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• W2039639938 abstract "Interleukin-17 (IL-17) is essential in host defense against extracellular bacteria and fungi, especially at mucosal sites, but it also contributes significantly to inflammatory and autoimmune disease pathologies. Binding of IL-17 to its receptor leads to recruitment of adaptor protein CIKS/Act1 via heterotypic association of their respective SEFIR domains and activation of transcription factor NF-κB; it is not known whether CIKS and/or NF-κB are required for all gene induction events. Here we report that CIKS is essential for all IL-17-induced immediate-early genes in primary mouse embryo fibroblasts, whereas NF-κB is profoundly involved. We also identify a novel subdomain in the N terminus of CIKS that is essential for IL-17-mediated NF-κB activation. This domain is both necessary and sufficient for interaction between CIKS and TRAF6, an adaptor required for NF-κB activation. The ability of decoy peptides to block this interaction may provide a new therapeutic strategy for intervention in IL-17-driven autoimmune and inflammatory diseases. Interleukin-17 (IL-17) is essential in host defense against extracellular bacteria and fungi, especially at mucosal sites, but it also contributes significantly to inflammatory and autoimmune disease pathologies. Binding of IL-17 to its receptor leads to recruitment of adaptor protein CIKS/Act1 via heterotypic association of their respective SEFIR domains and activation of transcription factor NF-κB; it is not known whether CIKS and/or NF-κB are required for all gene induction events. Here we report that CIKS is essential for all IL-17-induced immediate-early genes in primary mouse embryo fibroblasts, whereas NF-κB is profoundly involved. We also identify a novel subdomain in the N terminus of CIKS that is essential for IL-17-mediated NF-κB activation. This domain is both necessary and sufficient for interaction between CIKS and TRAF6, an adaptor required for NF-κB activation. The ability of decoy peptides to block this interaction may provide a new therapeutic strategy for intervention in IL-17-driven autoimmune and inflammatory diseases. The discovery of the inflammatory T helper cell type-17 (Th17), a subset distinct from the classical Th1 and Th2 populations, has revolutionized our understanding of T-cell mediated immunity (1Dong C. Nat. Rev. Immunol. 2006; 6: 329-333Crossref PubMed Scopus (441) Google Scholar, 2Weaver C.T. Hatton R.D. Mangan P.R. Harrington L.E. Annu. Rev. Immunol. 2007; 25: 821-852Crossref PubMed Scopus (1557) Google Scholar, 3McGeachy M.J. Cua D.J. Immunity. 2008; 28: 445-453Abstract Full Text Full Text PDF PubMed Scopus (569) Google Scholar). Th17 cells are critical in host defense against many pathogens, in particular extracellular bacteria and fungi. When improperly controlled, however, Th17 responses can also feature prominently in a number of inflammatory and autoimmune diseases, such as rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, and psoriasis (reviewed in Refs. 4Annunziato F. Cosmi L. Liotta F. Maggi E. Romagnani S. Nat. Rev. Rheumatol. 2009; 5: 325-331Crossref PubMed Scopus (186) Google Scholar, 5Di Cesare A. Di Meglio P. Nestle F.O. J. Invest. Dermatol. 2009; 129: 1339-1350Abstract Full Text Full Text PDF PubMed Scopus (848) Google Scholar, 6Miossec P. Korn T. Kuchroo V.K. N. Engl. J. Med. 2009; 361: 888-898Crossref PubMed Scopus (1168) Google Scholar). The discovery of the Th17 cell type has also focused attention on its signature cytokine IL-17 (also known as IL-17A). A critical, although by no means exclusive biologic corollary of IL-17 expression is the recruitment of neutrophils to sites of inflammation (7Korn T. Bettelli E. Oukka M. Kuchroo V.K. Annu. Rev. Immunol. 