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Rip1 Mediates the Trif-dependent Toll-like Receptor 3- and 4-induced NF-κB Activation but Does Not Contribute to Interferon Regulatory Factor 3 Activation *

Rip1 Mediates the Trif-dependent Toll-like Receptor 3- and 4-induced NF-κB Activation but Does... THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 280, NO. 44, pp. 36560 –36566, November 4, 2005 © 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Rip1 Mediates the Trif-dependent Toll-like Receptor 3- and 4-induced NF-B Activation but Does Not Contribute to Interferon Regulatory Factor 3 Activation Received for publication, June 23, 2005, and in revised form, August 17, 2005 Published, JBC Papers in Press, August 22, 2005, DOI 10.1074/jbc.M506831200 ‡ ‡ ‡ § ‡1 Nicole Cusson-Hermance , Smriti Khurana , Thomas H. Lee , Katherine A. Fitzgerald , and Michelle A. Kelliher ‡ § From the Departments of Cancer Biology and Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605 Rip1 is required for IB kinase activation in response to tumor (TLR3)-mediated NF-B response to dsRNA (8). Rip1 interacts with the necrosis factor  (TNF-) and has been implicated in the Toll-like TLR3- and TLR4-specific adapter Trif (Toll-interleukin-1 receptor receptor 3 (TLR3) response to double-stranded RNA. Cytokine pro- domain-containing adaptor inducing IFN-). Trif is a TIR domain-con- duction is impaired when rip1/ cells are treated with TNF-, taining adapter protein that is essential for all signaling by TLR3 and poly(I-C), or lipopolysaccharide, implicating Rip1 in the Trif-de- some signaling by TLR4. Rip1 binds the C terminus of the Trif protein pendent TLR3 and TLR4 pathways. To examine the role of Rip1 in via a Rip homotypic interaction motif (8). Trif also binds Traf6 (tumor the Trif-dependent TLR4 pathway, we generated rip1/ necrosis factor receptor-associated factor-6) and TBK-1 via its N termi- MyD88/ cells. Lipopolysaccharide failed to stimulate NF-B nus, and these interactions result in interferon regulatory factor 3 activation in rip1/MyD88/ cells, revealing that Rip1 is also (IRF-3) activation. The TLR4 ligand LPS induces NF-B activation by required for the Trif-dependent TLR4-induced NF-B pathway. In engaging the MyD88-dependent and Trif-dependent pathways. Thus, addition to activating NF-B, TLR3/4 pathways also stimulate we reasoned that Rip1 may also participate in late phase NF-B activa- interferon regulatory factor 3 activation. However, we find that tion induced by TLR4 Trif-dependent pathway. Rip1 expression stimulates NF-B but not interferon regulatory We find NF-B responses and cytokine production ablated when factor 3 activity. In the TNF- pathway, Rip1 interacts with the E3 rip1/ murine embryonic fibroblasts (MEF) or splenocytes are stim- ubiquitin ligase Traf2 and is modified by polyubiquitin chains. ulated with dsRNA or TNF-. Although the NF-B responses to TLR4 Upon TLR3 activation, Rip1 is also modified by polyubiquitin appear unaffected due to activation of the MyD88-dependent pathway, chains and is recruited to TLR3 along with Traf6 and the ubiquitin- LPS-induced cytokine production was impaired in the absence of Rip1. activated kinase Tak1. These studies suggest that Rip1 uses a simi- Importantly, LPS-induced NF-B activation was completely ablated in lar, ubiquitin-dependent mechanism to activate IB kinase- in rip1/MyD88/ MEF, providing genetic evidence that Rip1 con- response to TNF- and TLR3 ligands. tributes to the Trif-dependent, TLR4-induced NF-B pathway. More- over, we find Rip1 phosphorylated and polyubiquitinated in TLR3-stim- ulated cells and demonstrate that Rip1, Traf6, and the ubiquitin- The death domain kinase Rip1 (receptor-interacting protein 1) medi- activated kinase Tak1 are recruited to TLR3 in response to poly(I-C) treatment. Taken together, these studies suggest that Rip1 may use sim- ates TNF- -induced NF-B and p38 mitogen-activated protein kinase ilar ubiquitin-dependent mechanisms to activate IKK- in TLR3-stim- activation (1–4). Although Rip1 is required for IKK activation (5), its ulated cells. kinase activity is dispensable (3, 6), suggesting that Rip1 may mediate IKK activation by recruiting other MAP kinases such as Mekk3 or by employing other mechanisms to achieve IKK activation. Upon recruit- EXPERIMENTAL PROCEDURES ment to the TNF receptor (TNFR1), Rip1 is initially modified by K63- Generation of rip1/ and rip1/MyD88/ Murine Embry- linked polyubiquitin chains. These K63-linked polyubiquitin chains are onic Fibroblasts—Rip1/ mice were interbred, females were sacri- recognized by the ubiquitin receptor Tab2 (7), resulting in the recruit- ficed between embryonic days 15.5 and 17, and MEF were prepared as ment of the ubiquitin-activated Tak1 (TGF--activating kinase 1) described in Ref. 1. For other studies, rip1/tnfr1/ mice were enzyme to the TNFR1. intercrossed, and splenocytes were isolated from day 2 rip1// Recently, Rip1 has also been implicated in the Toll-like receptor 3 tnfr1/ or rip1//tnfr1/ mice. MyD88/ mice were gifts from S. Akira (Osaka, Japan). The MyD88/ mice used for this study were backcrossed into the C57BL/6 background for 11 generations. To * This work was supported in part by National Institutes of Health Grant GM061298 (to generate rip1/MyD88/ MEF, rip1/ mice were mated with M. K.) and by the Wellcome Trust (to K. A. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be MyD88/ mice, and then rip1/MyD88/ mice were inter- hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to crossed, and females were sacrificed to generate rip1/ or rip1/ indicate this fact. Scholar of the Leukemia and Lymphoma Society of America. To whom correspondence MyD88/ or rip1/MyD88/ MEF. Traf6/ MEF were should be addressed: Dept. of Cancer Biology, University of Massachusetts Medical provided by Dr. J.-I. Inoue (Tokyo, Japan). School, Lazare Research Bldg., 364 Plantation St., Worcester, MA 01605. E-mail: Cell Lines and Reagents—HEK293 cells were stably transfected with [email protected]. The abbreviations used are: TNF-, tumor necrosis factor ; IRF3, interferon-regulatory FLAG-tagged human TLR3 as described in Ref. 9. LPS derived from factor 3; TLR, Toll-like receptor; TNFR1, tumor necrosis factor receptor 1; MEF, mouse Escherichia coli strain 0111.B4 was purchased from Sigma, dissolved in embryonic fibroblast(s); MyD88, myeloid differentiation factor 88; IFN, interferon; IKK, IB kinase; LPS, lipopolysaccharide; MAP, mitogen-activated protein; dsRNA, double- deoxycholate, and re-extracted by phenol/chloroform as described in stranded RNA; RANTES, regulated on activation normal T cell expressed and secreted; Ref. 10. Poly(I-C) was purchased from Amersham Biosciences, and ELISA, enzyme-linked immunosorbent assay; ISRE, interferon-stimulated response element. MALP2 and peptidoglycan were purchased from EMC Microcollec- 36560 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 280 • NUMBER 44 •NOVEMBER 4, 2005 This is an Open Access article under the CC BY license. Rip1 Mediates TLR3 and TLR4 NF-B Pathways FIGURE 1. Rip1 mediates TLR3-induced NF-B activation but does not appear to contribute to TLR2 or TLR4-induced NF-B activation. A,an impaired TLR3 response in the absence of Rip1. Wild type (wt) and rip1/ MEF were left untreated or stimulated with poly(I-C) (100 g/ml) or IL-1 (10 ng/ml) for the time periods indicated, and NF-B activity was measured using a phos- phospecific IB antibody. B, normal LPS-induced NF-B activation in rip1/ MEF. Wild type and rip1/ MEF were left untreated or stimulated with purified LPS (100 ng/ml) or IL-1 (10 ng/ml). Cell lysates were probed with a phosphospecific IB antibody. C, normal TLR2-induced NF-B responses in rip1/ MEF. Wild type and rip1/ MEF were left untreated or treated with the TLR2 ligand peptidoglycan (PGN at 10 g/ml) or IL-1 (10 ng/ml) for the time periods indicated. Activa- tion of NF-B was measured by probing the cell lysates with a phosphospecific IB antibody. To ensure that equal amounts of total protein was achieved, cell lysates were probed with an IKK- antibody. Three independent rip1/ MEF lines were examined, and a representative experiment is shown. tions (Tuebingen, Germany). Sendai virus was purchased from Charles Coimmunoprecipitation and Western Blotting—HEK293 cells stably River Laboratories (Wilmington, MA). Anti-Rip1 antibody was pur- transfected with FLAG-tagged human TLR3 left unstimulated or chased from BD Biosciences (catalog number 601459), and anti-phos- treated with poly(I-C) (100 g/ml) were lysed in endogenous lysis buffer pho-IB was from Cell Signaling (catalog number 9246s). Anti-IB (150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 1% Nonidet P-40, 0.5% deoxy- (catalog number sc-7183), anti-Traf6 (catalog number sc-6224), anti- cholate, 0.1% SDS) and immunoprecipitated with anti-FLAG antibody, IKK (catalog number sc-371), anti-extracellular signal-regulated