NF-κB Suppression by the Deubiquitinating Enzyme Cezanne

Transcription factors belonging to the NF-κB family regulate inflammation by inducing pro-inflammatory molecules (e.g. interleukin (IL)-8) in response to cytokines (e.g. tumor necrosis factor (TNF) α, IL-1) or other stimuli. Several negative regulators of NF-κB, including the ubiquitin-editing enzyme A20, participate in the resolution of inflammatory responses. We report that Cezanne, a member of the A20 family of the deubiquitinating cysteine proteases, can be induced by TNFα in cultured cells. Silencing of endogenous Cezanne using small interfering RNA led to elevated NF-κB luciferase reporter gene activity and enhanced expression of IL-8 transcripts in TNFα-treated cells. Thus we conclude that endogenous Cezanne can attenuate NF-κB activation and the induction of pro-inflammatory transcripts in response to TNF receptor (TNFR) signaling. Overexpression studies revealed that Cezanne suppressed NF-κB nuclear translocation and transcriptional activity by targeting the TNFR signaling pathway at the level of the IκB kinase complex or upstream from it. These effects were not observed in a form of Cezanne that was mutated at the catalytic cysteine residue (Cys209), indicating that the deubiquitinating activity of Cezanne is essential for NF-κB regulation. Finally, we demonstrate that Cezanne can be recruited to activated TNFRs where it suppresses the build-up of polyubiquitinated RIP1 signal adapter proteins. Thus we conclude that Cezanne forms a novel negative feedback loop in pro-inflammatory signaling and that it suppresses NF-κB activation by targeting RIP1 signaling intermediaries for deubiquitination. Transcription factors belonging to the NF-κB family regulate inflammation by inducing pro-inflammatory molecules (e.g. interleukin (IL)-8) in response to cytokines (e.g. tumor necrosis factor (TNF) α, IL-1) or other stimuli. Several negative regulators of NF-κB, including the ubiquitin-editing enzyme A20, participate in the resolution of inflammatory responses. We report that Cezanne, a member of the A20 family of the deubiquitinating cysteine proteases, can be induced by TNFα in cultured cells. Silencing of endogenous Cezanne using small interfering RNA led to elevated NF-κB luciferase reporter gene activity and enhanced expression of IL-8 transcripts in TNFα-treated cells. Thus we conclude that endogenous Cezanne can attenuate NF-κB activation and the induction of pro-inflammatory transcripts in response to TNF receptor (TNFR) signaling. Overexpression studies revealed that Cezanne suppressed NF-κB nuclear translocation and transcriptional activity by targeting the TNFR signaling pathway at the level of the IκB kinase complex or upstream from it. These effects were not observed in a form of Cezanne that was mutated at the catalytic cysteine residue (Cys209), indicating that the deubiquitinating activity of Cezanne is essential for NF-κB regulation. Finally, we demonstrate that Cezanne can be recruited to activated TNFRs where it suppresses the build-up of polyubiquitinated RIP1 signal adapter proteins. Thus we conclude that Cezanne forms a novel negative feedback loop in pro-inflammatory signaling and that it suppresses NF-κB activation by targeting RIP1 signaling intermediaries for deubiquitination. The transcription factor NF-κB drives inflammatory responses by inducing pro-inflammatory molecules including adhesion proteins, cytokines, and chemokines (e.g. IL-8) 2The abbreviations used are:ILinterleukinTNFtumor necrosis factorTNFRTNF receptorsiRNAsmall interfering RNAIKKIκB kinaseE1ubiquitin-activating enzymeE2ubiquitin carrier proteinE3ubiquitin-protein isopeptide ligaseHAhemagglutininGFPgreen fluorescent proteinHUVEChuman umbilical vein endothelial cellCTnumber of thermal cycles that were necessary to generate threshold amounts of product. (1Hayden M.S. Ghosh S. Genes Dev. 2004; 18: 2195-2224Crossref PubMed Scopus (3380) Google Scholar). It is regulated by diverse stimuli including pro-inflammatory cytokines (e.g. TNFα, IL-1) (1Hayden M.S. Ghosh S. Genes Dev. 2004; 18: 2195-2224Crossref PubMed Scopus (3380) Google Scholar) and