2009; 27: 485-517Crossref PubMed Scopus (3859) Google Scholar, 8Xu S. Cao X. Cell Mol. Immunol. 2010; 7: 164-174Crossref PubMed Scopus (242) Google Scholar). IL-17A is a member of a family of cytokines that also includes IL-17B, -C, -D, -E (also known as IL-25), and -F. IL-17A signals via a receptor composed of the IL-17RA and RC chains; these receptor chains are members of a family that also includes RB, RD, and RE (reviewed by Ref. 9Gaffen S.L. Nat. Rev. Immunol. 2009; 9: 556-567Crossref PubMed Scopus (1079) Google Scholar). All receptor polypeptides encode a so-called SEFIR domain (similar expression to fibroblast growth factor genes and IL-17Rs) in their cytoplasmic tails (10Gaffen S.L. Cytokine. 2008; 43: 402-407Crossref PubMed Scopus (286) Google Scholar, 11Novatchkova M. Leibbrandt A. Werzowa J. Neubüser A. Eisenhaber F. Trends Biochem. Sci. 2003; 28: 226-229Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar). Such a domain is also present on the adaptor protein CIKS 2The abbreviations used are: CIKS, connection to IκB kinase and stress-activated protein kinases; MEF, mouse embryo fibroblasts; IP, immunoprecipitate; AA, amino acid; JNK, c-Jun N-terminal kinase. (connection to IκB Kinase and Stress-activated protein kinases (12Leonardi A. Chariot A. Claudio E. Cunningham K. Siebenlist U. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 10494-10499Crossref PubMed Scopus (132) Google Scholar); also known as Act1 (13Li X. Commane M. Nie H. Hua X. Chatterjee-Kishore M. Wald D. Haag M. Stark G.R. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 10489-10493Crossref PubMed Scopus (142) Google Scholar) or TRAF3IP2). IL-17A and -F, as well as IL-25 have been shown to signal by recruitment of CIKS to their cognate receptors, mediated via heterotypic SEFIR domain associations (14Chang S.H. Park H. Dong C. J. Biol. Chem. 2006; 281: 35603-35607Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar, 15Qian Y. Liu C. Hartupee J. Altuntas C.Z. Gulen M.F. Jane-Wit D. Xiao J. Lu Y. Giltiay N. Liu J. Kordula T. Zhang Q.W. Vallance B. Swaidani S. Aronica M. Tuohy V.K. Hamilton T. Li X. Nat. Immunol. 2007; 8: 247-256Crossref PubMed Scopus (459) Google Scholar, 16Claudio E. Sønder S.U. Saret S. Carvalho G. Ramalingam T.R. Wynn T.A. Chariot A. Garcia-Perganeda A. Leonardi A. Paun A. Chen A. Ren N.Y. Wang H. Siebenlist U. J. Immunol. 2009; 182: 1617-1630Crossref PubMed Scopus (19) Google Scholar, 17Swaidani S. Bulek K. Kang Z. Liu C. Lu Y. Yin W. Aronica M. Li X. J. Immunol. 2009; 182: 1631-1640Crossref PubMed Scopus (122) Google Scholar). A number of downstream effectors can be activated by IL-17, the best studied member of this cytokine family, including the transcription factors NF-κB, C/EBP, and AP-1, as well as MAP kinases; in addition, IL-17 can potently stabilize mRNAs, although mechanisms remain to be discovered (reviewed by Ref. 9Gaffen S.L. Nat. Rev. Immunol. 2009; 9: 556-567Crossref PubMed Scopus (1079) Google Scholar). Mere overexpression of CIKS profoundly activates p50/p65 NF-κB complexes via the classical activation pathway (12Leonardi A. Chariot A. Claudio E. Cunningham K. Siebenlist U. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 10494-10499Crossref PubMed Scopus (132) Google Scholar, 13Li X. Commane M. Nie H. Hua X. Chatterjee-Kishore M. Wald D. Haag M. Stark G.R. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 10489-10493Crossref PubMed Scopus (142) Google Scholar, 18Mauro C. Vito P. Mellone S. Pacifico F. Chariot A. Formisano S. Leonardi A. Biochem. Biophys. Res. Commun. 