and associated proteins were detected by immunoblotting with anti- kinase (catalog number sc-154), anti-Tak1 (catalog number sc-7162), Rip1, -Traf6, or -Tak1 antibodies. To measure NF-B activity, wild type and anti-ubiquitin (catalog number sc-8017) were from Santa Cruz Bio- or rip1/ MEF were left untreated or stimulated with IL-1 (10 technology, Inc. (Santa Cruz, CA). Anti--actin was from Sigma (cata- ng/ml), TNF- (10 ng/ml), or TLR ligands poly(I-C) (100 g/ml), puri- log number A5441), and antibodies were used according to the manu- fied LPS (100 ng/ml), peptidoglycan, or MALP2 or infected with Sendai facturer’s suggestions. The RANTES enzyme-linked immunosorbent virus, and total cell lysates were examined for evidence of phosphoryl- assay (ELISA) kit and recombinant TNF- and IL-1 were purchased ated IB using a phosphospecific IB antibody (Cell Signaling). To from R & D Systems. The IRF3–5D plasmid was from John Hiscott ensure that an equivalent amount of total protein was examined, lysates (Montreal, Canada). The NF-B-luciferase, the ISG54 ISRE luciferase, were probed with either an IKK- antibody (Santa Cruz) or anti-extra- and the Trif and p65 expression constructs are described in Ref. 11. cellular signal-regulated kinase antibody. To measure TNF- and LPS- Real Time PCR—RNA from wild type and rip1/ MEF was iso- induced NF-B activation in the rip1/MyD88/ MEF, IB deg- lated, and cDNA was synthesized using the Superscript First Strand radation was measured by probing equivalent amounts of total protein System (Invitrogen). Real time PCR was performed using the DNA with an anti-IB antibody (Santa Cruz Biotechnology). Polyubiquiti- Engine-Opticon 2 PCR Machine (MJ Research). The reaction included nated Rip1 was detected by stimulating the RAW264.7 macrophage or 2 SYBR Green PCR Master Mix, forward and reverse primer (10 M), U373 astrocytoma cell lines with poly(I-C) or TNF- and immunopre- and the appropriate amount of cDNA. The thermal cycling parameter cipitating the lysates with anti-Rip1 antibody, followed by immunoblot- was 95 °C for 15 min, 95 °C for 15 s, 58.5 °C for 30 s, 72 °C for 30 s for 45 ting with an anti-ubiquitin antibody. cycles followed by 72 °C for 10 min. The primers for real time PCR were RESULTS murine IFN- (5-AGC TCC AAG AAA GGA CGA ACA T-3), murine IFN- (3-GCC CTG TAG GTG AG TTG ATC T-5); murine Rip1 Mediates the Trif-dependent NF-B Pathway—Recently, the -actin (5-TTG AAC ATG GCA TTG TTA CCA A-3); and murine Rip1 protein has been implicated in the innate immune response to -actin (3-TGG CAT AGA GGT CTT TAC GGA-5). double-stranded RNA viruses (8, 12). Mouse embryonic fibroblasts that Transfection Assays—HEK 293 cells (1.5  10 cells/well) were lack Rip1 fail to activate NF-B when stimulated with poly(I-C) (8); seeded into 96-well plates and transfected on the following day with 40 however, the response to other Toll-like ligands has not been examined. ng of luciferase reporter genes (NF-B, ISRE, or Viperin promoter) To test whether Rip1, like Rip2, mediates multiple Toll-like receptor using Genejuice (Novagen). The Renilla luciferase reporter gene (Pro- pathways, we treated wild type and rip1/ MEF with IL-1, poly(I-C), mega) was cotransfected for normalization. In all cases, cell lysates were purified LPS, and the TLR2 ligand peptidoglycan. To measure NF-B prepared, and reporter gene activity was measured using the Dual Lucif- activity, we assayed cell lysates for phosphorylated IB by immuno- erase Assay System (Promega). Data are expressed as the mean relative blotting. As expected, we found poly(I-C)-induced NF-B activation stimulation  S.D. for a representative experiment from three separate impaired in rip1/ MEF (8) (Fig. 1A) yet observed normal NF-B experiments, each performed in triplicate. responses when rip1/ cells were stimulated with TLR2 or TLR4 NOVEMBER 4, 2005• VOLUME 280 • NUMBER 44 JOURNAL OF BIOLOGICAL CHEMISTRY 36561 Rip1 Mediates TLR3 and TLR4 NF-B Pathways FIGURE 2. Decreased IFN- expression and cytokine production in poly(I-C)-stimu- lated rip1/ MEF. A, IFN- expression is reduced in poly(I-C)-stimulated rip1/ cells. Wild type (wt) and rip1/ MEF were left untreated or treated with poly(I-C) (100 g/ml) for the time periods indicated, and real time PCR analysis was performed using primers specific for IFN- and -actin. B, poly(I-C) or TNF- treatment of rip1/ cells results in impaired cytokine production. Wild type and rip1/ MEF were left untreated or stim- ulated with poly(I-C) (100, 10, or 1 g/ml), Sendai virus (200, 20, or 2 HAU), or murine TNF- (100, 10, or 1 ng/ml). Twenty-four hours later, culture supernatants were exam- ined for RANTES production by ELISA. Three independent rip1/ MEF lines were exam- ined in triplicate, and one representative experiment is shown. FIGURE 3. Poly(I-C) and LPS-induced cytokine production is severely impaired in ligands (Fig. 1, B and C). Thus, the NF-B responses to Toll-like recep- Rip1-deficient cells. A, Rip1-deficient MEF are impaired in cytokine response to poly(I-C) and LPS. Wild type and rip1/ MEF were left unstimulated or stimulated with poly(I-C) tor 2 and 4 ligands appear unaffected by a Rip1 deficiency (Fig. 1, B and (100 or 10 mg/ml), LPS (100 or 10 ng/ml), or mTNF- (10 ng/ml). Culture supernatants C), indicating that, unlike Rip2, Rip1 may only mediate the Trif-depend- were examined for Rantes production by ELISA. B, poly(I-C)- and LPS-induced cytokine production is impaired in rip1/tnfr1/ splenocytes. rip1/tnfr1/ or ent pathways. rip1/tnfr1/ splenocytes were left untreated or stimulated with poly(I-C) (100 or The lack of TLR3 responsiveness in rip1/ MEF could reflect 10 g/ml) or purified LPS (100 or 10 ng/ml). Twenty-four hours later, culture superna- decreased TLR3 expression in Rip1-deficient cells. Thus, we examined tants were examined for RANTES production by ELISA. A minimum of three rip1/ MEF and splenocytes from three rip1/tnfr1/ and two rip1/tnfr1/ mice were wild type and rip1/ MEF for expression of TLR3 and TLR4 using examined in triplicate. A representative experiment for each is shown. wt, wild type. reverse transcription-PCR. We found similar TLR3 and TLR4 expres- sion levels in wild type and rip1/ cells (not shown), suggesting that points, induction of IFN- expression was observed in poly(I-C)-stim- the TLR3 signaling defect is not due to decreased TLR3 receptor levels ulated, wild type MEF but not in rip1/ MEF stimulated with in Rip1-deficient cells. poly(I-C) (Fig. 2A). In contrast, late phase (24-h) IFN- expression Decreased IFN- and RANTES Production in Poly(I-C)-stimulated appears less affected, although expression remained significantly rip1/ Cells—To foster antiviral responses, TLR3 and TLR4 activate reduced in poly(I-C)-treated rip1/ MEF (Fig. 2A). Similarly, the transcription factors NF-B and IRF-3 and induce IFN- expres- RANTES production was reduced when rip1/ cells were stimulated sion. The transcriptional enhancer of the IFN- promoter contains four with either poly(I-C) or TNF- (Fig. 2B). However, cytokine production positive regulatory domains, which function cooperatively to induce was unaffected when rip1/ MEF were infected with Sendai virus, a IFN- expression in response to viral infection. The transcription fac- single-stranded RNA virus, that induces IFN- expression via a Trif- tors that regulate IFN- expression include NF-B, IRF-3, and the ATF- and TLR3-independent pathway (14). These data suggest that Rip1 con- 2-c-Jun heterodimer (9, 13). Thus, the initial induction of IFN- expres- tributes to anti-viral responses mediated by TLR3 but is not required for sion requires the activation of NF-B, yet in our previous published NF-B activation by the intracellular retinoic acid-inducible gene I studies, we observed induction of IFN- in poly(I-C)-treated rip1/ pathway (15). cells (8). Since these studies were performed using reverse transcrip- Decreased RANTES Production When rip1/ Splenocytes Are tion-PCR (13), we reexamined IFN- expression in poly(I-C)-stimu- Stimulated with Purified LPS—Although Rip1 has been shown to inter- lated rip1/ MEF using quantitative real time PCR. At early time act with the TLR3 and TLR4 adapter Trif, cytokine responses to 36562 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 280 • NUMBER 44 •NOVEMBER 4, 2005 Rip1 Mediates TLR3 and TLR4 NF-B Pathways FIGURE 4. Rip1 mediates the Trif-dependent TLR4-induced NF-B pathway. Wild (wt) type MEF (A), rip1/MyD88/ (B) and double knockout rip1/MyD88/ MEF (C) were left untreated or stimulated with LPS (10 g/ml) or TNF- (10 ng/ml) for the time periods indicated, and NF-B activity was measured by immunoblotting with an anti- IB antibody. To ensure that equal amounts of total protein were loaded, cell lysates were probed with an anti-extracellular signal-regulated kinase (-ERK) antibody. One wild type, one rip1/MyD88/, and three rip1/MyD88/ MEF were examined. One representative