcription of target genes together with other transcription factors. Polyomavirus enhancer binding protein 2/core binding factor (PEBP2/CBF) is a transcription factor complex composed of α and β subunits. The α subunits of PEBP2/CBF, which contain the highly conserved Runt domain, play essential roles in hematopoiesis and osteogenesis. Here we show that three mammalian α subunits of PEBP2/CBF form complexes with R-Smads that act in TGF-β/activin pathways as well as those acting in bone morphogenetic protein (BMP) pathways. Among them, PEBP2αC/CBFA3/AML2 forms a complex with Smad3 and stimulates transcription of the germline Ig Cα promoter in a cooperative manner, for which binding of both factors to their specific binding sites is essential. PEBP2 may thus be a nuclear target of TGF-β/BMP signaling. Smads are signal transducers for members of the transforming growth factor-β (TGF-β) superfamily. Upon ligand stimulation, receptor-regulated Smads (R-Smads) are phosphorylated by serine/threonine kinase receptors, form complexes with common-partner Smad, and translocate into the nucleus, where they regulate the transcription of target genes together with other transcription factors. Polyomavirus enhancer binding protein 2/core binding factor (PEBP2/CBF) is a transcription factor complex composed of α and β subunits. The α subunits of PEBP2/CBF, which contain the highly conserved Runt domain, play essential roles in hematopoiesis and osteogenesis. Here we show that three mammalian α subunits of PEBP2/CBF form complexes with R-Smads that act in TGF-β/activin pathways as well as those acting in bone morphogenetic protein (BMP) pathways. Among them, PEBP2αC/CBFA3/AML2 forms a complex with Smad3 and stimulates transcription of the germline Ig Cα promoter in a cooperative manner, for which binding of both factors to their specific binding sites is essential. PEBP2 may thus be a nuclear target of TGF-β/BMP signaling. transforming growth factor-β bone morphogenetic protein receptor-regulated Smad common-partner Smad polyomavirus enhancer binding protein 2 core binding factor immunoglobulin Cα TGF-β type I receptor BMP type IB receptor wild-type TGF-β-responsive element glutathione S-transferase Mad homology electrophoretic mobility shift assay activation domain Smad proteins are signal transducers for members of the transforming growth factor-β (TGF-β)1 superfamily, which includes TGF-βs, activins, and bone morphogenetic proteins (BMPs) (1Heldin C.-H. Miyazono K. ten Dijke P. Nature. 1997; 390: 465-471Crossref PubMed Scopus (3358) Google Scholar,2Massagué J. Annu. Rev. Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3999) Google Scholar). Smads are classified into three subgroups, i.e.receptor-regulated Smads (R-Smads), common-partner Smads (Co-Smads), and inhibitory Smads. Smad2 and Smad3 are R-Smads that transmit TGF-β/activin signals, whereas Smad1, Smad5, and Smad8 act as R-Smads mediating BMP signals. Smad4 is the only Co-Smad identified in mammals. Upon ligand stimulation, R-Smads are phosphorylated by the serine/threonine kinase receptors, form complexes with Co-Smad, and translocate into the nucleus, where they cooperatively regulate the transcription of target genes with other transcription factors, including Xenopus FAST1 and its mammalian homologues (3Chen X. Rubock M.J. Whitman M. Nature. 1996; 383: 691-696Crossref PubMed Scopus (634) Google Scholar, 4Labbé E. Silvestri C. Hoodless P.A. Wrana J.L. Attisano L. Mol. Cell. 1998; 2: 109-120Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar, 5Zhou S. Zawel L. Lengauer C. Kinzler K.W. Vogelstein B. Mol. Cell. 1998; 2: 121-127Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar) and also the c-Jun/c-Fos complex (6Zhang Y. Feng X.-H. Derynck R. Nature. 1998; 394: 909-913Crossref PubMed Scopus (688) Google Scholar, 7Liberati N.T. Datto M.B. Frederick J.P. Shen X. Wong C. Rougier-Chapman E.M. Wang X.-F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4844-4849Crossref PubMed Scopus (275) Google Scholar). TGF-β is a potent growth inhibitor for most cell types, including hematopoietic cells and lymphocytes. In addition, TGF-β directs class switching to IgA in splenic B cells (8Coffman R.L. Lebman D.A. Shrader B. J. Exp. Med. 1989; 170: 1039-1044Crossref PubMed Scopus (483) Google Scholar, 9Sonoda E. Matsumoto R. Hitoshi Y. Ishii T. Sugimoto M. Araki S. Tominaga A. Yamaguchi N. Takatsu K. J. Exp. Med. 1989; 170: 1415-1420Crossref PubMed Scopus (348) Google Scholar). BMPs play important roles in early embryogenesis and in the induction of bone formation in vivo(10Hogan B.L. Genes & Dev. 1996; 10: 1580-1594Crossref PubMed Scopus (1725) Google Scholar). It is thus important to identify and classify transcription factors that serve as nuclear targets of TGF-β/BMP signals and regulate these biological events. Polyomavirus enhancer binding protein 2/core binding factor (PEBP2/CBF) is a transcription factor complex composed of α and β subunits (11Ito Y. Bae S.-C. Yaniv M. Ghysdael J. Oncogenes as Transcriptional Regulators. Birkäuser Verlag, Basel1997: 107-132Crossref Google Scholar,12Speck N.A. Stacy T. Crit. Rev. Eukaryotic Gene Expression. 