2003; 309: 84-90Crossref PubMed Scopus (21) Google Scholar). By contrast, stimulation of cells with IL-17, which signals via CIKS, activates NF-κB quite weakly (15Qian Y. Liu C. Hartupee J. Altuntas C.Z. Gulen M.F. Jane-Wit D. Xiao J. Lu Y. Giltiay N. Liu J. Kordula T. Zhang Q.W. Vallance B. Swaidani S. Aronica M. Tuohy V.K. Hamilton T. Li X. Nat. Immunol. 2007; 8: 247-256Crossref PubMed Scopus (459) Google Scholar, 19Awane M. Andres P.G. Li D.J. Reinecker H.C. J. Immunol. 1999; 162: 5337-5344PubMed Google Scholar), calling into question the physiologic significance of NF-κB activation. Indeed, IL-17 can synergize with TNFα, which has been ascribed to the fact that TNFα, unlike IL-17, strongly activates NF-κB, whereas IL-17 stabilizes some short-lived mRNAs induced by TNFα (20Hartupee J. Liu C. Novotny M. Li X. Hamilton T. J. Immunol. 2007; 179: 4135-4141Crossref PubMed Scopus (242) Google Scholar). This in turn has fostered the notion that mRNA stabilization may be a primary function of IL-17. Another unsettled question concerning IL-17 signaling is whether the CIKS adaptor is essential for expression of all target genes, especially since some reports suggest the possibility of CIKS-independent signaling events (15Qian Y. Liu C. Hartupee J. Altuntas C.Z. Gulen M.F. Jane-Wit D. Xiao J. Lu Y. Giltiay N. Liu J. Kordula T. Zhang Q.W. Vallance B. Swaidani S. Aronica M. Tuohy V.K. Hamilton T. Li X. Nat. Immunol. 2007; 8: 247-256Crossref PubMed Scopus (459) Google Scholar, 21Huang F. Kao C.Y. Wachi S. Thai P. Ryu J. Wu R. J. Immunol. 2007; 179: 6504-6513Crossref PubMed Scopus (181) Google Scholar, 22Shen F. Li N. Gade P. Kalvakolanu D.V. Weibley T. Doble B. Woodgett J.R. Wood T.D. Gaffen S.L. Sci. Signal. 2009; 2: ra8Crossref PubMed Scopus (110) Google Scholar). Precisely how CIKS transmits signals to its downstream effectors, including NF-κB, is only beginning to be elucidated. Recently a central domain of CIKS has been reported to function as an E3-ubiqutin ligase, capable of adding Lys63-linked polyubiquitin chains to the adaptor protein TRAF6 (23Liu C. Qian W. Qian Y. Giltiay N.V. Lu Y. Swaidani S. Misra S. Deng L. Chen Z.J. Li X. Sci. Signal. 2009; 2: ra63Crossref PubMed Scopus (71) Google Scholar). TRAF6 have previously been found essential for IL-17/CIKS-mediated activation of NF-κB (14Chang S.H. Park H. Dong C. J. Biol. Chem. 2006; 281: 35603-35607Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar, 15Qian Y. Liu C. Hartupee J. Altuntas C.Z. Gulen M.F. Jane-Wit D. Xiao J. Lu Y. Giltiay N. Liu J. Kordula T. Zhang Q.W. Vallance B. Swaidani S. Aronica M. Tuohy V.K. Hamilton T. Li X. Nat. Immunol. 2007; 8: 247-256Crossref PubMed Scopus (459) Google Scholar, 24Kanamori M. Kai C. Hayashizaki Y. Suzuki H. FEBS Lett. 2002; 532: 241-246Crossref PubMed Scopus (38) Google Scholar). The E3-ubiquitin ligase function was also reported necessary for mRNA stabilization, but in this case via a TRAF6 independent mechanism (23Liu C. Qian W. Qian Y. Giltiay N.V. Lu Y. Swaidani S. Misra S. Deng L. Chen Z.J. Li X. Sci. Signal. 2009; 2: ra63Crossref PubMed Scopus (71) Google Scholar, 25Hartupee J. Liu C. Novotny M. Sun D. Li X. Hamilton T.A. J. Immunol. 2009; 182: 1660-1666Crossref PubMed Scopus (80) Google Scholar). Ubiquitination of TRAF6 is secondary to recruitment to CIKS, and CIKS reportedly encodes two TRAF6 binding sites; these sites may be redundant because signaling was only impaired when both sites on CIKS were rendered non-functional through mutagenesis (23Liu C. Qian W. Qian Y. Giltiay N.V. Lu Y. Swaidani S. Misra S. Deng L. Chen Z.J. Li X. Sci. Signal. 2009; 2: ra63Crossref PubMed Scopus (71) Google Scholar, 24Kanamori M. Kai C. Hayashizaki Y. Suzuki H. FEBS Lett. 2002; 532: 241-246Crossref PubMed Scopus (38) Google Scholar). Once polyubiquitinated by CIKS, the TRAF6 adaptor may activate NF-κB via mechanisms already established for signaling by Toll, IL-1, and CD40 receptors, i.e. by activation of Tak1 and IKK (9Gaffen S.L. Nat. Rev. Immunol. 