experiment is shown. poly(I-C) or purified LPS were not examined in our previous study (8). Thus, Rip1 may contribute to NF-B activation when MEF are treated with poly(I-C) but may not contribute significantly to cytokine produc- tion in vivo, particularly when the TLR4 pathway is stimulated, since NF-B can be activated by the MyD88-dependent pathway. Due to the lethality associated with a Rip1 deficiency (1), we were unable to exam- ine TLR3/4 responses in Rip1-deficient macrophages or dendritic cells. To evaluate the contribution of Rip1 to TLR3- and TLR4-induced innate immune responses, we stimulated neonatal day 2 splenocytes from rip1/tnfr1/ mice and control littermates with poly(I-C), purified LPS, or TNF-. We found RANTES production significantly FIGURE 5. Rip1 expression stimulates NF-B but not IRF-3 activation. HEK 293 cells were transfected with an NF-B reporter plasmid (A) or with IRF3 reporter plasmids (B)or reduced when neonatal rip1/tnfr1/ splenocytes are stimulated with a Viperin-dependent reporter construct (C), together with expression constructs with poly(I-C) and LPS (Fig. 3). These findings suggest that Rip1 con- encoding p65, Rip1, Trif, or IRF-3. After 24 h, luciferase gene activity was measured. The luciferase assays were done in triplicate and repeated three times. tributes to both the Trif-dependent TLR3 and TLR4 pathways and dem- onstrate that activation of both the Trif-Rip1 and MyD88-dependent TLR4 pathways is required for robust cytokine production. double knock out cells failed to respond to TNF-, LPS, and poly(I-C) Rip1 Mediates the Trif-dependent TLR4 Pathway—The decreased but remained capable of activating NF-B in response to treatment with cytokine responses to the TLR4 ligand LPS observed in rip1/ MEF phorbol 12-myristate 13-acetate/ionomycin (not shown). These studies and splenocytes suggested that Rip1 may also mediate the Trif-depend- provide genetic evidence that Rip1 contributes to the Trif-dependent ent, TLR4-induced NF-B pathway. To test this possibility, we mated TLR4-induced NF-B pathway. our rip1/ mice with MyD88/ mice (generously provided by Rip1 Expression Stimulates NF-B but Not IRF-3 Activation—The S. Akira) and then stimulated wild type, rip1/MyD88/,or presence of a Rip homotypic interaction domain in Trif and Rip1 sug- rip1/MyD88/ and rip1/MyD88/ MEF with TNF- or gests that Rip1 and Trif may interact to mediate both NF-B and IRF-3 LPS and examined cytokine-induced IB degradation. TNF--in- activation. Deletion of the Rip homotypic interaction motif in Trif duced IB degradation was observed in wild type cells and in rip1/ ablates Trif-mediated NF-B activation but has no effect on IRF-3 acti- MyD88/ 10 min following cytokine treatment (Fig. 4, A and B). As vation (8). To test whether Rip1 can stimulate both NF-B and IRF-3 expected, no TNF--induced IB degradation is observed in the dou- activation, we transfected HEK293 cells with a NF-B or IRF reporter ble knockout rip1/MyD88/ cells due to the absence of Rip1 (Fig. constructs (ISG54 and ISRE) with expression plasmids containing p65 4C). Wild type MEF exhibit IB degradation 15 min following LPS (RelA), Rip1, or Trif or with a constitutively active IRF-3 expression treatment, whereas IB degradation is delayed in rip1/ construct, the phosphomimetic IRF3-5D. Twenty-four hours later, MyD88/ cells with evidence of IB degradation detected luciferase reporter gene activity was measured. RelA, Rip1, and Trif between 45 min and 1 h poststimulation (Fig. 4, A and B). In contrast, no expression all stimulated NF-B reporter activity (Fig. 5A), yet only Trif LPS-induced IB degradation was observed in cells deficient for both and IRF3-5D were capable of stimulating IRF-3-dependent reporter Rip1 and MyD88 even at 120 min following LPS treatment (Fig. 4C). The activity (ISRE reporter (Fig. 5B) and Viperin reporter (Fig. 5C)). This NOVEMBER 4, 2005• VOLUME 280 • NUMBER 44 JOURNAL OF BIOLOGICAL CHEMISTRY 36563 Rip1 Mediates TLR3 and TLR4 NF-B Pathways FIGURE 7. Poly(I-C) treatment stimulates the recruitment of Rip1, Traf6, and Tak1 to the TLR3. HEK293 cells expressing a FLAG-tagged version of human TLR3 were left untreated or stimulated with 100 g/ml poly(I-C) for the time periods indicated. Cell lysates were immunoprecipitated (IP) with anti-FLAG antibody, and TLR3-associated proteins were isolated. The presence of Rip1, Traf6, and Tak1 was detected by immuno- blotting cell lysates with an anti-Rip1, Traf6, or Tak1 antibody. Total cell lysates were then probed with an anti-Rip1 antibody to assure equal loading. The coimmunoprecipitation experiments were repeated twice. Rip1, Traf6, and Tak1 Are Recruited to TLR3 upon Poly(I-C) Stimulation—Our data suggest that Rip1 and Traf6 proteins contribute to the Trif-dependent NF-B pathway. To test whether the endogenous proteins are recruited to TLR3 upon receptor activation, we stimulated HEK 293 that express a FLAG-tagged TLR3 with poly(I-C) for 30, 60, and 120 min. We immunoprecipitated TLR3 using the anti-FLAG anti- FIGURE 6. Poly(I-C) and LPS-induced cytokine production is impaired in traf6/ body and tested whether Rip1 or Traf6 associated with TLR3. We MEF. A, poly(I-C) or LPS treatment does not stimulate NF-B activation in traf6/ MEF. Wild type (wt) and traf6/ MEF were left untreated or stimulated with poly(I-C) (100 detected Rip1 and Traf6 recruitment to TLR3 30 min following g/ml), LPS (100 ng/ml), or TNF- (10 ng/ml) for the indicated time periods. Cell lysates poly(I-C) treatment (Fig. 7, A and B). We also examined the TLR3- were separated on a 12% SDS-polyacrylamide gel, and NF-B activity was assessed by immunoblotting with an anti-phospho-IB antibody. B, poly(I-C) or LPS treatment fails associated proteins for evidence of Tak1 recruitment. Tak1 recruitment to stimulate cytokine production in traf6/ MEF. Wild type and traf6/ MEF were left to TLR3 was observed at 60 min when maximal phosphorylated IB untreated or stimulated with various concentrations of poly(I-C) (100, 10, or 1 g/ml), protein is also detected (Fig. 7C). Interestingly, Rip1 and Traf6 recruit- LPS (100, 10, or 1 ng/ml), 10 nM MALP2, or 10 ng/ml murine TNF-. Twenty-four hours later, culture supernatants were examined in triplicate for RANTES production. Each ment appears to precede Tak1 recruitment, suggesting that Traf6 may experiment was repeated a minimum of three times. modify Rip1 and thereby signal Tak1 recruitment. experiment suggests that Rip1 may not participate in the Trif-depend- Rip1 Is Phosphorylated and Polyubiquitinated in TLR3-stimulated ent IRF-3 pathway and is recruited by Trif to mediate IKK- activation. Cells—We have shown that the kinase activity of Rip1 is responsive to LPS- and Poly(I-C)-induced Cytokine Production Is Diminished in TNFR1 activation (6), suggesting that the kinase activity of Rip1 is traf6/ MEF—The MyD88-dependent NF-B pathway is mediated responsive to specific ligands or stresses. Studies in cells expressing by the recruitment of IRAK kinases and Traf6 and the Tak1-Tab1-Tab2 kinase-inactive Rip1 have revealed that Rip1 autophosphorylation is complex. Thus, we reasoned that Traf6 may also contribute to the Trif- induced in TNF--stimulated cells (6) (Fig. 8, A and B). Similarly, the dependent NF-B pathway. To test this possibility, we stimulated wild related Rip2 kinase and IRAK kinases are responsive to TLR and IL-1 type and traf6/ MEF with TLR ligand poly(I-C) or LPS or with receptor activation (16). To provide additional evidence that Rip1 TNF- and examined IKK activity using a phospho-IB antibody. In responds to TLR3/4 activation, we stimulated wild type cells with contrast to wild type MEF, traf6/ MEF responded only to TNF- TNF- or with the TLR ligand LPS or poly(I-C) and examined cell and failed to phosphorylate IB when stimulated with TLR3 or TLR4 lysates for phosphorylated Rip1. As expected, phosphorylated Rip1 was ligands (Fig. 6A). Culture supernatants were also harvested 24 h post- detected as early as 5 min following TNF- treatment. We find Rip1 stimulation, and RANTES production was measured by ELISA (Fig. 6B). autophosphorylation also rapidly induced upon poly(I-C) treatment or NF-B activation and cytokine production in response to TLR2, -3, and LPS treatment (Fig. 8, A and B). These studies reveal that the Rip1 kinase -4 ligands was ablated in traf6/ MEF, whereas TNF-induced IKK activity responds to TNFR1, TLR3, and TLR4 activation and further activation and cytokine production remained unaffected by a Traf6 defi- implicates Rip1 in a TLR3 and TLR4 Trif-dependent pathway. ciency (Fig. 6, A and B). Taken together, these studies suggest that Traf6 In addition to phosphorylation, TNF- stimulates the conjugation of and Rip1 contribute to the Trif-dependent TLR3 and TLR4 NF-B K63-linked polyubiquitin chains on Rip1 (6, 17–19). K63-linked poly- pathways. ubiquitin chains on Rip1, Traf6, or IKK- (NEMO) are recognized by 36564 