1995; 5: 337-364Crossref PubMed Scopus (150) Google Scholar). Three mammalian α subunits have been identified, termed PEBP2αA/CBFA1/AML3 (referred to as αA in this report), PEBP2αB/CBFA2/AML1 (αB), and PEBP2αC/CBFA3/AML2 (αC), whereas only a single β subunit (PEBP2β/CBFB) with several spliced variants is present in mammals. The α subunits of PEBP2, which contain the highly conserved Runt domain, are responsible for binding to DNA and transcription activity. In contrast, the β subunit does not bind to DNA by itself, but it enhances the DNA binding activity of the α subunits by interacting via the Runt domain. PEBP2/CBF plays critical roles in growth and differentiation of cells in certain specific tissues, i.e. αA in bone formation (13Ducy P. Zhang R. Geoffroy V. Ridall A.L. Karsenty G. Cell. 1997; 89: 747-754Abstract Full Text Full Text PDF PubMed Scopus (3668) Google Scholar, 14Komori T. Yagi H. Nomura S. Yamaguchi A. Sasaki K. Deguchi K. Shimizu Y. Bronson R.T. Gao Y.H. Inada M. Sato M. Okamoto R. Kitamura Y. Yoshiki S. Kishimoto T. Cell. 1997; 89: 755-764Abstract Full Text Full Text PDF PubMed Scopus (3678) Google Scholar, 15Otto F. Thornell A.P. Crompton T. Denzel A. Gilmour K.C. Rosewell I.R. Stamp G.W. Beddington R.S. Mundlos S. Olsen B.R. Selby P.B. Owen M.J. Cell. 1997; 89: 765-771Abstract Full Text Full Text PDF PubMed Scopus (2430) Google Scholar) and αB in definitive hematopoiesis (16Okuda T. van Deursen J. Hiebert S.W. Grosveld G. Downing J.R. Cell. 1996; 84: 321-330Abstract Full Text Full Text PDF PubMed Scopus (1613) Google Scholar, 17Wang Q. Stacy T. Binder M. Marin-Padilla M. Sharpe A.H. Speck N.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3444-3449Crossref PubMed Scopus (1036) Google Scholar); αC appears to be important in class switching to IgA because of its ability to activate the germline Ig Cα promoter (18Shi M.J. Stavnezer J. J. Immunol. 1998; 161: 6751-6760PubMed Google Scholar). Abnormalities of the PEBP2 genes are linked to human diseases. Mutations in one allele of the humanPEBP2αA/CBFA1 gene cause human cleidocranial dysplasia syndrome (19Mundlos S. Otto F. Mundlos C. Mulliken J.B. Aylsworth A.S. Albright S. Lindhout D. Cole W.G. Henn W. Knoll J.H. Owen M.J. Mertelsmann R. Zabel B.U. Olsen B.R. Cell. 1997; 89: 773-779Abstract Full Text Full Text PDF PubMed Scopus (1285) Google Scholar, 20Zhang Y.W. Bae S.C. Takahashi E. Ito Y. Oncogene. 1997; 15: 367-371Crossref PubMed Scopus (30) Google Scholar), whereas PEBP2αB/AML1 gene is frequently disrupted by chromosomal translocations in several types of human leukemia (11Ito Y. Bae S.-C. Yaniv M. Ghysdael J. Oncogenes as Transcriptional Regulators. Birkäuser Verlag, Basel1997: 107-132Crossref Google Scholar, 12Speck N.A. Stacy T. Crit. Rev. Eukaryotic Gene Expression. 1995; 5: 337-364Crossref PubMed Scopus (150) Google Scholar). PEBP2 has been shown to interact with several transcription factors and co-activators and support context-dependent transcription of target genes (21Wotton D. Ghysdael J. Wang S. Speck N.A. Owen M.J. Mol. Cell. Biol. 1994; 14: 840-850Crossref PubMed Scopus (199) Google Scholar, 22Kim W.-Y. Sieweke M. Ogawa E. Wee H.-J. Englmeier U. Graf T. Ito Y. EMBO J. 1999; 18: 1609-1620Crossref PubMed Scopus (197) Google Scholar, 23Yagi R. Chen L.-F. Shigesada K. Murakami Y. Ito Y. EMBO J. 1999; 18: 2551-2562Crossref PubMed Scopus (453) Google Scholar). Because BMPs and αA play critical roles in bone formation, and TGF-β and αC in transcription of germline Ig α transcripts required for IgA class switching, we examined the functional cooperation between the PEBP2α subunits and Smads. Our findings suggest that PEBP2α subunits and R-Smads cooperate to synergistically activate transcription in both the TGF-β and BMP signaling pathways, thereby regulating the function of cells in specific tissues upon activation by TGF-β-like factors. FLAG-pcDEF3 and 6Myc-pcDEF3 containing six tandem copies of the Myc-epitope tag were previously described (24Imamura T. Takase M. Nishihara A. Oeda E. Hanai J.-i. Kawabata M. Miyazono K. Nature. 1997; 389: 622-626Crossref PubMed Scopus (873) Google Scholar,25Kawabata M. Inoue H. Hanyu A. Imamura T. Miyazono K. EMBO J. 1998; 17: 4056-4065Crossref PubMed Scopus (249) Google Scholar). The constructions of constitutively active forms of TGF-β type I receptor (TβR-I(TD)) and BMP-type IB receptor (BMPR-IB(QD)), TβR-II, wild-type (WT) Smads, and Smad3(DE) were reported (24Imamura T. Takase M. Nishihara A. Oeda E. Hanai J.-i. Kawabata M. Miyazono K. Nature. 1997; 389: 622-626Crossref PubMed Scopus (873) Google Scholar, 25Kawabata M. Inoue H. Hanyu A. Imamura T. Miyazono K. EMBO J. 1998; 17: 4056-4065Crossref PubMed Scopus (249) Google Scholar, 26Goto D. Yagi K. Inoue H. Iwamoto I. Kawabata M. Miyazono K. Kato M. FEBS Lett. 1998; 430: 201-204Crossref PubMed Scopus (58) Google Scholar). The constructions of αA, αB, αC, and β2 have been described elsewhere (27Kanno T. Kanno Y. Chen L.F. Ogawa E. Kim W.Y. Ito Y. Mol. Cell. Biol. 1998; 18: 2444-2454Crossref PubMed Google Scholar, 28Bae S.-C. Takahashi E. Zhang Y.W. Ogawa E. Shigesada K. Namba Y. Satake M. Ito Y. Gene ( Amst. ). 1995; 159: 245-248Crossref PubMed Scopus (152) Google Scholar, 29Ogawa E. Maruyama M. Kagoshima H. Inuzuka M. Lu J. Satake M. Shigesada K. Ito Y. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6859-6863Crossref PubMed Scopus (563) Google Scholar). 2Y.-W. Zhang and Y. Ito, unpublished data. Deletion constructs of αC were prepared by a polymerase chain reaction-based approach. For construction of the isolated Ig Cα/TGF-β-responsive element (TβRE) promoter reporter construct ((TβRE)3-Lux) and its mutants, three tandemly repeated TβREs (WT or mutant versions) of the Ig Cα promoter were fused to the heterologous c-Fos (30Gilman M.Z. Wilson R.N. Weinberg R.A. Mol. Cell. Biol. 1986; 6: 4305-4316Crossref PubMed Scopus (301) Google Scholar) and luciferase reporters. All of the polymerase chain reaction products were sequenced. COS7 cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and antibiotics. A20.3 B lymphoma cells (18Shi M.J. Stavnezer J. J. Immunol. 1998; 161: 6751-6760PubMed Google Scholar, 31Xu M.Z. Stavnezer J. EMBO J. 1992; 11: 145-155Crossref PubMed Scopus (93) Google Scholar) were cultured in RPMI 1640 with 10% fetal bovine serum, 50 μm2-mercaptoethanol, 0.1 mm nonessential amino acids, 1 mm sodium pyruvate, 2 mml-glutamine, and antibiotics. P19 murine embryonal carcinoma cells were cultured in a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 supplemented with 10% fetal bovine serum and antibiotics (32Ogawa E. Inuzuka M. Maruyama M. Satake M. Naito-Fujimoto M. Ito Y. Shigesada K. Virology. 