mechanical forces (e.g. shear stress) (2Ganguli A. Persson L. Palmer I.R. Evans I. Yang L. Smallwood R. Black R. Qwarnstrom E.E. Circ. Res. 2005; 96: 626-634Crossref PubMed Scopus (36) Google Scholar) and plays a central role in numerous diseases including atherosclerosis, rheumatoid arthritis, and rejection of transplanted organs. interleukin tumor necrosis factor TNF receptor small interfering RNA IκB kinase ubiquitin-activating enzyme ubiquitin carrier protein ubiquitin-protein isopeptide ligase hemagglutinin green fluorescent protein human umbilical vein endothelial cell number of thermal cycles that were necessary to generate threshold amounts of product. In unstimulated cells, NF-κB is sequestered in the cytoplasm by inhibitory IκB molecules that bind its nuclear localization sequence. Signaling through TNF receptors (TNFR) leads to the recruitment of several signaling intermediaries including RIP1, TRAF2/5, and NEMO (IKKγ), which control the activation of TAK1, a mitogen-activated protein kinase kinase kinase that can activate IKKs through phosphorylation. Activation of IKKβ triggers phosphorylation of IκBα, which is subsequently destabilized, thus releasing free NF-κB for nuclear translocation and stimulation of transcription (1Hayden M.S. Ghosh S. Genes Dev. 2004; 18: 2195-2224Crossref PubMed Scopus (3380) Google Scholar). Recent studies have revealed that pro-inflammatory signaling is regulated by ubiquitin, a small protein that can be covalently attached to lysine residues of cellular proteins through a chain reaction co-ordinated by E1, E2, and E3 proteins (3Evans P.C. Exp. Rev. Mol. Med. 2005; 7: 1-19Crossref PubMed Scopus (29) Google Scholar, 4Sun L.J. Chen Z.J. Curr. Opin. Cell Biol. 2004; 16: 119-126Crossref PubMed Scopus (375) Google Scholar). Multiple rounds of ubiquitination, where ubiquitin is itself ubiquitinated on lysine residues, generate isopeptide-linked branched chains that play a key role in signaling to NF-κB. Specifically, activation of NF-κB in response to TNFR or IL-1/Toll-like receptor signaling relies on modification of RIP1 or TRAF6 signaling intermediaries, respectively, with a distinct structural form of polyubiquitin that contains ubiquitin Lys63 linkages (5Wu C.J. Conze D.B. Li T. Srinivasula S.M. Ashwell J.D. Nat. Cell Biol. 2006; 8: 398-406Crossref PubMed Scopus (508) Google Scholar, 6Deng L. Wang C. Spencer E. Yang L.Y. Braun A. You J.X. Slaughter C. Pickart C. Chen Z.J. Cell. 2000; 103: 351-361Abstract Full Text Full Text PDF PubMed Scopus (1524) Google Scholar, 7Wang C. Deng L. Hong M. Akkaraju G.R. Inoue J. Chen Z.J.J. Nature. 2001; 412: 346-351Crossref PubMed Scopus (1651) Google Scholar, 8Wertz I.E. O'Rourke K.M. Zhou H.L. Eby M. Aravind L. Seshagiri S. Wu P. Wiesmann C. Baker R. Boone D.L. Ma A. Koonin E.V. Dixit V.M. Nature. 2004; 430: 694-699Crossref PubMed Scopus (1477) Google Scholar). Pro-inflammatory signaling also leads to Lys63-polyubiquitination of NEMO, an adaptor protein that binds IKK and is essential for NF-κB activation (9Tang E.D. Wang C.Y. Xiong Y. Guan K.L. J. Biol. Chem. 2003; 278: 37297-37305Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). It has been suggested that Lys63 chains signal to NF-κB by interacting with TAB2 signaling intermediaries, thus triggering the oligomerization and activation of TAK1 and IKKβ (10Kanayama A. Seth R.B. Sun L.J. Ea C.K. Hong M. Shaito A. Chiu Y.H. Deng L. Chen Z.J. Mol. Cell. 2004; 15: 535-548Abstract Full Text Full Text PDF PubMed Scopus (703) Google Scholar). Polyubiquitin chains linked through Lys48 also play a vital role during NF-κB activation by targeting phosphorylated IκB proteins for proteasomal degradation, thus liberating NF-κB for nuclear entry (1Hayden M.S. Ghosh S. Genes Dev. 2004; 18: 2195-2224Crossref PubMed Scopus (3380) Google Scholar). TNFα induces several negative regulators of NF-κB including A20, a protein that interacts with TRAFs (11Song H.Y. Rothe M. Goeddel D.V. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6721-6725Crossref PubMed Scopus (376) Google Scholar, 12Heyninck K. Valck De D. Berghe Vanden W. Criekinge Van W. Contreras R. Fiers W. Haegeman G. Beyaert R. J. Cell Biol. 1999; 145: 1471-1482Crossref PubMed Scopus (255) Google Scholar), NEMO (13Zhang S.Q. Kovalenko A. Cantarella G. Wallach D. Immunity. 