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 280 • NUMBER 44 •NOVEMBER 4, 2005 Rip1 Mediates TLR3 and TLR4 NF-B Pathways DISCUSSION The TLR3 and TLR4 pathways employ the adapter protein Trif that signals both NF-B and IRF-3 activation. Trif recruits Traf6 and TBK-1 via its N terminus, and TBK-1 is responsible for IRF-3 phosphorylation and activation (11). However, it remains unclear how Trif activates the IKK- and JNK2 pathways. Data base screening for proteins containing regions homologous to Trif revealed that Trif-induced NF-B activa- tion may be mediated by Rip1 (8). Studies in Rip1-deficient MEF then confirmed defects in poly(I-C)-induced NF-B activation, implicating Rip1, a mediator of TNF--induced IKK activation, in the Trif-depend- ent NF-B pathway (8). In this study, we address the biologic contribution of Rip1 to innate immune responses by examining poly(I-C)- and LPS-induced cytokine production in rip1/ MEF and splenocytes. Consistent with the pub- lished studies on Trif-deficient and MyD88-deficient cells (20, 21), we find poly(I-C)- and LPS-induced cytokine production impaired in Rip1- deficient MEF and splenocytes. Taken together, these studies support the model that TLR4-induced cytokine responses to bacterial patho- gens require activation of both the Trif-dependent and MyD88-depend- ent pathways. The involvement of Rip1 in the Trif-dependent TLR3 NF-B path- way suggested that Rip1 may also contribute to the Trif-dependent TLR4-induced NF-B activation. Consistent with this idea, we find that LPS, like TNF- and poly(I-C), stimulates the kinase activity of Rip1, and we find LPS-induced cytokine levels diminished in rip1/ cells. To test the contribution of Rip1 to the Trif-dependent TLR4 NF-B FIGURE 8. Treatment with TLR3 or TLR4 ligands stimulates Rip1 phosphorylation pathway, we generated rip1/MyD88/ cells and stimulated them and polyubiquitination. A, the kinase activity of Rip1 is induced when cells are treated with either TNF- or LPS. As expected, NF-B activation is delayed in with TNF- or poly(I-C). Wild type MEF were left untreated or stimulated with 100 g/ml poly(I-C) or 10 ng/ml murine TNF- for the time periods indicated. Cell lysates were LPS-treated rip1/MyD88/ cells; however, in the absence of both separated on an 8% gel, and Rip1 protein was detected by immunoblotting with an MyD88 and Rip1, LPS-induced NF-B activation is not observed. Taken anti-Rip1 antibody. B, the TLR4 ligand LPS stimulates Rip1 phosphorylation. Wild type together, these studies demonstrate that Rip1 is responsive to TLR4 MEF were left untreated or stimulated with 100 ng/ml LPS or 10 ng/ml murine TNF- for the time periods indicated. Cell lysates were separated on an 8% gel and examined by activation and demonstrates that Rip1 mediates both the Trif-depend- immunoblotting with an anti-Rip1 antibody. C, TNF- and poly(I-C) stimulation induces ent TLR3 and TLR4 NF-B pathways. Interestingly, the dsRNA-acti- Rip1 polyubiquitination. RAW264.7 macrophages or U373 astrocytoma cells were left untreated or stimulated with 200 g/ml poly(I-C) or 20 ng/ml mouse or human TNF-. vated protein kinase PKR has also been implicated in poly(I-C)-induced Cell lysates were immunoprecipitated with anti-Rip1 antibody and separated on a 3– 8% NF-B activation and in apoptotic responses to poly(I-C), TNF-, and gradient gel. Polyubiquitinated Rip1 was detected by immunoblotting with an anti- ubiquitin antibody. LPS (22, 23), raising the possibility that dsRNA-induced NF-B activa- tion may involve PKR and Rip1. Consistent with this model, Li and co-workers (24) find PKR recruited to Trif upon poly(I-C) stimulation. the ubiquitin receptor protein Tab2, a component of the ubiquitin- However, unlike PKR-deficient cells that are resistant to poly(I-C)- and activated Tak1 complex (7). Thus, Rip1 may use similar mechanisms to TNF--induced apoptosis, Rip1-deficient cells are sensitive to TNF-- mediate IKK- activation in TNF-- and TLR3- or TLR4-stimulated and poly(I-C)-induced cell death, suggesting that Rip1 and PKR have cells. To test this possibility, we stimulated the macrophage cell line opposing functions in the TNF-- and dsRNA-activated pathways. RAW264.7 and the U373 astrocytoma line with TNF- for 10 min or We demonstrate that, like TNFR1 activation, TLR3 activation stim- treated the cells with poly(I-C) for 30, 60, or 120 min. We immunopre- ulates Rip1 recruitment and Rip1 polyubiquitination. In TNF--treated cipitated Rip1 from the untreated and treated cells and examined the cells, ubiquitin chain conjugation is mediated by the Ubc13-Uev1a com- lysates for polyubiquitinated Rip1 protein by immunoblotting with an plex and facilitated by Traf2, the E3 ubiquitin ligase recruited to the anti-ubiquitin antibody. We observed evidence of Rip1 polyubiquitina- TNFR1 that also interacts with Rip1 (6, 19). We find TLR3 and TLR4 tion in both the poly(I-C) and TNF--treated cell lysates (Fig. 8C), sug- responses diminished in traf6/ cells and observe ligand-dependent gesting that polyubiquitinated Rip1 may contribute to IKK- activation recruitment of Rip1, Traf6, and Tak1 to TLR3 (Fig. 7). Our studies suggest that Traf6 also contributes to TLR3/4-induced antimicrobial in both the TNFR1 and TLR3 pathway. It remains unclear whether Rip1 responses. Traf6 has been shown by others to be recruited to TLR3 and is modified by K63-linked and/or K48-linked polyubiquitin chains in the bind Trif (24, 25) and has been implicated in TLR signaling with TLR3-stimulated cells; however, Rip1 degradation does not appear decreased cytokine responses observed when traf6/ macrophages induced in the poly(I-C)-treated cells, suggesting that Rip1 may be mod- are stimulated with TLR2, -4, -7, and -9 ligands (26). ified by K63-linked polyubiquitin chains. Moreover, the recruitment of Rip1 polyubiquitination is induced upon TNF- or poly(I-C) treat- Traf6 and Tak1 would support the idea that K63-linked polyubiquiti- ment of cells, suggesting that Rip1 may use similar mechanisms to acti- nated Rip1 stimulates IKK- activation in TLR3/4-stimulated cells. vate NF-B in the TNFR1- and Trif-dependent TLR pathways. Thus, These studies provide genetic and biochemical evidence that Rip1 is an polyubiquitinated Rip1 may be recognized by the Tab2 protein (7), a essential mediator of the TLR3 and TLR4 NF-B responses and suggest that the Trif-dependent NF-B pathway may be mediated by polyubiq- uitinated Rip1 and the ubiquitin-activated Tak1 complex. G. Wen and M. A. Kelliher, unpublished data. NOVEMBER 4, 2005• VOLUME 280 • NUMBER 44 JOURNAL OF BIOLOGICAL CHEMISTRY 36565 Rip1 Mediates TLR3 and TLR4 NF-B Pathways 33185–33191 component of the Tak1 complex, and thereby, the ubiquitin-activated 7. Kanayama, A., Seth, R. B., Sun, L., Ea, C. K., Hong, M., Shaito, A., Chiu, Y. H., Deng, L., kinase Tak1 may be recruited to the TLR3. Consistently, we find Traf6 and Chen, Z. J. (2004) Mol. Cell 15, 535–548 and Tak1 recruited to TLR3, and we find TLR3/4-induced cytokine 8. Meylan, E., Burns, K., Hofmann, K., Blancheteau, V., Martinon, F., Kelliher, M., and production ablated in traf6/ cells. Additionally, TLR3 and TLR4 Tschopp, J. (2004) Nat. Immunol. 5, 503–507 9. Yamamoto, M., Sato, S., Mori, K., Hoshino, K., Takeuchi, O., Takeda, K., and Akira, S. NF-B and IRF-3 responses have been shown to be negatively regulated (2002) J. Immunol. 169, 6668–6672 by the ubiquitin-modifying enzyme A20 (27–29). 10. Hirschfeld, M., Ma, Y., Weis, J. H., Vogel, S. N., and Weis, J. J. (2000) J. Immunol. 165, Parallels can also be made to Drosophila melanogaster, where the Imd 618–622 protein, the mammalian counterpart to Rip1, mediates the antimicro- 11. Fitzgerald, K. A., McWhirter, S. M., Faia, K. L., Rowe, D. C., Latz, E., Golenbock, D. T., Coyle, A. J., Liao, S. M., and Maniatis, T. (2003) Nat. Immunol. 4, 491–496 bial immune response to Gram-negative bacteria. Imd is required for 12. Balachandran, S., Thomas, E., and Barber, G. N. (2004) Nature 432, 401–405 activation of Drosophila Tak1 and acts through a pathway that includes 13. Wathelet, M. G., Lin, C. H., Parekh, B. S., Ronco, L. V., Howley, P. M., and Maniatis, the Drosophila IB kinase complex (30). Recent studies suggest that Imd T. (1998) Mol. Cell 1, 507–518 signaling requires the fly homologues of the ubiquitin-conjugating 14. Li, K., Chen, Z., Kato, N., Gale, M., Jr., and Lemon, S. M. (2005) J. Biol. Chem. 280, 16739–16747 enzymes Ubc13/Uev1A and a Drosophila homologue of the human 15. Yoneyama, M., Kikuchi, M., Natsukawa, T., Shinobu, N., Imaizumi, T., Miyagishi, M., Tab2 protein. A Drosophila Tab2 mutant that is defective in activating Taira, K., Akira, S., and Fujita, T. (2004) Nat. Immunol. 