1993; 194: 314-331Crossref PubMed Scopus (443) Google Scholar, 33Bae S.-C. Ogawa E. Maruyama M. Oka H. Satake M. Shigesada K. Jenkins N.A. Gilbert D.J. Copeland N.G. Ito Y. Mol. Cell. Biol. 1994; 14: 3242-3252Crossref PubMed Google Scholar). For transient transfection, cells were transfected using FuGENE6 (Roche Molecular Biochemicals). COS7 cells were transiently transfected with expression constructs for PEBP2α subunits, Smads and constitutively active forms of type I receptors. Cells were then washed, scraped, and solubilized (25Kawabata M. Inoue H. Hanyu A. Imamura T. Miyazono K. EMBO J. 1998; 17: 4056-4065Crossref PubMed Scopus (249) Google Scholar). Immunoprecipitation and immunoblotting using an enhanced chemiluminescence detection system (Amersham Pharmacia Biotech) were performed as described (25Kawabata M. Inoue H. Hanyu A. Imamura T. Miyazono K. EMBO J. 1998; 17: 4056-4065Crossref PubMed Scopus (249) Google Scholar). A GST pull-down assay was performed as described previously (22Kim W.-Y. Sieweke M. Ogawa E. Wee H.-J. Englmeier U. Graf T. Ito Y. EMBO J. 1999; 18: 1609-1620Crossref PubMed Scopus (197) Google Scholar). GST-fusion proteins containing the full-length Smad3 or the Mad homology (MH)1 or MH2 domain of Smad3 were expressed and purified as described (32Ogawa E. Inuzuka M. Maruyama M. Satake M. Naito-Fujimoto M. Ito Y. Shigesada K. Virology. 1993; 194: 314-331Crossref PubMed Scopus (443) Google Scholar).In vitro transcription and translation of C-terminal deletion constructs of αC were done using the TNT system (Promega) in the presence of [35S]methionine. GST-Smad3 (full-length), Smad3 (MH1), Smad3 (MH2), or GST bound to glutathione-Sepharose was mixed with αC proteins in 500 μl of Tris-buffered saline, pH 7.4, containing 0.5% Nonidet P-40 for 1 h and washed vigorously three times with 1 ml of the in the they were by by A20.3 B were transfected with the germline Ig Cα promoter (18Shi M.J. Stavnezer J. J. Immunol. 1998; 161: 6751-6760PubMed Google Scholar) together with the expression constructs for αC, Smads, and P19 murine embryonal carcinoma cells were transfected with or mutant of together with αC, Smads, and and luciferase were with the luciferase assay system (Promega) using luciferase activity was with to the luciferase activity. was performed as described (27Kanno T. Kanno Y. Chen L.F. Ogawa E. Kim W.Y. Ito Y. Mol. Cell. Biol. 1998; 18: 2444-2454Crossref PubMed Google Scholar) with COS7 cells were transfected with a mixture of expression TβR-II, Smads, αC, and were mixed in vitro in as and for with a complex formation between αA and R-Smads by αA with and Smad5, which were a constitutively active form of Smad4 was with R-Smads by the receptors. αA also with Smad2 and Smad3 by an active examined the other PEBP2α subunits with Smad3 by complexes not only with αA but also with αB and αC 1 by also complexes with αA, αB, and i.e. Smad2 by and by also with three α subunits not αB and αC complexes with and Smad3 whereas αA with and with Smad3 A that three mammalian PEBP2α subunits form complexes with Because αC is by TGF-β in B and is critical for the induction of the promoter for germline Ig Cα transcripts upon TGF-β (18Shi M.J. Stavnezer J. J. Immunol. 1998; 161: 6751-6760PubMed Google Scholar), complex formation between αC and Smad3 was in The complex was in the presence and of and Smad4 with Smad3 upon by The of between αC and Smad3 was by GST pull-down using deletion constructs of these a of constructs of αC was deletion of a C-terminal 2 to a of the activation domain identified in αB (27Kanno T. Kanno Y. Chen L.F. Ogawa E. Kim W.Y. Ito Y. Mol. Cell. Biol. 1998; 18: 2444-2454Crossref PubMed Google Scholar) in a of with and by deletion of Smads have highly conserved and MH2 in their and C-terminal (1Heldin C.-H. Miyazono K. ten Dijke P. Nature. 1997; 390: 465-471Crossref PubMed Scopus (3358) Google Scholar, J. Annu. Rev. Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3999) Google Scholar). A GST pull-down assay that the MH2 domain bound to αC 2 In addition, the domain with αC, but the in αC where not be because of the of the the functional of using the Ig Cα The promoter for germline Ig Cα transcripts has been shown to contain a TGF-β-responsive Stavnezer J. J. Immunol. 1992; Google Scholar), in which PEBP2α binding sites have been identified (18Shi M.J. Stavnezer J. J. Immunol. 1998; 161: 6751-6760PubMed Google Scholar). The human germline Ig Cα promoter was also shown to contain PEBP2α binding sites in its E. M. P. T. J. Immunol. 1999; PubMed Scopus Google Scholar). In addition, Smad binding L. J.L. P. S. Kinzler K.W. Vogelstein B. Mol. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, S. S. D. ten Dijke P. S. EMBO J. 1998; 17: PubMed Scopus Google Scholar, S. C.-H. ten Dijke P. W. J. Biol. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar) are in the an PEBP2α binding and one Smad binding are between the and the transcription the functional of these binding were into the and a assay was performed using A20.3 B lymphocytes. previously reported (18Shi M.J. Stavnezer J. J. Immunol. 1998; 161: 6751-6760PubMed Google Scholar), TGF-β the which is enhanced by the presence of Mutations in the Smad binding in the and those in the PEBP2α binding sites in in activity A of was in the mutant with in PEBP2α and Smad binding that both of these binding are essential for A form of which the activation of both Smad2 and Smad3 by D. Yagi K. Inoue H. Iwamoto I. Kawabata M. Miyazono K. Kato M. FEBS Lett. 1998; 430: 201-204Crossref PubMed Scopus (58) Google Scholar), the transcription by and αC that transcription may be by the R-Smads by of Smad3 with αC transcription the Ig Cα promoter but not the Ig Cα promoter containing in the as shown in not Smad2 not the because Smad2 is to bind to the Smad binding L. J.L. P. S. Kinzler K.W. Vogelstein B. Mol. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, S. S. D. ten Dijke P. S. EMBO J. 1998; 17: PubMed Scopus Google Scholar, S. C.