2009; 9: 556-567Crossref PubMed Scopus (1079) Google Scholar, 26Li X. Cytokine. 2008; 41: 105-113Crossref PubMed Scopus (60) Google Scholar). Here we investigate initial transcriptional responses of IL-17 and its molecular signaling mechanisms with the use of primary mouse embryo fibroblasts. We demonstrate that CIKS is absolutely essential for all initial IL-17-induced transcription and we, furthermore, show that classical activation of NF-κB is especially critical for these responses. We also identify a novel domain in the N terminus of CIKS that is required for interaction with TRAF6 and activation of NF-κB. We discuss these findings in terms or their potential to open new avenues for therapeutic intervention in diseases dependent on IL-17 cytokines. Primary mouse embryo fibroblast cultures (MEFs) were established from wild-type (WT) and CIKS-deficient (knockout) mice as described previously (16Claudio E. Sønder S.U. Saret S. Carvalho G. Ramalingam T.R. Wynn T.A. Chariot A. Garcia-Perganeda A. Leonardi A. Paun A. Chen A. Ren N.Y. Wang H. Siebenlist U. J. Immunol. 2009; 182: 1617-1630Crossref PubMed Scopus (19) Google Scholar). Mice were bred and housed in National Institute of Allergy and Infectious Diseases facilities, and all experiments were done with approval of the National Institute of Allergy and Infectious Diseases Animal Care and Use Committee and in accordance with all relevant institutional guidelines. Immortalized NEMO-deficient MEFs were kindly donated by Dr. Manolis Pasparakis. Cycloheximide and actinomycin D were purchased from Sigma; SB203580, JNK inhibitor II, JAK inhibitor I, and PD98059 were from Calbiochem; and MLN120b was kindly supplied by Millennium Pharmaceuticals. FITC-tagged cell penetrating peptides, TAT wild-type CIKS, GRKKRRQRRRPPQMNRSIPVEVDESEPYP, and TAT E17A CIKS, GRKKRRQRRRPPQMNRSIPVAVDESEPYP, were purchased from American Peptide. MEFs were treated for 30 min with these peptides in serum-free medium prior to stimulation. Uptake of FITC-labeled cell penetrating peptide was confirmed by FACS analysis. Recombinant IL-17 (100 ng/ml, R&D Systems) and/or TNFα (2 ng/ml, Peprotech) were used for stimulation. RNA was isolated using the RNeasy RNA isolation kit (Qiagen) according to the manufacturer's instructions. cDNA was synthesized using oligo(dT) and SuperScript III (Invitrogen). Expression of Iκbζ, Ccl2, c/Ebpδ, Zc3h12a, Il-6, Cxc1, and β-actin was quantified by TaqMan qPCR using primers from Applied Biosystems. All gene expression results are expressed as 2−ΔΔCt, where ΔΔCt = (Ct,target − Ct,β-actin) for stimulated samples − (Ct,target − Ct,β-actin) for unstimulated controls. Data are shown as the mean ± S.E. For the mRNA stability experiment, expression of Cxcl1 was calculated as 2−ΔCt, where ΔCt = (Ct,Cxcl1 − Ct,β-actin). Expression at 0 h was set to 100% and the remaining samples were normalized accordingly. RNAs were extracted and DNA microarray targets were prepared as described previously (27Shea P.R. Virtaneva K. Kupko 3rd, J.J. Porcella S.F. Barry W.T. Wright F.A. Kobayashi S.D. Carmody A. Ireland R.M. Sturdevant D.E. Ricklefs S.M. Babar I. Johnson C.A. Graham M.R. Gardner D.J. Bailey J.R. Parnell M.J. Deleo F.R. Musser J.M. Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 4693-4698Crossref PubMed Scopus (29) Google Scholar). Gene expression was measured using the Affymetrix 430 2.0 Genechip containing the mouse genome and data analysis were carried out as described previously (28Li M. Lai Y. Villaruz A.E. Cha D.J. Sturdevant D.E. Otto M. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 9469-9474Crossref PubMed Scopus (226) Google Scholar) with the following modifications. An analysis of variance was performed using the Partek Genomics Suite (Partek, Inc., St. Louis, MO, version 6.3 6080414) to obtain multiple test corrected p values using the false discovery rate method at the 0.05 significance level combined with a fold-change value of 1.5. The data discussed in this article have been deposited in the NCBI Gene Expression Omnibus (29Edgar R. Domrachev M. Lash A.E. Nucleic Acids Res. 2002; 30: 207-210Crossref PubMed Scopus (8711) Google Scholar) and are accessible through GEO Series accession number GSE24873. Whole cell extracts were isolated, loaded on to 10% SDS-polyacrylamide gel, electrophoresed, and transferred to PVDF (Millipore) membranes. The following antibodies were used: anti-FLAG (Sigma); anti-HA, anti-TRAF6, anti-β-actin, and anti-IκBα (all from Santa Cruz); and anti-NF-κB p65, anti-phospho-Ser536 NF-κB p65, anti-ERK, and anti-phospho-ERK (all from Cell Signaling). Sequence alignment was carried out using ClustalW2 (30Larkin M.A. Blackshields G. Brown N.P. Chenna R. McGettigan P.A. McWilliam H. Valentin F. Wallace I.M. Wilm A. Lopez R. Thompson J.D. Gibson T.J. Higgins D.G. Bioinformatics. 2007; 23: 2947-2948Crossref PubMed Scopus (22841) Google Scholar) using default settings. Full-length human CIKS and CIKS deletion/point mutants were cloned into a Gateway Entry vector (Invitrogen) and subcloned into a lentiviral vector or into pcDNA3.1 HA or FLAG Tag destination vectors by Gateway LR recombination using the manufacturer's protocols to generate expression clones. In these vectors the standard cytomegalovirus promoter was replaced by the PolII promoter to ensure low level constitutive expression, with the exception of the vectors used in Fig. 2A and the IL-17RA vector used in Fig. 4B. The TRAF6 expression vector has been described previously (31Leonardi A. Ellinger-Ziegelbauer H. Franzoso G. Brown K. Siebenlist U. J. Biol. Chem. 2000; 275: 271-278Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Plasmid constructs were confirmed by sequencing. Lentivirus preparations used for transduction of wild-type and mutant CIKS proteins into CIKS-deficient primary MEFs were generated with the ViraPower Lentiviral Expression System (Invitrogen) following the manufacturer's instructions.FIGURE 4The N-terminal domain of CIKS is essential for interaction with TRAF6, but not for interaction with self or with IL-17RA. A, HeLa cells were co-transfected with tagged wild-type or ΔN50 mutant CIKS together and with differently tagged wild-type or mutant CIKS constructs as indicated. Cell lysates were IP to evaluate association of co-expressed CIKS proteins in immunoblots (IB) as indicated. B, FLAG-tagged wild-type or one of several mutant CIKS constructs were co-transfected together with HA-tagged IL-17RA. Cell lysates were IP with anti-HA IL-17RA and analyzed with immunoblots for the presence of co-precipitating CIKS proteins as well as endogenous TRAF6. Analyses shown in A and B are representative of at least three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) HeLa cells were transfected using Lipofectamine 2000 (Invitrogen). Whole cell extracts were isolated 48 h after transfection. Immunoprecipitations (IPs) were carried out using IP kits (Sigma) according to the manufacturer's instructions and analyzed by Western blotting as described above. For luciferase assays, HeLa cells were co-transfected with the IgκB Luc reporter as described previously (18Mauro C. Vito P. Mellone S. Pacifico F. Chariot A. Formisano S. Leonardi A. Biochem. Biophys. Res. Commun. 