NF-κB Is Essential for Induction of CYLD, the Negative Regulator of NF-κB

Abstract

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