-H. ten Dijke P. W. J. Biol. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, K. D. T. S. Kato M. Miyazono K. J. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). the roles of αC and in three tandemly repeated TβREs (WT or mutant versions) of the Ig Cα promoter were fused to the heterologous c-Fos and activity was using transfected P19 embryonal carcinoma which have of PEBP2α activity (32Ogawa E. Inuzuka M. Maruyama M. Satake M. Naito-Fujimoto M. Ito Y. Shigesada K. Virology. 1993; 194: 314-331Crossref PubMed Scopus (443) Google Scholar, 33Bae S.-C. Ogawa E. Maruyama M. Oka H. Satake M. Shigesada K. Jenkins N.A. Gilbert D.J. Copeland N.G. Ito Y. Mol. Cell. Biol. 1994; 14: 3242-3252Crossref PubMed Google Scholar). to the with the Ig Cα promoter using A20.3 B activity of was by Smad3 and whereas the of and αC in cells by transcription In contrast, mutant of and which have in the PEBP2 binding sites and Smad binding not to or αC, that both of these binding are essential for activation by the the in αC critical in the activation in with Smads, a of C-terminal of αC was for transcription activity. αC containing the activation in the presence of and deletion of of the in a in of was with the the that the between Smad3 and αC may be critical for the activation 2 The formation of complexes containing αC and Smad3 the germline Cα DNA was by The β subunit of PEBP2 was in this assay to the DNA binding of Smad3 by and αC which be as complexes in 2 and and In the presence of Smad3 and a complex was both in vitro and complexes were in the presence of to the or an to the β that and Smad3 bind to DNA as a Mutations in the Smad binding or in the or of Smad3 and but the binding of the PEBP2α sites were a in but not in disrupted the of and but binding of Smad3 was The Smad binding and PEBP2α binding sites thus to be specific and for the binding of but both are required for the binding of the complex to the and for activation of the promoter by αC and The findings shown in the present that PEBP2α subunits and R-Smads specific for both TGF-β and BMP signaling pathways form complexes together with Smad4 and that the complex formation appears to be critical for activation of target including the germline Ig Cα Our findings suggest that PEBP2 may function as a nuclear target of TGF-β/BMP signaling pathways and that the biological of TGF-β/BMP may be by cooperation between Smads and Smads have been reported to interact with proteins as well as the and R. Zhang Y. Feng X.-H. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, D. R.S. S. J. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). Because members of the TGF-β have with transcription factors may be required for Smads to specific in certain cell of these interacting including and the receptor (6Zhang Y. Feng X.-H. Derynck R. Nature. 1998; 394: 909-913Crossref PubMed Scopus (688) Google Scholar, J. Y. Y. M. T. K. M. Kawabata M. Miyazono K. Kato S. 1999; PubMed Scopus Google Scholar), interact with FAST1 and murine have been shown to with Smad2 as well (3Chen X. Rubock M.J. Whitman M. Nature. 1996; 383: 691-696Crossref PubMed Scopus (634) Google Scholar, 4Labbé E. Silvestri C. Hoodless P.A. Wrana J.L. Attisano L. Mol. Cell. 1998; 2: 109-120Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar, K. D. T. S. Kato M. Miyazono K. J. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). a transcription factor has been shown to bind to X. X. Chen D. X. J. Biol. 1999; Full Text Full Text PDF PubMed Scopus (152) Google Scholar). PEBP2 is with these factors, because three mammalian α subunits of PEBP2 interact with R-Smads in the present has been shown to interact with in to the PEBP2 α subunits, is a and with R-Smads may to of of target genes by K. C. H. P. L. G. R. D. J. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). Smad3 with αC the MH2 domain, whereas the domain to by C-terminal deletion of αC that the C-terminal including the of αC, is required for with the MH2 domain of PEBP2 is a context-dependent transcription interacting for including (21Wotton D. Ghysdael J. Wang S. Speck N.A. Owen M.J. Mol. Cell. Biol. 1994; 14: 840-850Crossref PubMed Scopus (199) Google Scholar, 22Kim W.-Y. Sieweke M. Ogawa E. Wee H.-J. Englmeier U. Graf T. Ito Y. EMBO J. 1999; 18: 1609-1620Crossref PubMed Scopus (197) Google Scholar). In the germline Ig Cα both PEBP2 and Smad binding sites are essential for In contrast, FAST1 to the gene promoter with and binding of Smads to DNA may be important in the Ig Cα promoter K. D. T. S. Kato M. Miyazono K. J. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). in certain other to which PEBP2 with a together with other transcription factors, DNA binding of Smads may not be critical for cooperative activation by PEBP2 and Smads. Our present that PEBP2α subunits interact with R-Smads by as well as with those by and that functional cooperation between αC and Smad3 is required for transcription by the germline Cα Ig α transcripts are required for IgA class switching J. Immunol. 1996; PubMed Scopus (275) Google Scholar). Because members of the TGF-β a of biological it be important to PEBP2 is in these biological as a nuclear target of Smads. are to Y. and A. Nishihara for and and to Y. Inada and Y. for