2000; 12: 301-311Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar), and IKKα (14Zetoune F.S. Murthy A.R. Zhao Z.H. Hlaing T. Zeidler M.G. Li Y. Vincenz C. Cytokine. 2001; 15: 282-298Crossref PubMed Scopus (38) Google Scholar) and inhibits the catalytic activity of IKKβ (15Lee E.G. Boone D.L. Chai S. Libby S.L. Chien M. Lodolce J.P. Ma A. Science. 2000; 289: 2350-2354Crossref PubMed Scopus (1200) Google Scholar). Recent work demonstrated that A20 is a deubiquitinating cysteine protease that can cleave ubiquitin monomers from modified proteins (8Wertz I.E. O'Rourke K.M. Zhou H.L. Eby M. Aravind L. Seshagiri S. Wu P. Wiesmann C. Baker R. Boone D.L. Ma A. Koonin E.V. Dixit V.M. Nature. 2004; 430: 694-699Crossref PubMed Scopus (1477) Google Scholar, 16Evans P.C. Ovaa H. Hamon M. Kilshaw P.J. Hamm S. Bauer S. Ploegh H.L. Smith T.S. Biochem. J. 2004; 378: 727-734Crossref PubMed Scopus (197) Google Scholar). Thus it has been suggested that A20 suppresses signaling to NF-κB by targeting signaling intermediaries for ubiquitin editing. We have previously described a novel protein called Cezanne that belongs to the A20 family of deubiquitinating enzymes and can modulate TNFR signaling when overexpressed (17Evans P.C. Taylor E.R. Coadwell J. Heyninck K. Beyaert R. Kilshaw P.J. Biochem. J. 2001; 357: 617-623Crossref PubMed Scopus (77) Google Scholar, 18Evans P.C. Smith T.S. Lai M.J. Williams M.G. Burke D.F. Heyninck K. Kreike M.M. Beyaert R. Blundell T.L. Kilshaw P.J. J. Biol. Chem. 2003; 278: 23180-23186Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). Here we demonstrate that Cezanne can be induced by TNFα in cultured epithelial or endothelial cells. Gene silencing revealed that endogenous Cezanne attenuates NF-κB activation and the induction of pro-inflammatory transcripts in TNFα-treated cells, thus completing a novel negative feedback loop. Finally, we provide evidence that Cezanne suppresses NF-κB upstream of IKK activation by targeting RIP1 signaling intermediaries for deubiquitination. Reagents and Antibodies—Human recombinant TNFα was obtained commercially (R Santa Cruz Biotechnology, Santa Cruz, CA), anti-IκBα (sc-847; Santa Cruz Biotechnology), anti-phosphoIκBα Ser32/36 (Cell Signaling Technology), anti-RIP (BD Biosciences Pharmingen, Oxford, UK), anti-HA (Roche Applied Science), anti-GFP (Santa Cruz Biotechnology), anti-IKKα (BD Biosciences Pharmingen, Oxford, UK), anti-IKKγ (BD Biosciences Pharmingen, Oxford, UK), anti-RIP (BD Biosciences Pharmingen, Oxford, UK), anti-TNFR (Invitrogen), and anti-tubulin (Sigma) were obtained commercially. Rabbit polyclonal antibodies were raised against a peptide that was conserved between human and mouse versions of Cezanne but not encoded by other mammalian genes (C+YSNGYREPPEPDGWA-CONH2) and were subsequently affinity purified (Eurogentech, Liege, Belgium). Anti-Cezanne antibodies were validated by Western blotting of cytosolic lysates made from with forms of Cezanne were from Cezanne Cezanne and forms mutated at the catalytic cysteine and have been described previously (17Evans P.C. Taylor E.R. Coadwell J. Heyninck K. Beyaert R. Kilshaw P.J. Biochem. J. 2001; 357: 617-623Crossref PubMed Scopus (77) Google Scholar, 18Evans P.C. Smith T.S. Lai M.J. Williams M.G. Burke D.F. Heyninck K. Kreike M.M. Beyaert R. Blundell T.L. Kilshaw P.J. J. Biol. Chem. 2003; 278: 23180-23186Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). ubiquitin or versions of ubiquitin residues or were described previously P.C. Ovaa H. Hamon M. Kilshaw P.J. Hamm S. Bauer S. Ploegh H.L. Smith T.S. Biochem. J. 2004; 378: 727-734Crossref PubMed Scopus (197) Google Scholar). and to umbilical vein endothelial were using and cultured described previously J. H. K. H. M. L. A. M. R. Evans P.C. J. PubMed Scopus Google Scholar). were to shear for using a described previously J. H. K. H. M. L. A. M. R. Evans P.C. J. PubMed Scopus Google Scholar). Cell and were cultured using modified with of was using to the expression of or