5, 730–737 the Imd pathway contains a nonsense mutation in the C-terminal zinc 16. Janssens, S., and Beyaert, R. (2003) Mol. Cell 11, 293–302 finger domain that recognizes K63-linked polyubiquitin chains. Thus, 17. Legler, D. F., Micheau, O., Doucey, M. A., Tschopp, J., and Bron, C. (2003) Immunity 18, 655–664 the antimicrobial response and mechanism of IKK activation in flies and 18. Zhang, S. Q., Kovalenko, A., Cantarella, G., and Wallach, D. (2000) Immunity 12, mammals appear highly conserved and may be dependent on the ubiq- 301–311 uitin modification of Rip1 and potentially Imd. 19. Wertz, I. E., O’Rourke, K. M., Zhou, H., Eby, M., Aravind, L., Seshagiri, S., Wu, P., Wiesmann, C., Baker, R., Boone, D. L., Ma, A., Koonin, E. V., and Dixit, V. M. (2004) Acknowledgments—We thank Annette Schoenmeyer for the real time PCR Nature 430, 694–699 20. Yamamoto, M., Sato, S., Hemmi, H., Hoshino, K., Kaisho, T., Sanjo, H., Takeuchi, O., assay for mouse IFN-, Neal Silverman for critical reading of the manuscript, Sugiyama, M., Okabe, M., Takeda, K., and Akira, S. (2003) Science 301, 640–643 and members of the University of Massachusetts Medical School Innate 21. Kawai, T., Adachi, O., Ogawa, T., Takeda, K., and Akira, S. (1999) Immunity 11, Immunity Data Club for helpful discussion. 115–122 22. Der, S. D., Yang, Y. L., Weissmann, C., and Williams, B. R. (1997) Proc. Natl. Acad. Sci. 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Genes Dev. 14, 2461–2471 36566 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 280 • NUMBER 44 •NOVEMBER 4, 2005 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry American Society for Biochemistry and Molecular Biology

Rip1 Mediates the Trif-dependent Toll-like Receptor 3- and 4-induced NF-κB Activation but Does Not Contribute to Interferon Regulatory Factor 3 Activation *

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American Society for Biochemistry and Molecular Biology
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0021-9258
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DOI
10.1074/jbc.m506831200
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Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 280, NO. 44, pp. 36560 –36566, November 4, 2005 © 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Rip1 Mediates the Trif-dependent Toll-like Receptor 3- and 4-induced NF-B Activation but Does Not Contribute to Interferon Regulatory Factor 3 Activation Received for publication, June 23, 2005, and in revised form, August 17, 2005 Published, JBC Papers in Press, August 22, 2005, DOI 10.1074/jbc.M506831200 ‡ ‡ ‡ § ‡1 Nicole Cusson-Hermance , Smriti Khurana , Thomas H. Lee , Katherine A. Fitzgerald , and Michelle A. Kelliher ‡ § From the Departments of Cancer Biology and Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605 Rip1 is required for IB kinase activation in response to tumor (TLR3)-mediated NF-B response to dsRNA (8). Rip1 interacts with the necrosis factor  (TNF-) and has been implicated in the Toll-like TLR3- and TLR4-specific adapter Trif (Toll-interleukin-1 receptor receptor 3 (TLR3) response to double-stranded RNA. Cytokine pro- domain-containing adaptor inducing IFN-). Trif is a TIR domain-con- duction is impaired when rip1/ cells are treated with TNF-, taining adapter protein that is essential for all signaling by TLR3 and poly(I-C), or lipopolysaccharide, implicating Rip1 in the Trif-de- some signaling by TLR4. Rip1 binds the C terminus of the Trif protein pendent TLR3 and TLR4 pathways. To examine the role of Rip1 in via a Rip homotypic interaction motif (8). Trif also binds Traf6 (tumor the Trif-dependent TLR4 pathway, we generated rip1/ necrosis factor receptor-associated factor-6) and TBK-1 via its N termi- MyD88/ cells. Lipopolysaccharide failed to stimulate NF-B nus, and these interactions result in interferon regulatory factor 3 activation in rip1/MyD88/ cells, revealing that Rip1 is also (IRF-3) activation. The TLR4 ligand LPS induces NF-B activation by required for the Trif-dependent TLR4-induced NF-B pathway. In engaging the MyD88-dependent and Trif-dependent pathways. Thus, addition to activating NF-B, TLR3/4 pathways also stimulate we reasoned that Rip1 may also participate in late phase NF-B activa- interferon regulatory factor 3 activation. However, we find that tion induced by TLR4 Trif-dependent pathway. Rip1 expression stimulates NF-B but not interferon regulatory We find NF-B responses and cytokine production ablated when factor 3 activity. In the TNF- pathway, Rip1 interacts with the E3 rip1/ murine embryonic fibroblasts (MEF) or splenocytes are stim- ubiquitin ligase Traf2 and is modified by polyubiquitin chains. ulated with dsRNA or TNF-. Although the NF-B responses to TLR4 Upon TLR3 activation, Rip1 is also modified by polyubiquitin appear unaffected due to activation of the MyD88-dependent pathway, chains and is recruited to TLR3 along with Traf6 and the ubiquitin- LPS-induced cytokine production was impaired in the absence of Rip1. activated kinase Tak1. These studies suggest that Rip1 uses a simi- Importantly, LPS-induced NF-B activation was completely ablated in lar, ubiquitin-dependent mechanism to activate IB kinase- in rip1/MyD88/ MEF, providing genetic evidence that Rip1 con- response to TNF- and TLR3 ligands. tributes to the Trif-dependent, TLR4-induced NF-B pathway. More- over, we find Rip1 phosphorylated and polyubiquitinated in TLR3-stim- ulated cells and demonstrate that Rip1, Traf6, and the ubiquitin- The death domain kinase Rip1 (receptor-interacting protein 1) medi- activated kinase Tak1 are recruited to TLR3 in response to poly(I-C) treatment. Taken together, these studies suggest that Rip1 may use sim- ates TNF- -induced NF-B and p38 mitogen-activated protein kinase ilar ubiquitin-dependent mechanisms to activate IKK- in TLR3-stim- activation (1–4). Although Rip1 is required for IKK activation (5), its ulated cells. kinase activity is dispensable (3, 6), suggesting that Rip1 may mediate IKK activation by recruiting other MAP kinases such as Mekk3 or by employing other mechanisms to achieve IKK activation. Upon recruit- EXPERIMENTAL PROCEDURES ment to the TNF receptor (TNFR1), Rip1 is initially modified by K63- Generation of rip1/ and rip1/MyD88/ Murine Embry- linked polyubiquitin chains. These K63-linked polyubiquitin chains are onic Fibroblasts—Rip1/ mice were interbred, females were sacri- recognized by the ubiquitin receptor Tab2 (7), resulting in the recruit- ficed between embryonic days 15.5 and 17, and MEF were prepared as ment of the ubiquitin-activated Tak1 (TGF--activating kinase 1) described in Ref. 1. For other studies, rip1/tnfr1/ mice were enzyme to the TNFR1. intercrossed, and splenocytes were isolated from day 2 rip1// Recently, Rip1 has also been implicated in the Toll-like receptor 3 tnfr1/ or rip1//tnfr1/ mice. MyD88/ mice were gifts from S. Akira (Osaka, Japan). The MyD88/ mice used for this study were backcrossed into the C57BL/6 background for 11 generations. To * This work was supported in part by National Institutes of Health Grant GM061298 (to generate rip1/MyD88/ MEF, rip1/ mice were mated with M. K.) and by the Wellcome Trust (to K. A. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be MyD88/ mice, and then rip1/MyD88/ mice were inter- hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to crossed, and females were sacrificed to generate rip1/ or rip1/ indicate this fact. Scholar of the Leukemia and Lymphoma Society of America. To whom correspondence MyD88/ or rip1/MyD88/ MEF. Traf6/ MEF were should be addressed: Dept. of Cancer Biology, University of Massachusetts Medical provided by Dr. J.-I. Inoue (Tokyo, Japan). School, Lazare Research Bldg., 364 Plantation St., Worcester, MA 01605. E-mail: Cell Lines and Reagents—HEK293 cells were stably transfected with [email protected]. The abbreviations used are: TNF-, tumor necrosis factor ; IRF3, interferon-regulatory FLAG-tagged human TLR3 as described in Ref. 9. LPS derived from factor 3; TLR, Toll-like receptor; TNFR1, tumor necrosis factor receptor 1; MEF, mouse Escherichia coli strain 0111.B4 was purchased from Sigma, dissolved in embryonic fibroblast(s); MyD88, myeloid differentiation factor 88; IFN, interferon; IKK, IB kinase; LPS, lipopolysaccharide; MAP, mitogen-activated protein; dsRNA, double- deoxycholate, and re-extracted by phenol/chloroform as described in stranded RNA; RANTES, regulated on activation normal T cell expressed and secreted; Ref. 10. Poly(I-C) was purchased from Amersham Biosciences, and ELISA, enzyme-linked immunosorbent assay; ISRE, interferon-stimulated response element. MALP2 and peptidoglycan were purchased from EMC Microcollec- 36560 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 280 • NUMBER 44 •NOVEMBER 4, 2005 This is an Open Access article under the CC BY license. Rip1 Mediates TLR3 and TLR4 NF-B Pathways FIGURE 1. Rip1 mediates TLR3-induced NF-B activation but does not appear to contribute to TLR2 or TLR4-induced NF-B activation. A,an impaired TLR3 response in the absence of Rip1. Wild type (wt) and rip1/ MEF were left untreated or stimulated