The transcription factor NF-κB regulates genes involved in inflammatory and immune responses, tumorigenesis, and apoptosis. In contrast to the pleiotropic stimuli that lead to its positive regulation, the known signaling mechanisms that underlie the negative regulation of NF-κB are very few. Recent studies have identified the tumor suppressor CYLD, loss of which causes a benign human syndrome called cylindromatosis, as a key negative regulator for NF-κB signaling by deubiquitinating tumor necrosis factor (TNF) receptor-associated factor (TRAF) 2, TRAF6, and NEMO (NF-κB essential modulator, also known as IκB kinase γ). However, how CYLD is regulated remains unknown. The present study revealed a novel autoregulatory feedback pathway through which activation of NF-κB by TNF-α and bacterium nontypeable Haemophilus influenzae (NTHi) induces CYLD that in turn leads to the negative regulation of NF-κB signaling. In addition, TRAF2 and TRAF6 appear to be differentially involved in NF-κB-dependent induction of CYLD by TNF-α and NTHi. These findings provide novel insights into the autoregulation of NF-κB activation. The transcription factor NF-κB regulates genes involved in inflammatory and immune responses, tumorigenesis, and apoptosis. In contrast to the pleiotropic stimuli that lead to its positive regulation, the known signaling mechanisms that underlie the negative regulation of NF-κB are very few. Recent studies have identified the tumor suppressor CYLD, loss of which causes a benign human syndrome called cylindromatosis, as a key negative regulator for NF-κB signaling by deubiquitinating tumor necrosis factor (TNF) receptor-associated factor (TRAF) 2, TRAF6, and NEMO (NF-κB essential modulator, also known as IκB kinase γ). However, how CYLD is regulated remains unknown. The present study revealed a novel autoregulatory feedback pathway through which activation of NF-κB by TNF-α and bacterium nontypeable Haemophilus influenzae (NTHi) induces CYLD that in turn leads to the negative regulation of NF-κB signaling. In addition, TRAF2 and TRAF6 appear to be differentially involved in NF-κB-dependent induction of CYLD by TNF-α and NTHi. These findings provide novel insights into the autoregulation of NF-κB activation. The transcription factor NF-κB plays critical roles in regulating inflammatory and immune responses, tumorigenesis, and protection against apoptosis (1Karin M. Cao Y. Greten F.R. Li Z.W. Nat. Rev. Cancer. 2002; 2: 301-310Crossref PubMed Scopus (2250) Google Scholar, 2Li Q. Verma I.M. Nat. Rev. Immunol. 2002; 2: 725-734Crossref PubMed Scopus (3307) Google Scholar, 3Orlowski R.Z. Baldwin Jr., A.S. Trends Mol. Med. 2002; 8: 385-389Abstract Full Text Full Text PDF PubMed Scopus (533) Google Scholar). Previous studies identified an inducible feedback inhibition pathway for controlling IκBα gene transcription and down-regulation of transient activation of NF-κB (4Sun S.C. Ganchi P.A. Ballard D.W. Greene W.C. Science. 1993; 259: 1912-1915Crossref PubMed Scopus (955) Google Scholar, 5Cheng Q. Cant C.A. Moll T. Hofer-Warbinek R. Wagner E. Birnstiel M.L. Bach F.H. de Martin R. J. Biol. Chem. 1994; 269: 13551-13557Abstract Full Text PDF PubMed Google Scholar, 6Chiao P.J. Miyamoto S. Verma I.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 28-32Crossref PubMed Scopus (390) Google Scholar). Recent studies have identified the tumor suppressor CYLD 1The abbreviations used are: CYLD, cylindromatosis; TNF, tumor necrosis factor; NTHi, nontypeable Haemophilus influenzae; IL, inter-leukin; MEF, mouse embryonic fibroblast; WT, wild-type; NHBE, normal human bronchial epithelial; siRNA, small interfering RNA; IKK, IκB kinase; NEMO, NF-κB essential modulator.1The abbreviations used are: CYLD, cylindromatosis; TNF, tumor necrosis factor; NTHi, nontypeable Haemophilus influenzae; IL, inter-leukin; MEF, mouse embryonic fibroblast; WT, wild-type; NHBE, normal human bronchial epithelial; siRNA, small interfering RNA; IKK, IκB kinase; NEMO, NF-κB essential modulator. as a key negative regulator for NF-κB signaling by deubiquitinating tumor necrosis factor (TNF) receptor-associated factor (TRAF) 2, TRAF6, and NEMO (7Trompouki E. Hatzivassiliou E. Tsichritzis T. Farmer H. Ashworth A. Mosialos G. Nature. 2003; 424: 793-796Crossref PubMed Scopus (799) Google Scholar, 8Brummelkamp T.R. Nijman S.M. Dirac A.M. Bernards R. Nature. 2003; 424: 797-801Crossref PubMed Scopus (831) Google Scholar, 9Kovalenko A. Chable-Bessia C. Cantarella G. Israel A. Wallach D. Courtois G. Nature. 2003; 424: 801-805Crossref PubMed Scopus (848) Google Scholar). However, how CYLD is regulated is totally unknown. It is still unclear whether activation of NF-κB induces CYLD transcription that in turn leads to the inhibition of NF-κB especially in more delayed or persistent phase in an autoregulatory feedback manner. To determine whether CYLD is induced during inflammation, we first sought to evaluate the effects on CYLD expression of a variety of inflammation stimuli such as proinflammatory cytokines and bacteria. Having demonstrated that CYLD is indeed induced by TNF-α, interleukin-1β (IL-1β) and nontypeable Haemophilus influenzae (NTHi), an important Gram-negative bacterial pathogen for respiratory infections, we next sought to determine whether activation of NF-κB is required for CYLD induction based on the fact that all of the above CYLD inducers are also potent inducers for NF-κB. Here we showed that activation of NF-κB is indeed required for CYLD induction by TNF-α, IL-1β, and NTHi and that TRAF2 and TRAF6 are differentially involved in NF-κB-dependent induction of CYLD by TNF-α and NTHi. The present study thus revealed a novel autoregulatory feedback pathway through which activation of NF-κB by TNF-α and NTHi induces CYLD that in turn leads to the inhibition of NF-κB signaling. These findings should enhance our understanding of the negative feedback regulation of NF-κB activation during inflammation. Reagents—MG-132 was purchased from Calbiochem. Recombinant mTNF-α, hTNF-α, and hIL-1β were purchased from R&D Systems. NTHi strain 12 was described previously (10Shuto T. Xu H. Wang B. Han J. Kai H. Gu X.X. Murphy T. Lim D.J. Li J.D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8774-8779Crossref PubMed Scopus (233) Google Scholar, 11Watanabe T. Jono H. Han J. Lim D.J. Li J.D. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 3563-3568Crossref PubMed Scopus (73) Google Scholar). Cell Culture—Human cervix epithelial cell line HeLa was maintained as described (10Shuto T. Xu H. Wang B. Han J. Kai H. Gu X.X. Murphy T. Lim D.J. Li J.D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8774-8779Crossref PubMed Scopus (233) Google Scholar, 11Watanabe T. Jono H. Han J. Lim D.J. Li J.D. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 3563-3568Crossref PubMed Scopus (73) Google Scholar) and was used for all experiments unless otherwise indicated. All mouse embryonic fibroblast (MEF) cells were maintained as described (12Li Q. Lu Q. Hwang J.Y. Buscher D. Lee K.F. Izpisua-Belmonte J.C. Verma I.M. Genes Dev. 1999; 13: 1322-1328Crossref PubMed Scopus (417) Google Scholar, 13Li Q. Estepa G. Memet S. Israel A. Verma I.M. Genes Dev. 2000; 14: 1729-1733Crossref PubMed Google Scholar, 14Li Z.W. Chu W. Hu Y. Delhase M. Deerinck T. Ellisman M. Johnson R. Karin M. J. Exp. Med. 1999; 189: 1839-1845Crossref PubMed Scopus (820) Google Scholar, 15Yang F. Tang E. Guan K. Wang C.Y. J. Immunol. 2003; 170: 5630-5635Crossref PubMed Scopus (333) Google Scholar). Wild-type (WT), IKK1–/–, IKK2–/–, and IKK1/2–/– MEFs were provided by Dr. I. Verma (12Li Q. Lu Q. Hwang J.Y. Buscher D. Lee K.F. Izpisua-Belmonte J.C. Verma I.M. Genes Dev. 1999; 13: 1322-1328Crossref PubMed Scopus (417) Google Scholar, 13Li Q. Estepa G. Memet S. Israel A. Verma I.M. Genes Dev. 2000; 14: 1729-1733Crossref PubMed Google Scholar); p65–/– and reconstituted p65–/– MEFs were provided by Dr. C. Y. Wang (15Yang F. Tang E. Guan K. Wang C.Y. J. Immunol. 2003; 170: 5630-5635Crossref PubMed Scopus (333) Google Scholar). WT Rat-1 cells and IKKγ (also known as NEMO (NF-κB essential modulator))-deficient cells were provided by Dr. S. Yamaoka (11Watanabe T. Jono H. Han J. Lim D.J. Li J.D. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 3563-3568Crossref PubMed Scopus (73) Google Scholar, 16Yamaoka S. Courtois G. Bessia C. Whiteside S.T. Weil R. Agou F. Kirk H.E. Kay R.J. Israel A. Cell. 1998; 93: 1231-1240Abstract Full Text Full Text PDF PubMed Scopus (949) Google Scholar). Primary normal human bronchial epithelial (NHBE) cells were described previously (10Shuto T. Xu H. Wang B. Han J. Kai H. Gu X.X. Murphy T. Lim D.J. Li J.D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8774-8779Crossref PubMed Scopus (233) Google Scholar, 11Watanabe T. Jono H. Han J. Lim D.J. Li J.D. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 3563-3568Crossref PubMed Scopus (73) Google Scholar). Real-time Quantitative Reverse Transcriptase-PCR Analysis—Real-time quantitative PCR (Q-PCR) was performed using an ABI 7700 Sequence Detection System (Applied Biosystems) as described (11Watanabe T. Jono H. Han J. Lim D.J. Li J.D. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 3563-3568Crossref PubMed Scopus (73) Google Scholar). The sequences of primers and probes were as follows: human CYLD (GenBank™ accession number NM015247), 5′-ACGCCACAATCTTCATCACACT-3′ (forward primer) and 5′-AGGTCGTGGTCAAGGTTTCACT-3′ (reverse primer); TaqMan probe, 5′-6-carboxyfluorescein-AAAAAGCTGTTTCCCTTGGTACACCCCG-6-carboxytetramethylrhodamine-3′); mouse CYLD (GenBank™ accession number NM173369, 5′-CTC AGC CTA TTT AGA AAC AGA CT-3′ (forward primer) and 5′-TCT CCT GGG CCT GCA AAA T-3′ (reverse primer); rat CYLD (GenBank™ accession number XM232642), 5′-CTC AGC CTA TTT AGA AAC AGA AT-3′ (forward primer) and 5′-TCT CCT GGG CCT GCA AAA T-3′ (reverse primer). Plasmids, Transfections, and Luciferase Assays—The plasmids WT-CYLD, IκBα(S32/36A), IKK2(K49A), p65, and NF-κB luciferase were described previously (7Trompouki E. Hatzivassiliou E. Tsichritzis T. Farmer H. Ashworth A. Mosialos G. Nature. 2003; 424: 793-796Crossref PubMed Scopus (799) Google Scholar, 10Shuto T. Xu H. Wang B. Han J. Kai H. Gu X.X. Murphy T. Lim D.J. Li J.D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8774-8779Crossref PubMed Scopus (233) Google Scholar, 11Watanabe T. Jono H. Han J. Lim D.J. Li J.D. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 3563-3568Crossref PubMed Scopus (73) Google Scholar). All transient transfections were carried out in triplicate using TransIT-LT1 reagent (Panvera, Madison, WI). The transfected cells were treated with TNF-α, IL-1β, or NTHi for 5 h before being harvested for luciferase assay. Luciferase activity was normalized with respect to β-galactosidase activity. RNA-mediated Interference—RNA-mediated interference for down-regulating CYLD expression was done using small interfering RNA (siRNA)-CYLD (pSUPER-CYLD) as described previously (8Brummelkamp T.R. Nijman S.M. Dirac A.M. Bernards R. Nature. 2003; 424: 797-801Crossref PubMed Scopus (831) Google Scholar). Western Blot Analysis—Western blot analysis was performed as described (8Brummelkamp T.R. Nijman S.M. Dirac A.M. Bernards R. Nature. 2003; 424: 797-801Crossref PubMed Scopus (831) Google Scholar). The rabbit polyclonal antibody against CYLD, developed in the laboratory of Dr. Mosialos, was raised against amino acids 538–953 of human CYLD. Antibodies against IKK1, IKK2, and NEMO were purchased from Cell Signaling, p65 was from Santa Cruz Biotechnology, and β-actin was from Sigma. In Vivo Study—7–8-Week-old BALB/c mice (Charles River Laboratories) were used in this study. After the trachea was surgically exposed by middle line incision in the skin, TNF-α or NTHi was directly injected into the trachea. Lung tissues were collected and then stored at –80 °C; total RNA was isolated from the frozen tissue. For inhibition study, mice were pretreated with 1 mg/kg MG-132 interperitoneally 1 h before inoculation of TNF-α or NTHi. Three mice were used for each inoculation group. The House Ear Institute Institution's Animal Care and Use Committee (IACUC) approved all of the animal protocols used in this study. CYLD Is a General Negative Regulator for NF-κB Activation—We first sought to determine whether CYLD indeed acts as a negative regulator for NF-κB activation by a variety of stress stimuli using a siRNA approach (8Brummelkamp T.R. Nijman S.M. Dirac A.M. Bernards R. Nature. 