in was by using or respectively, with amounts of or were by cell and used in RNA was using a that is to Cezanne in the expression of T. and M. for with or control were Cell that were were with using the and in for were by using for Cezanne IL-8 and RNA was and described previously J. H. K. H. M. L. A. M. R. Evans P.C. J. PubMed Scopus Google Scholar). was using the and green to the The were at for thermal at for and for The were in gene expression was by the number of thermal cycles that were necessary to generate threshold amounts of described previously J. H. K. H. M. L. A. M. R. Evans P.C. J. PubMed Scopus Google Scholar). was for the genes of and for the gene the for was from the for gene of to the thus the of RNA The of was where is the between the of the to be to was by using for to the of antibodies and antibodies by to NF-κB and localization of NF-κB was by of using antibodies and antibodies by was using to the of in the with the of were by of using anti-IκBα antibodies and antibodies by of NF-κB transcriptional activity was in using an NF-κB reporter by The were with and luciferase to using and for The were with TNFα for of NF-κB and luciferase activity was using a luciferase reporter and IKK were using The was from lysates by IKKβ kinase was purified from lysates by using anti-IKKγ antibodies (BD Pharmingen, Oxford, UK), and its activity was using described previously S. L. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of TNFR were with TNFα for and using a of protease (Roche Applied The lysates were by and using protein of TNFR using (Roche Applied The were using or lysates were by Western blotting using antibodies and of in of Cezanne on ubiquitinated proteins was using cultured in were with of mammalian expression ubiquitin or mutated forms of ubiquitin that residues for polyubiquitin chain Lys63 or Lys48 were with of or Polyubiquitin and Cezanne were in cytosolic lysates by Western blotting using anti-HA and anti-GFP respectively, and The of Cezanne on RIP1 was also were with expression or forms of Cezanne using the the were with TNFα for of TNFR or lysates were by Western blotting using and between were using a and of of Cezanne by Pro-inflammatory observed by that Cezanne transcripts were induced in response to TNFα in The expression of Cezanne was also in cultured in or of shear shear effects on Cezanne it for enhanced expression of Cezanne transcripts in response to TNFα The effects of TNFα on Cezanne transcripts were by and in Cezanne protein in demonstrated by Western blotting or using that Cezanne can be induced in cultured by TNFα or shear that it regulate cellular responses to stimuli. Cezanne Pro-inflammatory Activation by used RNA to the role of endogenous Cezanne in pro-inflammatory responses to We observed that suppressed Cezanne and protein by at in cells, a control and Silencing of Cezanne in on of IL-8 transcripts in unstimulated but enhanced the induction of IL-8 transcripts by TNFα and a control and reporter gene revealed that NF-κB transcriptional activation in response to TNFα was enhanced by in but was not by control and We observed that silencing of Cezanne using an also enhanced NF-κB activity in TNFα-treated and Thus we conclude that endogenous Cezanne a negative of NF-κB activation and pro-inflammatory transcriptional responses in to The of Cezanne for NF-κB the deubiquitinating cysteine protease activity of Cezanne is for its inhibitory effects on NF-κB. gene or of NF-κB localization were in with expression Cezanne or a form in which the cysteine has been mutated to We observed that Cezanne NF-κB transcriptional activity in a in with TNFα with or with with Cezanne, the form was in NF-κB activity in response to TNFα with or with the level at which Cezanne NF-κB transcriptional we it nuclear translocation of NF-κB in response to revealed that to the of unstimulated and to the in response to of with TNFα and Overexpression of Cezanne nuclear localization of in response to TNFα for a form of nuclear in for and It is that is from the to the cytoplasm in to pro-inflammatory stimuli for of M. G. E. R. D. D.B. Science. 