2003; 309: 84-90Crossref PubMed Scopus (21) Google Scholar). Luciferase activity was determined 24 h later using the Dual Luciferase assay system (Promega) according to the manufacturer's instructions and normalized to an ER Renilla internal control (Promega). To understand the significance of NF-κB and CIKS in the cellular response to IL-17 we made use of freshly isolated primary mouse embryo fibroblasts. We first re-examined whether and, if so, to what extent this cytokine can activate NF-κB in primary, freshly isolated, wild-type or CIKS-deficient MEFs. IL-17 induced the degradation of IκBα in wild-type cells, albeit much less so than TNFα, and only TNFα, not IL-17 was able to do so in CIKS-deficient cells (Fig. 1A). Thus IL-17 was able to liberate NF-κB in a CIKS-dependent manner in primary cells. The transcriptional activity of the dimeric NF-κB complexes in the nucleus is also determined by phosphorylation, especially of p65/RelA. Various potential phosphorylation sites have been reported for p65; their occurrence, relative significance, and precise function are not fully understood and appear to be context-dependent (32Neumann M. Naumann M. FASEB J. 2007; 21: 2642-2654Crossref PubMed Scopus (213) Google Scholar, 33Perkins N.D. Oncogene. 2006; 25: 6717-6730Crossref PubMed Scopus (558) Google Scholar). Here we show that both IL-17 and TNFα were able to induce phosphorylation of p65 at Ser536 in wild-type MEFs, which was fully dependent on CIKS in the case of IL-17 but not TNFα, as expected. No synergistic effect was observed when stimulating with both IL-17 and TNFα (Fig. 1B). To further explore the role of NF-κB and CIKS in the transcriptional response to IL-17 in primary MEFs, we first performed a genome-wide microarray analysis to identify genes that were significantly induced by IL-17 within 2 h (see supplemental Fig. S1 for full results). After applying stringent criteria to the results from 6 independently performed experiments, we identified 9 such genes in primary wild-type MEFs; by contrast, IL-17 failed to induce any genes in CIKS-deficient MEFs. These results were confirmed with qPCR analyses (supplemental Fig. S1C). A set of 9 genes encodes for chemokines Cxcl1, Ccl2, and Ccl7, the cytokines Lif and Il-6, transcription factors Iκbζ, c/Ebpδ, and RelB, and RNA-binding protein Zc3hl2a; most of these genes have been identified previously in various screens for IL-17-induced genes (21Huang F. Kao C.Y. Wachi S. Thai P. Ryu J. Wu R. J. Immunol. 2007; 179: 6504-6513Crossref PubMed Scopus (181) Google Scholar, 34Kao C.Y. Chen Y. Thai P. Wachi S. Huang F. Kim C. Harper R.W. Wu R. J. Immunol. 2004; 173: 3482-3491Crossref PubMed Scopus (356) Google Scholar, 35Shen F. Ruddy M.J. Plamondon P. Gaffen S.L. J. Leukocyte Biol. 2005; 77: 388-399Crossref PubMed Scopus (224) Google Scholar), although Zc3h12a has been noted only once in a recent screen of liver cells (36Sparna T. Rétey J. Schmich K. Albrecht U. Naumann K. Gretz N. Fischer H.P. Bode J.G. Merfort I. BMC Genomics. 2010; 11: 226Crossref PubMed Scopus (48) Google Scholar), whereas RelB has never been identified previously. Whereas Iκbζ, c/Ebpδ, and Zc3h12a were induced only with IL-17, not TNFα, the remaining genes were induced with either cytokine and are all known potential targets of NF-κB (37Ueda A. Okuda K. Ohno S. Shirai A. Igarashi T. Matsunaga K. Fukushima J. Kawamoto S. Ishigatsubo Y. Okubo T. J. Immunol. 1994; 153: 2052-2063PubMed Google Scholar, 38Bren G.D. Solan N.J. Miyoshi H. Pennington K.N. Pobst L.J. Paya C.V. Oncogene. 2001; 20: 7722-7733Crossref PubMed Scopus (164) Google Scholar, 39Hoffmann A. Leung T.H. Baltimore D. EMBO J. 2003; 22: 5530-5539Crossref PubMed Scopus (292) Google Scholar). Within the latter group, Cxcl1, Lif, and Il-6 appeared to be synergistically induced by the two cytokines, which may be due to post-transcriptional regulation (20Hartupee J. Liu C. Novotny M. Li X. Hamilton T. J. Immunol. 