with poly(I-C) (100 g/ml) or IL-1 (10 ng/ml) for the time periods indicated, and NF-B activity was measured using a phos- phospecific IB antibody. B, normal LPS-induced NF-B activation in rip1/ MEF. Wild type and rip1/ MEF were left untreated or stimulated with purified LPS (100 ng/ml) or IL-1 (10 ng/ml). Cell lysates were probed with a phosphospecific IB antibody. C, normal TLR2-induced NF-B responses in rip1/ MEF. Wild type and rip1/ MEF were left untreated or treated with the TLR2 ligand peptidoglycan (PGN at 10 g/ml) or IL-1 (10 ng/ml) for the time periods indicated. Activa- tion of NF-B was measured by probing the cell lysates with a phosphospecific IB antibody. To ensure that equal amounts of total protein was achieved, cell lysates were probed with an IKK- antibody. Three independent rip1/ MEF lines were examined, and a representative experiment is shown. tions (Tuebingen, Germany). Sendai virus was purchased from Charles Coimmunoprecipitation and Western Blotting—HEK293 cells stably River Laboratories (Wilmington, MA). Anti-Rip1 antibody was pur- transfected with FLAG-tagged human TLR3 left unstimulated or chased from BD Biosciences (catalog number 601459), and anti-phos- treated with poly(I-C) (100 g/ml) were lysed in endogenous lysis buffer pho-IB was from Cell Signaling (catalog number 9246s). Anti-IB (150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 1% Nonidet P-40, 0.5% deoxy- (catalog number sc-7183), anti-Traf6 (catalog number sc-6224), anti- cholate, 0.1% SDS) and immunoprecipitated with anti-FLAG antibody, IKK (catalog number sc-371), anti-extracellular signal-regulated and associated proteins were detected by immunoblotting with anti- kinase (catalog number sc-154), anti-Tak1 (catalog number sc-7162), Rip1, -Traf6, or -Tak1 antibodies. To measure NF-B activity, wild type and anti-ubiquitin (catalog number sc-8017) were from Santa Cruz Bio- or rip1/ MEF were left untreated or stimulated with IL-1 (10 technology, Inc. (Santa Cruz, CA). Anti--actin was from Sigma (cata- ng/ml), TNF- (10 ng/ml), or TLR ligands poly(I-C) (100 g/ml), puri- log number A5441), and antibodies were used according to the manu- fied LPS (100 ng/ml), peptidoglycan, or MALP2 or infected with Sendai facturer’s suggestions. The RANTES enzyme-linked immunosorbent virus, and total cell lysates were examined for evidence of phosphoryl- assay (ELISA) kit and recombinant TNF- and IL-1 were purchased ated IB using a phosphospecific IB antibody (Cell Signaling). To from R & D Systems. The IRF3–5D plasmid was from John Hiscott ensure that an equivalent amount of total protein was examined, lysates (Montreal, Canada). The NF-B-luciferase, the ISG54 ISRE luciferase, were probed with either an IKK- antibody (Santa Cruz) or anti-extra- and the Trif and p65 expression constructs are described in Ref. 11. cellular signal-regulated kinase antibody. To measure TNF- and LPS- Real Time PCR—RNA from wild type and rip1/ MEF was iso- induced NF-B activation in the rip1/MyD88/ MEF, IB deg- lated, and cDNA was synthesized using the Superscript First Strand radation was measured by probing equivalent amounts of total protein System (Invitrogen). Real time PCR was performed using the DNA with an anti-IB antibody (Santa Cruz Biotechnology). Polyubiquiti- Engine-Opticon 2 PCR Machine (MJ Research). The reaction included nated Rip1 was detected by stimulating the RAW264.7 macrophage or 2 SYBR Green PCR Master Mix, forward and reverse primer (10 M), U373 astrocytoma cell lines with poly(I-C) or TNF- and immunopre- and the appropriate amount of cDNA. The thermal cycling parameter cipitating the lysates with anti-Rip1 antibody, followed by immunoblot- was 95 °C for 15 min, 95 °C for 15 s, 58.5 °C for 30 s, 72 °C for 30 s for 45 ting with an anti-ubiquitin antibody. cycles followed by 72 °C for 10 min. The primers for real time PCR were RESULTS murine IFN- (5-AGC TCC AAG AAA GGA CGA ACA T-3), murine IFN- (3-GCC CTG TAG GTG AG TTG ATC T-5); murine Rip1 Mediates the Trif-dependent NF-B Pathway—Recently, the -actin (5-TTG AAC ATG GCA TTG TTA CCA A-3); and murine Rip1 protein has been implicated in the innate immune response to -actin (3-TGG CAT AGA GGT CTT TAC GGA-5). double-stranded RNA viruses (8, 12). Mouse embryonic fibroblasts that Transfection Assays—HEK 293 cells (1.5  10 cells/well) were lack Rip1 fail to activate NF-B when stimulated with poly(I-C) (8); seeded into 96-well plates and transfected on the following day with 40 however, the response to other Toll-like ligands has not been examined. ng of luciferase reporter genes (NF-B, ISRE, or Viperin promoter) To test whether Rip1, like Rip2, mediates multiple Toll-like receptor using Genejuice (Novagen). The Renilla luciferase reporter gene (Pro- pathways, we treated wild type and rip1/ MEF with IL-1, poly(I-C), mega) was cotransfected for normalization. In all cases, cell lysates were purified LPS, and the TLR2 ligand peptidoglycan. To measure NF-B prepared, and reporter gene activity was measured using the Dual Lucif- activity, we assayed cell lysates for phosphorylated IB by immuno- erase Assay System (Promega). Data are expressed as the mean relative blotting. As expected, we found poly(I-C)-induced NF-B activation stimulation  S.D. for a representative experiment from three separate impaired in rip1/ MEF (8) (Fig. 1A) yet observed normal NF-B experiments, each performed in triplicate. responses when rip1/ cells were stimulated with TLR2 or TLR4 NOVEMBER 4, 2005• VOLUME 280 • NUMBER 44 JOURNAL OF BIOLOGICAL CHEMISTRY 36561 Rip1 Mediates TLR3 and TLR4 NF-B Pathways FIGURE 2. Decreased IFN- expression and cytokine production in poly(I-C)-stimu- lated rip1/ MEF. A, IFN- expression is reduced in poly(I-C)-stimulated rip1/ cells. Wild type (wt) and rip1/ MEF were left untreated or treated with poly(I-C) (100 g/ml) for the time periods indicated, and real time PCR analysis was performed using primers specific for IFN- and -actin. B, poly(I-C) or TNF- treatment of rip1/ cells results in impaired cytokine production. Wild type and rip1/ MEF were left untreated or stim- ulated with poly(I-C) (100, 10, or 1 g/ml), Sendai virus (200, 20, or 2 HAU), or murine TNF- (100, 10, or 1 ng/ml). Twenty-four hours later, culture supernatants were exam- ined for RANTES production by ELISA. Three independent rip1/ MEF lines were exam- ined in triplicate, and one representative experiment is shown. FIGURE 3. Poly(I-C) and LPS-induced cytokine production is severely impaired in ligands (Fig. 1, B and C). Thus, the NF-B responses to Toll-like recep- Rip1-deficient cells. A, Rip1-deficient MEF are impaired in cytokine response to poly(I-C) and LPS. Wild type and rip1/ MEF were left unstimulated or stimulated with poly(I-C) tor 2 and 4 ligands appear unaffected by a Rip1 deficiency (Fig. 1, B and (100 or 10 mg/ml), LPS (100 or 10 ng/ml), or mTNF- (10 ng/ml). Culture supernatants C), indicating that, unlike Rip2, Rip1 may only mediate the Trif-depend- were examined for Rantes production by ELISA. B, poly(I-C)- and LPS-induced cytokine production is impaired in rip1/tnfr1/ splenocytes. rip1/tnfr1/ or ent pathways. rip1/tnfr1/ splenocytes were left untreated or stimulated with poly(I-C) (100 or The lack of TLR3 responsiveness in rip1/ MEF could reflect 10 g/ml) or purified LPS (100 or 10 ng/ml). Twenty-four hours later, culture superna- decreased TLR3 expression in Rip1-deficient cells. Thus, we examined tants were examined for RANTES production by ELISA. A minimum of three rip1/ MEF and splenocytes from three rip1/tnfr1/ and two rip1/tnfr1/ mice were wild type and rip1/ MEF for expression of TLR3 and TLR4 using examined in triplicate. A representative experiment for each is shown. wt, wild type. reverse transcription-PCR. We found similar TLR3 and TLR4 expres- sion levels in wild type and rip1/ cells (not shown), suggesting that points, induction of IFN- expression was observed in poly(I-C)-stim- the TLR3 signaling defect is not due to decreased TLR3 receptor levels ulated, wild type MEF but not in rip1/ MEF stimulated with in Rip1-deficient cells. poly(I-C) (Fig. 2A). In contrast, late phase (24-h) IFN- expression Decreased IFN- and RANTES Production in Poly(I-C)-stimulated appears less affected, although expression remained significantly rip1/ Cells—To foster antiviral responses, TLR3 and TLR4 activate reduced in poly(I-C)-treated rip1/ MEF (Fig. 2A). Similarly, the transcription factors NF-B and IRF-3 and induce IFN- expres- RANTES production was reduced when rip1/ cells were stimulated sion. The transcriptional enhancer of the IFN- promoter contains four with either poly(I-C) or TNF- (Fig. 2B). However, cytokine production positive regulatory domains, which function cooperatively to induce was unaffected when rip1/ MEF were infected with Sendai virus, a IFN- expression in response to viral infection. The transcription fac- single-stranded RNA virus, that induces IFN- expression