2004; PubMed Scopus Google Scholar). is with that nuclear were in or Cezanne of stimulation with TNFα and and and nuclear was not from that overexpressed Cezanne of stimulation and of the by Cezanne of NF-κB transcriptional which is essential for that the catalytic of Cezanne for of NF-κB nuclear localization and transcriptional activation in response to pro-inflammatory stimuli. Cezanne of NF-κB by IKK level at which Cezanne TNFR signaling to NF-κB was by studies using that of or Western blotting of cytosolic lysates revealed that was by of TNFα in and with and in that and with but it was in that and with and the role of IKKβ in IκBα, we its activation by TNFα can be by We observed that IKK activity was elevated by TNFα in control that of IKK activation in response to TNFα was in that and We conclude that Cezanne the TNFR signaling pathway at the level of the IKK complex or upstream from thus NF-κB by We the deubiquitinating activity of Cezanne is for its inhibitory effects on phosphorylation and using with expression Cezanne or a form We observed by and that Cezanne suppressed of IκB in response to TNFα, of Cezanne and and and Western blotting revealed that Cezanne suppressed phosphorylation of in response to TNFα, Cezanne not and Thus we conclude that the deubiquitinating activity of Cezanne is essential for of phosphorylation and in activated cells. Cezanne or the effects of Cezanne on the modification of cellular proteins with or which essential during NF-κB activation Polyubiquitin chains linked through or residues were in using expression versions of ubiquitin or ubiquitin, We observed that the of cellular proteins modified with or chains were in that with that with and with In the build-up of or chains was suppressed in that Cezanne with that a and and and that Cezanne has the to proteins modified with or in cultured cells. Cezanne to the TNFR It RIP1 for that Lys63-polyubiquitination of RIP1 is essential for the activation of IKKβ in response to TNFR signaling. that Cezanne suppresses IKK we it be recruited to the TNFR to RIP1 or Cezanne was overexpressed in cultured cells, and its to be recruited to the TNFR and its effects on polyubiquitinated RIP1 proteins were of TNFR using Western blotting revealed of Cezanne with TNFα in that were for but not in unstimulated that Cezanne can be recruited to in response to We observed the of polyubiquitinated forms of RIP1 in TNFR in response to of TNFα previously (5Wu C.J. Conze D.B. Li T. Srinivasula S.M. Ashwell J.D. Nat. Cell Biol. 2006; 8: 398-406Crossref PubMed Scopus (508) Google Scholar, 8Wertz I.E. O'Rourke K.M. Zhou H.L. Eby M. Aravind L. Seshagiri S. Wu P. Wiesmann C. Baker R. Boone D.L. Ma A. Koonin E.V. Dixit V.M. Nature. 2004; 430: 694-699Crossref PubMed Scopus (1477) Google Scholar) and with Cezanne suppressed polyubiquitinated RIP1 and enhanced the of or RIP1, a form or with and and with and studies have TNFα leads to modification of RIP1 with polyubiquitin chains linked through Lys63 (5Wu C.J. Conze D.B. Li T. Srinivasula S.M. Ashwell J.D. Nat. Cell Biol. 2006; 8: 398-406Crossref PubMed Scopus (508) Google Scholar). the effects of Cezanne on Lys63-polyubiquitination at the we with Cezanne and with a mutated of ubiquitin that forms polyubiquitin chains linked through Cezanne suppressed the build-up of chains at the the form not and that Cezanne can be recruited to the TNFR in response to TNFα where it from can be by deubiquitinating enzymes that cleave ubiquitin from modified proteins, thus or activity M. 2004; PubMed Scopus Google Scholar). deubiquitinating enzymes that regulate signaling have in and diverse including Cell 2004; PubMed Scopus Google Scholar), M. V.M. G. E.R. Li Nature. PubMed Scopus Google Scholar), and Genes Dev. 16: PubMed Scopus Google Scholar) has been Recent studies have the family of deubiquitinating cysteine proteases, which Cezanne, A20, and The family including NF-κB activity (8Wertz I.E. O'Rourke K.M. Zhou H.L. Eby M. Aravind L. Seshagiri S. Wu P. Wiesmann C. Baker R. Boone D.L. Ma A. Koonin E.V. Dixit V.M. Nature. 2004; 430: 694-699Crossref PubMed Scopus (1477) Google Scholar, S.Q. Kovalenko A. Cantarella G. Wallach D. Immunity. 