2007; 179: 4135-4141Crossref PubMed Scopus (242) Google Scholar, 40Schluns K.S. Cook J.E. Le P.T. J. Immunol. 1997; 158: 2704-2712PubMed Google Scholar). Despite the early time point after stimulation chosen in our analyses, Il-6 and c/Ebpδ are not immediate-early genes; they have previously been reported to depend on induced expression of IκBζ, and their peak of expression occurred well after 2 h (41Yamamoto M. Yamazaki S. Uematsu S. Sato S. Hemmi H. Hoshino K. Kaisho T. Kuwata H. Takeuchi O. Takeshige K. Saitoh T. Yamaoka S. Yamamoto N. Yamamoto S. Muta T. Takeda K. Akira S. Nature. 2004; 430: 218-222Crossref PubMed Scopus (397) Google Scholar, 42Yamazaki S. Matsuo S. Muta T. Yamamoto M. Akira S. Takeshige K. J. Biol. Chem. 2008; 283: 32404-32411Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar) (see also supplemental Fig. S2A). We conclude that CIKS is essential for expression of all IL-17-induced genes in primary MEFs within 2 h. The expression profiling study also identified immediate-early targets of IL-17 in primary cells that could be investigated further for their dependence on NF-κB. To address what downstream effectors may contribute to immediate-early IL-17-induced gene expression, we pre-treated primary wild-type MEFs with inhibitors for NF-κB, JAK, and MAP kinases p38, JNK, and ERK prior to stimulation with IL-17. Induced expression of immediate-early genes Cxcl1, Ccl2 and, to a lesser extent Iκbζ and Zc3h12a, was dependent on NF-κB, but was not significantly dependent on p38, JNK, JAK, or ERK (Fig. 1C, see also supplemental Fig. S2B). To verify the role of NF-κB, we also investigated the IL-17-induced expression of the above genes in NEMO-deficient MEFs, as the classical NF-κB activation pathway is completely abrogated in these cells. Consistent with the inhibitor studies, Ccl2 and Cxcl1 failed to be induced in the absence of NEMO, whereas induced expression of Iκbζ and Zc3h12a was significantly decreased, albeit not abolished (Fig. 1D). We conclude that NF-κB is critically involved in the IL-17-induced expression of immediate-early genes, albeit to a variable degree. To identify novel domains within CIKS critical for activation of NF-κB we generated and initially screened a number of CIKS mutants by assessing their ability to induce expression from an NF-κB-dependent luciferase reporter plasmid upon overexpression in HeLa cells (18Mauro C. Vito P. Mellone S. Pacifico F. Chariot A. Formisano S. Leonardi A. Biochem. Biophys. Res. Commun. 2003; 309: 84-90Crossref PubMed Scopus (21) Google Scholar). As expected, deletion of the SEFIR domain (ΔSEFIR), which is required for self-association (18Mauro C. Vito P. Mellone S. Pacifico F. Chariot A. Formisano S. Leonardi A. Biochem. Biophys. Res. Commun. 2003; 309: 84-90Crossref PubMed Scopus (21) Google Scholar), or the central region (Δ200–400), which carries the E3 ubiquitin-ligase domain (23Liu C. Qian W. Qian Y. Giltiay N.V. Lu Y. Swaidani S. Misra S. Deng L. Chen Z.J. Li X. Sci. Signal. 2009; 2: ra63Crossref PubMed Scopus (71) Google Scholar), significantly reduced the ability of exogenously introduced CIKS to activate NF-κB (Fig. 2A). By contrast, deletion of one of the two purported TRAF6 binding sites on CIKS (Δ38–42) had no effect on NF-κB activation (23Liu C. Qian W. Qian Y. Giltiay N.V. Lu Y. Swaidani S. Misra S. Deng L. Chen Z.J. Li X. Sci. Signal. 