2003; 424: 797-801Crossref PubMed Scopus (831) Google Scholar). We first confirmed the efficiency of CYLD-specific siRNA (siRNA-CYLD) in reducing CYLD expression in HeLa cells co-transfected with WT CYLD and siRNA-CYLD or empty vector. As expected, the CYLD protein was markedly reduced by siRNA-CYLD (Fig. 1A, left). Consistent with this result, the endogenous CYLD protein was also greatly reduced (Fig. 1A, right). We then assessed the effect of siRNA-CYLD on NF-κB activation by TNF-α, IL-1β, and bacterium NTHi (10Shuto T. Xu H. Wang B. Han J. Kai H. Gu X.X. Murphy T. Lim D.J. Li J.D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8774-8779Crossref PubMed Scopus (233) Google Scholar, 11Watanabe T. Jono H. Han J. Lim D.J. Li J.D. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 3563-3568Crossref PubMed Scopus (73) Google Scholar). As shown in Fig. 1B, CYLD knockdown by siRNA-CYLD greatly enhanced NF-κB activation. To determine whether CYLD knockdown also enhances NF-κB-dependent transcription of several key inflammatory mediators, we next assessed the effect of siRNA-CYLD on TNF-α- and NTHi-induced up-regulation of TNF-α, IL-1β, and IL-8 using Q-PCR analysis. As shown in Fig. 1C, CYLD knock-down greatly enhanced induction of TNF-α, IL-1β, and IL-8 by TNF-α and NTHi (upper and lower panels), respectively. To further confirm whether CYLD knockdown also enhances NF-κB activation in primary epithelial cells, we then examined the effect of siRNA-CYLD on NF-κB activation in primary NHBE cells. As evidenced in Fig. 1D, NF-κB activation was markedly enhanced by siRNA-CYLD in NHBE cells. Similarly, activation of NF-κB induced by other known NF-κB inducers phorbol ester (phorbol 12-myristate 13-acetate) and peptidoglycan was also enhanced by siRNA-CYLD (data not shown). Taken together, these data indicate that CYLD is indeed a negative regulator for NF-κB activation induced by a variety of known NF-κB stimuli. CYLD Is Induced by a Variety of NF-κB Stimuli in Vitro and in Vivo—Because a variety of genes involved in inflammatory response undergo changes in expression pattern after initiation of inflammation (1Karin M. Cao Y. Greten F.R. Li Z.W. Nat. Rev. Cancer. 2002; 2: 301-310Crossref PubMed Scopus (2250) Google Scholar, 2Li Q. Verma I.M. Nat. Rev. Immunol. 2002; 2: 725-734Crossref PubMed Scopus (3307) Google Scholar, 3Orlowski R.Z. Baldwin Jr., A.S. Trends Mol. Med. 2002; 8: 385-389Abstract Full Text Full Text PDF PubMed Scopus (533) Google Scholar), and the endogenous expression of CYLD is relatively low in epithelial cells, we hypothesized that CYLD is induced by a variety of inflammation stimuli such as proinflammatory cytokines and bacteria. We thus tested our hypothesis by assessing the effects on CYLD expression of TNF-α, IL-1β, and NTHi that are known as highly potent NF-κB inducers. As shown in Fig. 2A, TNF-α, IL-1β, and NTHi strongly induced CYLD expression at the mRNA level in HeLa (left) and NHBE cells (right) by Q-PCR analysis. Consistent with this result, induction of CYLD by TNF-α and NTHi was also observed at the protein level (Fig. 2B). Similarly, CYLD induction by peptidoglycan, phorbol 12-myristate 13-acetate, and Gram-positive bacterium Streptococcus pneumoniae was also observed (data not shown), suggesting that induction of CYLD may be generalizable for a variety of NF-κB inducers. To further confirm whether CYLD is also induced in vivo, we next determined the effects of TNF-α and NTHi on CYLD expression in the lungs of the mice. As shown in Fig. 2C, both TNF-α and NTHi induced CYLD expression in a dose-dependent manner, respectively. The induction of CYLD became evident at 3 h, greatly up-regulated at 6 h, and returned to base-line level by 4 days after inoculation of either TNF-α or NTHi (Fig. 2D). Collectively, these data demonstrate that CYLD is induced by a variety of NF-κB stimuli including TNF-α and bacterium NTHi in vitro and in vivo. NF-κB Is Essential for Induction of CYLD by TNF-α and NTHi—On the basis of evidence that NF-κB controls expression of many genes involved in inflammatory response (1Karin M. Cao Y. Greten F.R. Li Z.W. Nat. Rev. Cancer. 2002; 2: 301-310Crossref PubMed Scopus (2250) Google Scholar, 2Li Q. Verma I.M. Nat. Rev. Immunol. 2002; 2: 725-734Crossref PubMed Scopus (3307) Google Scholar, 3Orlowski R.Z. Baldwin Jr., A.S. Trends Mol. Med. 2002; 8: 385-389Abstract Full Text Full Text PDF PubMed Scopus (533) Google Scholar) and CYLD, a key negative regulator for NF-κB(7Trompouki E. Hatzivassiliou E. Tsichritzis T. Farmer H. Ashworth A. Mosialos G. Nature. 2003; 424: 793-796Crossref PubMed Scopus (799) Google Scholar, 8Brummelkamp T.R. Nijman S.M. Dirac A.M. Bernards R. Nature. 2003; 424: 797-801Crossref PubMed Scopus (831) Google Scholar, 9Kovalenko A. Chable-Bessia C. Cantarella G. Israel A. Wallach D. Courtois G. Nature. 2003; 424: 801-805Crossref PubMed Scopus (848) Google Scholar), is induced by a variety of NF-κB inducers including TNF-α or NTHi, we next sought to determine whether NF-κB is also required for induction of CYLD in an inducible autoregulatory feedback manner. We first assessed the effects of blocking NF-κB signaling on CYLD induction by TNF-α and NTHi using various approaches. As shown in Fig. 3A, CYLD induction by either TNF-α and NTHi was greatly inhibited by blocking IKK2-IκBα signaling using MG-132 (left) and expressing a transdominant mutant of IκBα or a dominant-negative mutant of IKK2 (right) in HeLa cells (10Shuto T. Xu H. Wang B. Han J. Kai H. Gu X.X. Murphy T. Lim D.J. Li J.D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8774-8779Crossref PubMed Scopus (233) Google Scholar, 11Watanabe T. Jono H. Han J. Lim D.J. Li J.D. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 3563-3568Crossref PubMed Scopus (73) Google Scholar), suggesting the involvement of IKK2-IκBα signaling in CYLD induction. To confirm the requirement of IKK complex in CYLD induction, we next investigated CYLD induction in MEFs derived from WT and IKK1–/– and IKK2–/– mice. As shown in Fig. 3B, both TNF-α and NTHi induced CYLD expression in WT but not in IKK1–/–, IKK2–/–, and double knockout IKK1/2–/– MEFs (left) (12Li Q. Lu Q. Hwang J.Y. Buscher D. Lee K.F. Izpisua-Belmonte J.C. Verma I.M. Genes Dev. 1999; 13: 1322-1328Crossref PubMed Scopus (417) Google Scholar, 13Li Q. Estepa G. Memet S. Israel A. Verma I.M. Genes Dev. 2000; 14: 1729-1733Crossref PubMed Google Scholar, 14Li Z.W. Chu W. Hu Y. Delhase M. Deerinck T. Ellisman M. Johnson R. Karin M. J. Exp. Med. 1999; 189: 1839-1845Crossref PubMed Scopus (820) Google Scholar). Likewise, TNF-α and NTHi induced CYLD expression in WT but not in NF-κB essential modulator (NEMO) or IKKγ-deficient cells (right) (16Yamaoka S. Courtois G. Bessia C. Whiteside S.T. Weil R. Agou F. Kirk H.E. Kay R.J. Israel A. Cell. 1998; 93: 1231-1240Abstract Full Text Full Text PDF PubMed Scopus (949) Google Scholar), thereby confirming the requirement of IKK complex signaling in CYLD induction. To determine whether NF-κB is required for CYLD induction, we then assessed the effects of TNF-α and NTHi on CYLD expression in WT and p65–/– MEFs (15Yang F. Tang E. Guan K. Wang C.Y. J. Immunol. 2003; 170: 5630-5635Crossref PubMed Scopus (333) Google Scholar). Fig. 3C shows that both TNF-α and NTHi induced CYLD expression in WT but not in p65–/– MEFs. When p65–/– MEFs were reconstituted with WT p65 expression plasmid, CYLD induction became evident in reconstituted p65–/– MEFs in response to both TNF-α and NTHi, indicating NF-κB is required for CYLD induction by TNF-α and NTHi. To further determine whether direct activation of NF-κB induces CYLD, we transfected HeLa cells with WT p65 expression plasmid. Interestingly, overexpression of WT p65 induced CYLD expression in a dose-dependent manner (Fig. 3D). Thus, it is clear that activation of NF-κB is indeed required for inducing CYLD expression. Similar to HeLa cells, induction of CYLD by TNF-α and NTHi was also inhibited by perturbing IκBα signaling using MG-132 in primary NHBE cells (Fig. 3E). Moreover, CYLD induction was also abolished by MG-132 in the lung of BALB/c thus confirming the involvement of NF-κB signaling in CYLD induction in (Fig. Taken together, our data that activation of NF-κB is essential for CYLD induction by TNF-α and NTHi in vitro and in vivo, thereby evidence for an inducible autoregulatory feedback TRAF2 and TRAF6 in NF-κB-dependent Induction of CYLD by TNF-α and demonstrated the requirement of the signaling pathway in CYLD induction in an inducible autoregulatory feedback manner, still are the signaling that CYLD induction by TNF-α and NTHi CYLD. In of the known signaling NF-κB activation of NEMO, receptor-associated factor and TRAF6 have shown to as important involved in TNF-α and signaling to NF-κB A. Cell. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, K. T. S. Rev. Immunol. 2003; PubMed Scopus Google Scholar). studies indicate that CYLD regulates NF-κB activation by deubiquitinating TRAF2 and TRAF6 (7Trompouki E. Hatzivassiliou E. Tsichritzis T. Farmer H. Ashworth A. Mosialos G. Nature. 2003; 424: 793-796Crossref PubMed Scopus (799) Google Scholar, 8Brummelkamp T.R. Nijman S.M. Dirac A.M. Bernards R. Nature. 2003; 424: 797-801Crossref PubMed Scopus (831) Google Scholar, 9Kovalenko A. Chable-Bessia C. Cantarella G. Israel A. Wallach D. Courtois G. Nature. 2003; 424: 801-805Crossref PubMed Scopus (848) Google Scholar). We investigated whether TRAF2 and TRAF6 are also involved in CYLD induction. As shown in Fig. a dominant-negative mutant of TRAF2 the induction of CYLD by TNF-α but not by NTHi. In the cells with a dominant-negative mutant TRAF6 inhibited the induction of CYLD by NTHi but not by These data that TRAF2 and TRAF6 are differentially involved in CYLD induction by TNF-α and NTHi. To confirm whether inhibition of NF-κB activity by CYLD through perturbing we assessed the effect of siRNA-CYLD on NF-κB activation induced by expressing WT TRAF2 and As expected, activation of NF-κB by expressing WT TRAF2 and TRAF6 was enhanced by CYLD knockdown (Fig. In activation of NF-κB by expressing WT p65 was by Thus, these data indicate that the inhibition of NF-κB by CYLD indeed through perturbing and signaling. In our findings revealed a novel autoregulatory feedback through which activation of NF-κB by TNF-α and bacterium NTHi induces CYLD, which in turn leads to the inhibition of NF-κB signaling (Fig. In addition, TRAF2 and TRAF6 appear to be differentially involved in NF-κB-dependent induction of CYLD by TNF-α and NTHi. Moreover, the inhibition of NF-κB by CYLD through perturbing and signaling. Previous studies identified NF-κB-dependent induction of its IκBα as an important to the transient of NF-κB induction. It remains unclear whether the autoregulatory feedback of NF-κB activation also at the level of The present studies thus identified an autoregulatory feedback that controls the more signaling pathway to NF-κB activation. In contrast to the that NF-κB-dependent induction of IκBα plays in controlling the transient of NF-κB induction, the NF-κB-dependent induction of CYLD may a more important in controlling the delayed activation of NF-κB induction. Thus, the involvement of the NF-κB-dependent induction of both IκBα and CYLD may be essential for the of NF-κB activation in the transient and the delayed or persistent C. B. Li S. J. M. P.J. Mol. Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, W. E. Greene W.C. Science. 2001; PubMed Scopus Google Scholar, T. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). It should also be that analysis revealed NF-κB the CYLD thereby further for the requirement of NF-κB in CYLD induction. studies on and the of CYLD gene that the In addition, the involvement of other signaling in CYLD induction should also be as our data not the involvement of other signaling We I. R. A. C. T. H. and S. Yamaoka for