2000; 12: 301-311Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar, F.S. Murthy A.R. Zhao Z.H. Hlaing T. Zeidler M.G. Li Y. Vincenz C. Cytokine. 2001; 15: 282-298Crossref PubMed Scopus (38) Google Scholar, 16Evans P.C. Ovaa H. Hamon M. Kilshaw P.J. Hamm S. Bauer S. Ploegh H.L. Smith T.S. Biochem. J. 2004; 378: 727-734Crossref PubMed Scopus (197) Google in A. G. J. Cell Biol. 2004; PubMed Scopus Google and cell L. C. H. P. E. Nat. 2004; PubMed Scopus (139) Google studies using cultured mammalian have revealed that Cezanne can with signaling molecules and has the to NF-κB transcriptional activity in response to TNFR or signaling (17Evans P.C. Taylor E.R. Coadwell J. Heyninck K. Beyaert R. Kilshaw P.J. Biochem. J. 2001; 357: 617-623Crossref PubMed Scopus (77) Google Scholar). the of endogenous Cezanne has not been previously Here we demonstrate by gene silencing that Cezanne can NF-κB activation and the induction of pro-inflammatory molecules in to In to a report from (17Evans P.C. Taylor E.R. Coadwell J. Heyninck K. Beyaert R. Kilshaw P.J. Biochem. J. 2001; 357: 617-623Crossref PubMed Scopus (77) Google Scholar), we have revealed using with that Cezanne can be induced in cultured by Thus we conclude that Cezanne to the resolution of inflammation by a negative feedback loop in pro-inflammatory Signaling through TNFR leads to modification of RIP1 signaling intermediaries with a that triggers IKKβ activation (8Wertz I.E. O'Rourke K.M. Zhou H.L. Eby M. Aravind L. Seshagiri S. Wu P. Wiesmann C. Baker R. Boone D.L. Ma A. Koonin E.V. Dixit V.M. Nature. 2004; 430: 694-699Crossref PubMed Scopus (1477) Google Scholar, A. Seth R.B. Sun L.J. Ea C.K. Hong M. Shaito A. Chiu Y.H. Deng L. Chen Z.J. Mol. Cell. 2004; 15: 535-548Abstract Full Text Full Text PDF PubMed Scopus (703) Google Scholar). several of evidence to that Cezanne suppresses TNFR signaling to NF-κB by targeting RIP1 for deubiquitination. Cezanne suppressed TNFR signaling at the level of the IKK complex or upstream from it. Cezanne can be recruited to activated TNFRs where it can polyubiquitinated forms of the of Cezanne to modified RIP1 proteins and NF-κB on its deubiquitinating The catalytic activity of Cezanne was also for of NF-κB activation in response to the for by Cezanne in pathway other deubiquitinating A20 and negative regulators of NF-κB. A20, a of Cezanne, also ubiquitin ligase which has been to a the (8Wertz I.E. O'Rourke K.M. Zhou H.L. Eby M. Aravind L. Seshagiri S. Wu P. Wiesmann C. Baker R. Boone D.L. Ma A. Koonin E.V. Dixit V.M. Nature. 2004; 430: 694-699Crossref PubMed Scopus (1477) Google Scholar). It has been suggested that A20 of its catalytic to NF-κB activation by deubiquitinating and subsequently pro-inflammatory signaling intermediaries RIP1 in the TNFR pathway and TRAF6 in the receptor pathway (8Wertz I.E. O'Rourke K.M. Zhou H.L. Eby M. Aravind L. Seshagiri S. Wu P. Wiesmann C. Baker R. Boone D.L. Ma A. Koonin E.V. Dixit V.M. 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Cezanne and A20 can RIP1 for and it is that molecules for the negative of TNFR signaling. it is that Cezanne and A20 have other TNFR signaling intermediaries, thus a for studies be to the of Cezanne, A20, and in pro-inflammatory signaling has effects on the of endothelial and from the of by inflammation 2005; PubMed Scopus Google Scholar, H. S. 2003; PubMed Scopus Google Scholar, E. G.R. S. C.J. Proc. Natl. Acad. Sci. U. S. A. 2004; PubMed Scopus Google Scholar). We have previously that of cultured endothelial to transcriptional responses to TNFα by the induction of pro-inflammatory IL-8) and transcripts J. H. K. H. M. L. A. M. R. Evans P.C. J. PubMed Scopus Google Scholar). In we report that led to the induction of Cezanne in endothelial and also for enhanced induction of Cezanne in response to Thus it is that Cezanne NF-κB activation in endothelial to in the of with a in which Cezanne transcripts were at in to of in the with to in the of the E. G.R. S. C.J. Proc. Natl. Acad. Sci. U. S. A. 2004; PubMed Scopus Google Scholar). Gene targeting studies to the of Cezanne in We of and of for kindly and Prof. Cohen (University of for kindly with