via a Trif- tors that regulate IFN- expression include NF-B, IRF-3, and the ATF- and TLR3-independent pathway (14). These data suggest that Rip1 con- 2-c-Jun heterodimer (9, 13). Thus, the initial induction of IFN- expres- tributes to anti-viral responses mediated by TLR3 but is not required for sion requires the activation of NF-B, yet in our previous published NF-B activation by the intracellular retinoic acid-inducible gene I studies, we observed induction of IFN- in poly(I-C)-treated rip1/ pathway (15). cells (8). Since these studies were performed using reverse transcrip- Decreased RANTES Production When rip1/ Splenocytes Are tion-PCR (13), we reexamined IFN- expression in poly(I-C)-stimu- Stimulated with Purified LPS—Although Rip1 has been shown to inter- lated rip1/ MEF using quantitative real time PCR. At early time act with the TLR3 and TLR4 adapter Trif, cytokine responses to 36562 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 280 • NUMBER 44 •NOVEMBER 4, 2005 Rip1 Mediates TLR3 and TLR4 NF-B Pathways FIGURE 4. Rip1 mediates the Trif-dependent TLR4-induced NF-B pathway. Wild (wt) type MEF (A), rip1/MyD88/ (B) and double knockout rip1/MyD88/ MEF (C) were left untreated or stimulated with LPS (10 g/ml) or TNF- (10 ng/ml) for the time periods indicated, and NF-B activity was measured by immunoblotting with an anti- IB antibody. To ensure that equal amounts of total protein were loaded, cell lysates were probed with an anti-extracellular signal-regulated kinase (-ERK) antibody. One wild type, one rip1/MyD88/, and three rip1/MyD88/ MEF were examined. One representative experiment is shown. poly(I-C) or purified LPS were not examined in our previous study (8). Thus, Rip1 may contribute to NF-B activation when MEF are treated with poly(I-C) but may not contribute significantly to cytokine produc- tion in vivo, particularly when the TLR4 pathway is stimulated, since NF-B can be activated by the MyD88-dependent pathway. Due to the lethality associated with a Rip1 deficiency (1), we were unable to exam- ine TLR3/4 responses in Rip1-deficient macrophages or dendritic cells. To evaluate the contribution of Rip1 to TLR3- and TLR4-induced innate immune responses, we stimulated neonatal day 2 splenocytes from rip1/tnfr1/ mice and control littermates with poly(I-C), purified LPS, or TNF-. We found RANTES production significantly FIGURE 5. Rip1 expression stimulates NF-B but not IRF-3 activation. HEK 293 cells were transfected with an NF-B reporter plasmid (A) or with IRF3 reporter plasmids (B)or reduced when neonatal rip1/tnfr1/ splenocytes are stimulated with a Viperin-dependent reporter construct (C), together with expression constructs with poly(I-C) and LPS (Fig. 3). These findings suggest that Rip1 con- encoding p65, Rip1, Trif, or IRF-3. After 24 h, luciferase gene activity was measured. The luciferase assays were done in triplicate and repeated three times. tributes to both the Trif-dependent TLR3 and TLR4 pathways and dem- onstrate that activation of both the Trif-Rip1 and MyD88-dependent TLR4 pathways is required for robust cytokine production. double knock out cells failed to respond to TNF-, LPS, and poly(I-C) Rip1 Mediates the Trif-dependent TLR4 Pathway—The decreased but remained capable of activating NF-B in response to treatment with cytokine responses to the TLR4 ligand LPS observed in rip1/ MEF phorbol 12-myristate 13-acetate/ionomycin (not shown). These studies and splenocytes suggested that Rip1 may also mediate the Trif-depend- provide genetic evidence that Rip1 contributes to the Trif-dependent ent, TLR4-induced NF-B pathway. To test this possibility, we mated TLR4-induced NF-B pathway. our rip1/ mice with MyD88/ mice (generously provided by Rip1 Expression Stimulates NF-B but Not IRF-3 Activation—The S. Akira) and then stimulated wild type, rip1/MyD88/,or presence of a Rip homotypic interaction domain in Trif and Rip1 sug- rip1/MyD88/ and rip1/MyD88/ MEF with TNF- or gests that Rip1 and Trif may interact to mediate both NF-B and IRF-3 LPS and examined cytokine-induced IB degradation. TNF--in- activation. Deletion of the Rip homotypic interaction motif in Trif duced IB degradation was observed in wild type cells and in rip1/ ablates Trif-mediated NF-B activation but has no effect on IRF-3 acti- MyD88/ 10 min following cytokine treatment (Fig. 4, A and B). As vation (8). To test whether Rip1 can stimulate both NF-B and IRF-3 expected, no TNF--induced IB degradation is observed in the dou- activation, we transfected HEK293 cells with a NF-B or IRF reporter ble knockout rip1/MyD88/ cells due to the absence of Rip1 (Fig. constructs (ISG54 and ISRE) with expression plasmids containing p65 4C). Wild type MEF exhibit IB degradation 15 min following LPS (RelA), Rip1, or Trif or with a constitutively active IRF-3 expression treatment, whereas IB degradation is delayed in rip1/ construct, the phosphomimetic IRF3-5D. Twenty-four hours later, MyD88/ cells with evidence of IB degradation detected luciferase reporter gene activity was measured. RelA, Rip1, and Trif between 45 min and 1 h poststimulation (Fig. 4, A and B). In contrast, no expression all stimulated NF-B reporter activity (Fig. 5A), yet only Trif LPS-induced IB degradation was observed in cells deficient for both and IRF3-5D were capable of stimulating IRF-3-dependent reporter Rip1 and MyD88 even at 120 min following LPS treatment (Fig. 4C). The activity (ISRE reporter (Fig. 5B) and Viperin reporter (Fig. 5C)). This NOVEMBER 4, 2005• VOLUME 280 • NUMBER 44 JOURNAL OF BIOLOGICAL CHEMISTRY 36563 Rip1 Mediates TLR3 and TLR4 NF-B Pathways FIGURE 7. Poly(I-C) treatment stimulates the recruitment of Rip1, Traf6, and Tak1 to the TLR3. HEK293 cells expressing a FLAG-tagged version of human TLR3 were left untreated or stimulated with 100 g/ml poly(I-C) for the time periods indicated. Cell lysates were immunoprecipitated (IP) with anti-FLAG antibody, and TLR3-associated proteins were isolated. The presence of Rip1, Traf6, and Tak1 was detected by immuno- blotting cell lysates with an anti-Rip1, Traf6, or Tak1 antibody. Total cell lysates were then probed with an anti-Rip1 antibody to assure equal loading. The coimmunoprecipitation experiments were repeated twice. Rip1, Traf6, and Tak1 Are Recruited to TLR3 upon Poly(I-C) Stimulation—Our data suggest that Rip1 and Traf6 proteins contribute to the Trif-dependent NF-B pathway. To test whether the endogenous proteins are recruited to TLR3 upon receptor activation, we stimulated HEK 293 that express a FLAG-tagged TLR3 with poly(I-C) for 30, 60, and 120 min. We immunoprecipitated TLR3 using the anti-FLAG anti- FIGURE 6. Poly(I-C) and LPS-induced cytokine production is impaired in traf6/ body and tested whether Rip1 or Traf6 associated with TLR3. We MEF. A, poly(I-C) or LPS treatment does not stimulate NF-B activation in traf6/ MEF. Wild type (wt) and traf6/ MEF were left untreated or stimulated with poly(I-C) (100 detected Rip1 and Traf6 recruitment to TLR3 30 min following g/ml), LPS (100 ng/ml), or TNF- (10 ng/ml) for the indicated time periods. Cell lysates poly(I-C) treatment (Fig. 7, A and B). We also examined the TLR3- were separated on a 12% SDS-polyacrylamide gel, and NF-B activity was assessed by immunoblotting with an anti-phospho-IB antibody. B, poly(I-C) or LPS treatment fails associated proteins for evidence of Tak1 recruitment. Tak1 recruitment to stimulate cytokine production in traf6/ MEF. Wild type and traf6/ MEF were left to TLR3 was observed at 60 min when maximal phosphorylated IB untreated or stimulated with various concentrations of poly(I-C) (100, 10, or 1 g/ml), protein is also detected (Fig. 7C). Interestingly, Rip1 and Traf6 recruit- LPS (100, 10, or 1 ng/ml), 10 nM MALP2, or 10 ng/ml murine TNF-. Twenty-four hours later, culture supernatants were examined in triplicate for RANTES production. Each ment appears to precede Tak1 recruitment, suggesting that Traf6 may experiment was repeated a minimum of three times. modify Rip1 and thereby signal Tak1 recruitment. experiment suggests that Rip1 may not participate in the Trif-depend- Rip1 Is Phosphorylated and Polyubiquitinated in TLR3-stimulated ent IRF-3 pathway and is recruited by Trif to mediate IKK- activation. Cells—We have shown that the kinase activity of Rip1 is responsive to LPS- and Poly(I-C)-induced Cytokine Production Is Diminished in TNFR1 activation (6), suggesting that the kinase activity of Rip1 is traf6/ MEF—The MyD88-dependent NF-B pathway is mediated responsive to specific ligands or stresses. Studies in cells expressing by the recruitment of IRAK kinases and Traf6 and the Tak1-Tab1-Tab2 kinase-inactive Rip1 have revealed that Rip1 autophosphorylation is complex. Thus, we reasoned that Traf6 may also contribute to the Trif- induced in TNF--stimulated cells (6) (Fig. 8, A and B). Similarly, the dependent NF-B pathway. To test this possibility, we stimulated wild