2009; 2: ra63Crossref PubMed Scopus (71) Google Scholar). Surprisingly, deletion of the N-terminal 50 amino acids (ΔN50) completely abolished NF-κB activation (Fig. 2B). We therefore generated additional mutants to identify sequences within the N terminus that may be required for NF-κB activation. Deletion of amino acids 1–35 (Δ1–35), 1–15 (Δ1–15), and 10–25 (Δ10–25) all completely eliminated the ability of overexpressed CIKS to activate NF-κB (numbering is based on the longer of the two human CIKS isoforms; the shorter isoform starts at position 10, but is otherwise identical) (Fig. 2B). Deletion of amino acids 20–35 (Δ20–35) resulted in partial impairment of CIKS to activate NF-κB, whereas deletion of amino acids 35–50 (Δ35–50), which contains a site previously thought to bind TRAF6 (23Liu C. Qian W. Qian Y. Giltiay N.V. Lu Y. Swaidani S. Misra S. Deng L. Chen Z.J. Li X. Sci. Signal. 2009; 2: ra63Crossref PubMed Scopus (71) Google Scholar, 24Kanamori M. Kai C. Hayashizaki Y. Suzuki H. FEBS Lett. 2002; 532: 241-246Crossref PubMed Scopus (38) Google Scholar) failed to impair this CIKS function. This suggested that a previously unknown N-terminal domain, most likely contained between positions 10 and 25, was crucial for activation of NF-κB. A comparison of CIKS sequences from various species showed a remarkable conservation of amino acids in the interval between positions 10 and 21; there is little or no conservation elsewhere in the larger N-terminal region of CIKS when more divergent species are compared (Fig. 2C). To further delimit the critical sequences, we generated four point mutations in the domain between positions 10 and 21, replacing Ser13, Val16, Glu17, or Asp19 with Ala (S13A, V16A, E17A and D19A); in addition we generated a combined mutant in which all four positions were replaced (“Quad”). As shown in Fig. 2D, the E17A mutation completely abrogated the ability of CIKS to activate NF-κB and the D19A mutation partially interfered, whereas the S13A and V16A mutations had no significant effect in this assay. Consistent with this, the Quad mutation completely blocked CIKS from activating NF-κB as well. We conclude that the N terminus of CIKS harbors a previously unidentified domain likely located between amino acid positions 10 and 21 that is required for activation of NF-κB in CIKS-transfected cells. To understand the importance of this newly identified N-terminal domain in a physiologic context, we employed a lentivirus transduction strategy to reconstitute CIKS-deficient primary MEFs with minimal levels of wild-type and mutant CIKS proteins (Δ10–25, E17A, and ΔN50), or with a GFP control, and then assayed for responses to stimulation with IL-17. We first tested for IL-17-induced expression of the immediate-early target test genes investigated above: Iκbζ, Zc3h12a, Cxcl1, and Ccl2. Reconstitution with the ΔN50, Δ10–25, and E17A CIKS mutants significantly impaired induction of Cxcl1, Ccl2, and Zc3h12a, and to a lesser extent Iκbζ, when compared with cells reconstituted with wild-type CIKS (Fig. 3, A and B) (see supplemental Fig. S2C for Il-6 and c/Ebpδ). To directly evaluate the ability of IL-17 to activate NF-κB in the primary MEFs reconstituted with the wild-type and CIKS mutants shown above (Fig. 3, A and B), we measured IL-17-induced degradation of IκBα. As shown in Fig. 3C, only CIKS-deficient MEFs reconstituted with wild-type CIKS were able to degrade IκBα in response to IL-17, whereas those reconstituted with CIKS mutants that crippled the critical N-terminal domain failed to respond to IL-17, as did those reconstituted with GFP only. Together these data demonstrate that the newly identified N-terminal CIKS domain is essential for IL-17-induced degradation of IκBα and thus for activation of NF-κB-dependent target genes. The ability of the N-terminal mutants to partially restore induced expression of, in particular, Iκbζ," @default.
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• W2039639938 title "IL-17-induced NF-κB Activation via CIKS/Act1" @default.
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