related Rip2 kinase and IRAK kinases are responsive to TLR and IL-1 type and traf6/ MEF with TLR ligand poly(I-C) or LPS or with receptor activation (16). To provide additional evidence that Rip1 TNF- and examined IKK activity using a phospho-IB antibody. In responds to TLR3/4 activation, we stimulated wild type cells with contrast to wild type MEF, traf6/ MEF responded only to TNF- TNF- or with the TLR ligand LPS or poly(I-C) and examined cell and failed to phosphorylate IB when stimulated with TLR3 or TLR4 lysates for phosphorylated Rip1. As expected, phosphorylated Rip1 was ligands (Fig. 6A). Culture supernatants were also harvested 24 h post- detected as early as 5 min following TNF- treatment. We find Rip1 stimulation, and RANTES production was measured by ELISA (Fig. 6B). autophosphorylation also rapidly induced upon poly(I-C) treatment or NF-B activation and cytokine production in response to TLR2, -3, and LPS treatment (Fig. 8, A and B). These studies reveal that the Rip1 kinase -4 ligands was ablated in traf6/ MEF, whereas TNF-induced IKK activity responds to TNFR1, TLR3, and TLR4 activation and further activation and cytokine production remained unaffected by a Traf6 defi- implicates Rip1 in a TLR3 and TLR4 Trif-dependent pathway. ciency (Fig. 6, A and B). Taken together, these studies suggest that Traf6 In addition to phosphorylation, TNF- stimulates the conjugation of and Rip1 contribute to the Trif-dependent TLR3 and TLR4 NF-B K63-linked polyubiquitin chains on Rip1 (6, 17–19). K63-linked poly- pathways. ubiquitin chains on Rip1, Traf6, or IKK- (NEMO) are recognized by 36564 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 280 • NUMBER 44 •NOVEMBER 4, 2005 Rip1 Mediates TLR3 and TLR4 NF-B Pathways DISCUSSION The TLR3 and TLR4 pathways employ the adapter protein Trif that signals both NF-B and IRF-3 activation. Trif recruits Traf6 and TBK-1 via its N terminus, and TBK-1 is responsible for IRF-3 phosphorylation and activation (11). However, it remains unclear how Trif activates the IKK- and JNK2 pathways. Data base screening for proteins containing regions homologous to Trif revealed that Trif-induced NF-B activa- tion may be mediated by Rip1 (8). Studies in Rip1-deficient MEF then confirmed defects in poly(I-C)-induced NF-B activation, implicating Rip1, a mediator of TNF--induced IKK activation, in the Trif-depend- ent NF-B pathway (8). In this study, we address the biologic contribution of Rip1 to innate immune responses by examining poly(I-C)- and LPS-induced cytokine production in rip1/ MEF and splenocytes. Consistent with the pub- lished studies on Trif-deficient and MyD88-deficient cells (20, 21), we find poly(I-C)- and LPS-induced cytokine production impaired in Rip1- deficient MEF and splenocytes. Taken together, these studies support the model that TLR4-induced cytokine responses to bacterial patho- gens require activation of both the Trif-dependent and MyD88-depend- ent pathways. The involvement of Rip1 in the Trif-dependent TLR3 NF-B path- way suggested that Rip1 may also contribute to the Trif-dependent TLR4-induced NF-B activation. Consistent with this idea, we find that LPS, like TNF- and poly(I-C), stimulates the kinase activity of Rip1, and we find LPS-induced cytokine levels diminished in rip1/ cells. To test the contribution of Rip1 to the Trif-dependent TLR4 NF-B FIGURE 8. Treatment with TLR3 or TLR4 ligands stimulates Rip1 phosphorylation pathway, we generated rip1/MyD88/ cells and stimulated them and polyubiquitination. A, the kinase activity of Rip1 is induced when cells are treated with either TNF- or LPS. As expected, NF-B activation is delayed in with TNF- or poly(I-C). Wild type MEF were left untreated or stimulated with 100 g/ml poly(I-C) or 10 ng/ml murine TNF- for the time periods indicated. Cell lysates were LPS-treated rip1/MyD88/ cells; however, in the absence of both separated on an 8% gel, and Rip1 protein was detected by immunoblotting with an MyD88 and Rip1, LPS-induced NF-B activation is not observed. Taken anti-Rip1 antibody. B, the TLR4 ligand LPS stimulates Rip1 phosphorylation. Wild type together, these studies demonstrate that Rip1 is responsive to TLR4 MEF were left untreated or stimulated with 100 ng/ml LPS or 10 ng/ml murine TNF- for the time periods indicated. Cell lysates were separated on an 8% gel and examined by activation and demonstrates that Rip1 mediates both the Trif-depend- immunoblotting with an anti-Rip1 antibody. C, TNF- and poly(I-C) stimulation induces ent TLR3 and TLR4 NF-B pathways. Interestingly, the dsRNA-acti- Rip1 polyubiquitination. RAW264.7 macrophages or U373 astrocytoma cells were left untreated or stimulated with 200 g/ml poly(I-C) or 20 ng/ml mouse or human TNF-. vated protein kinase PKR has also been implicated in poly(I-C)-induced Cell lysates were immunoprecipitated with anti-Rip1 antibody and separated on a 3– 8% NF-B activation and in apoptotic responses to poly(I-C), TNF-, and gradient gel. Polyubiquitinated Rip1 was detected by immunoblotting with an anti- ubiquitin antibody. LPS (22, 23), raising the possibility that dsRNA-induced NF-B activa- tion may involve PKR and Rip1. Consistent with this model, Li and co-workers (24) find PKR recruited to Trif upon poly(I-C) stimulation. the ubiquitin receptor protein Tab2, a component of the ubiquitin- However, unlike PKR-deficient cells that are resistant to poly(I-C)- and activated Tak1 complex (7). Thus, Rip1 may use similar mechanisms to TNF--induced apoptosis, Rip1-deficient cells are sensitive to TNF-- mediate IKK- activation in TNF-- and TLR3- or TLR4-stimulated and poly(I-C)-induced cell death, suggesting that Rip1 and PKR have cells. To test this possibility, we stimulated the macrophage cell line opposing functions in the TNF-- and dsRNA-activated pathways. RAW264.7 and the U373 astrocytoma line with TNF- for 10 min or We demonstrate that, like TNFR1 activation, TLR3 activation stim- treated the cells with poly(I-C) for 30, 60, or 120 min. We immunopre- ulates Rip1 recruitment and Rip1 polyubiquitination. In TNF--treated cipitated Rip1 from the untreated and treated cells and examined the cells, ubiquitin chain conjugation is mediated by the Ubc13-Uev1a com- lysates for polyubiquitinated Rip1 protein by immunoblotting with an plex and facilitated by Traf2, the E3 ubiquitin ligase recruited to the anti-ubiquitin antibody. We observed evidence of Rip1 polyubiquitina- TNFR1 that also interacts with Rip1 (6, 19). We find TLR3 and TLR4 tion in both the poly(I-C) and TNF--treated cell lysates (Fig. 8C), sug- responses diminished in traf6/ cells and observe ligand-dependent gesting that polyubiquitinated Rip1 may contribute to IKK- activation recruitment of Rip1, Traf6, and Tak1 to TLR3 (Fig. 7). Our studies suggest that Traf6 also contributes to TLR3/4-induced antimicrobial in both the TNFR1 and TLR3 pathway. It remains unclear whether Rip1 responses. Traf6 has been shown by others to be recruited to TLR3 and is modified by K63-linked and/or K48-linked polyubiquitin chains in the bind Trif (24, 25) and has been implicated in TLR signaling with TLR3-stimulated cells; however, Rip1 degradation does not appear decreased cytokine responses observed when traf6/ macrophages induced in the poly(I-C)-treated cells, suggesting that Rip1 may be mod- are stimulated with TLR2, -4, -7, and -9 ligands (26). ified by K63-linked polyubiquitin chains. Moreover, the recruitment of Rip1 polyubiquitination is induced upon TNF- or poly(I-C) treat- Traf6 and Tak1 would support the idea that K63-linked polyubiquiti- ment of cells, suggesting that Rip1 may use similar mechanisms to acti- nated Rip1 stimulates IKK- activation in TLR3/4-stimulated cells. vate NF-B in the TNFR1- and Trif-dependent TLR pathways. Thus, These studies provide genetic and biochemical evidence that Rip1 is an polyubiquitinated Rip1 may be recognized by the Tab2 protein (7), a essential mediator of the TLR3 and TLR4 NF-B responses and suggest that the Trif-dependent NF-B pathway may be mediated by polyubiq- uitinated Rip1 and the ubiquitin-activated Tak1 complex. G. Wen and M. A. Kelliher, unpublished data. NOVEMBER 4, 2005• VOLUME 280 • NUMBER 44 JOURNAL OF BIOLOGICAL CHEMISTRY 36565 Rip1 Mediates TLR3 and TLR4 NF-B Pathways 33185–33191 component of the Tak1 complex, and thereby, the ubiquitin-activated 7. Kanayama, A., Seth, R. B., Sun, L., Ea, C. K., Hong, M., Shaito, A., Chiu, Y. H., Deng, L., kinase Tak1 may be recruited to the TLR3. Consistently, we find Traf6 and Chen, Z. J. (2004) Mol. Cell 15, 535–548 and Tak1 recruited to TLR3, and we find TLR3/4-induced cytokine 8. Meylan, E., Burns, K., Hofmann, K., Blancheteau, V., Martinon, F., Kelliher, M., and production ablated in traf6/ cells. 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Journal

Journal of Biological Chemistry – American Society for Biochemistry and Molecular Biology

Published: Nov 4, 2005

References