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
Rip1 is required for I!B kinase activation in response to tumor
necrosis factor " (TNF-") and has been implicated in the Toll-like
receptor 3 (TLR3) response to double-stranded RNA. Cytokine production is impaired when rip1#/# cells are treated with TNF-",
poly(I-C), or lipopolysaccharide, implicating Rip1 in the Trif-dependent TLR3 and TLR4 pathways. To examine the role of Rip1 in
the Trif-dependent TLR4 pathway, we generated rip1#/#
MyD88#/# cells. Lipopolysaccharide failed to stimulate NF-!B
activation in rip1#/#MyD88#/# cells, revealing that Rip1 is also
required for the Trif-dependent TLR4-induced NF-!B pathway. In
addition to activating NF-!B, TLR3/4 pathways also stimulate
interferon regulatory factor 3 activation. However, we find that
Rip1 expression stimulates NF-!B but not interferon regulatory
factor 3 activity. In the TNF-" pathway, Rip1 interacts with the E3
ubiquitin ligase Traf2 and is modified by polyubiquitin chains.
Upon TLR3 activation, Rip1 is also modified by polyubiquitin
chains and is recruited to TLR3 along with Traf6 and the ubiquitinactivated kinase Tak1. These studies suggest that Rip1 uses a similar, ubiquitin-dependent mechanism to activate I!B kinase-$ in
response to TNF-" and TLR3 ligands.
The death domain kinase Rip1 (receptor-interacting protein 1) mediates TNF-!2-induced NF-"B and p38 mitogen-activated protein kinase
activation (1– 4). Although Rip1 is required for IKK activation (5), its
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 recruitment to the TNF receptor (TNFR1), Rip1 is initially modified by K63linked polyubiquitin chains. These K63-linked polyubiquitin chains are
recognized by the ubiquitin receptor Tab2 (7), resulting in the recruitment of the ubiquitin-activated Tak1 (TGF-#-activating kinase 1)
enzyme to the TNFR1.
Recently, Rip1 has also been implicated in the Toll-like receptor 3
* This work was supported in part by National Institutes of Health Grant GM061298 (to
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
hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
1
Scholar of the Leukemia and Lymphoma Society of America. To whom correspondence
should be addressed: Dept. of Cancer Biology, University of Massachusetts Medical
School, Lazare Research Bldg., 364 Plantation St., Worcester, MA 01605. E-mail:
[email protected].
2
The abbreviations used are: TNF-!, tumor necrosis factor !; IRF3, interferon-regulatory
factor 3; TLR, Toll-like receptor; TNFR1, tumor necrosis factor receptor 1; MEF, mouse
embryonic fibroblast(s); MyD88, myeloid differentiation factor 88; IFN, interferon; IKK,
I"B kinase; LPS, lipopolysaccharide; MAP, mitogen-activated protein; dsRNA, doublestranded RNA; RANTES, regulated on activation normal T cell expressed and secreted;
ELISA, enzyme-linked immunosorbent assay; ISRE, interferon-stimulated response
element.
36560 JOURNAL OF BIOLOGICAL CHEMISTRY
(TLR3)-mediated NF-"B response to dsRNA (8). Rip1 interacts with the
TLR3- and TLR4-specific adapter Trif (Toll-interleukin-1 receptor
domain-containing adaptor inducing IFN-#). Trif is a TIR domain-containing adapter protein that is essential for all signaling by TLR3 and
some signaling by TLR4. Rip1 binds the C terminus of the Trif protein
via a Rip homotypic interaction motif (8). Trif also binds Traf6 (tumor
necrosis factor receptor-associated factor-6) and TBK-1 via its N terminus, and these interactions result in interferon regulatory factor 3
(IRF-3) activation. The TLR4 ligand LPS induces NF-"B activation by
engaging the MyD88-dependent and Trif-dependent pathways. Thus,
we reasoned that Rip1 may also participate in late phase NF-"B activation induced by TLR4 Trif-dependent pathway.
We find NF-"B responses and cytokine production ablated when
rip1!/! murine embryonic fibroblasts (MEF) or splenocytes are stimulated with dsRNA or TNF-!. Although the NF-"B responses to TLR4
appear unaffected due to activation of the MyD88-dependent pathway,
LPS-induced cytokine production was impaired in the absence of Rip1.
Importantly, LPS-induced NF-"B activation was completely ablated in
rip1!/!MyD88!/! MEF, providing genetic evidence that Rip1 contributes to the Trif-dependent, TLR4-induced NF-"B pathway. Moreover, we find Rip1 phosphorylated and polyubiquitinated in TLR3-stimulated cells and demonstrate that Rip1, Traf6, and the ubiquitinactivated kinase Tak1 are recruited to TLR3 in response to poly(I-C)
treatment. Taken together, these studies suggest that Rip1 may use similar ubiquitin-dependent mechanisms to activate IKK-# in TLR3-stimulated cells.
EXPERIMENTAL PROCEDURES
Generation of rip1!/! and rip1!/!MyD88!/! Murine Embryonic Fibroblasts—Rip1"/! mice were interbred, females were sacrificed between embryonic days 15.5 and 17, and MEF were prepared as
described in Ref. 1. For other studies, rip1"/!tnfr1!/! mice were
intercrossed, and splenocytes were isolated from day 2 rip1"/"/
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
generate rip1!/!MyD88!/! MEF, rip1"/! mice were mated with
MyD88!/! mice, and then rip1"/!MyD88"/! mice were intercrossed, and females were sacrificed to generate rip1"/" or rip1"/
!MyD88!/! or rip1!/!MyD88!/! MEF. Traf6!/! MEF were
provided by Dr. J.-I. Inoue (Tokyo, Japan).
Cell Lines and Reagents—HEK293 cells were stably transfected with
FLAG-tagged human TLR3 as described in Ref. 9. LPS derived from
Escherichia coli strain 0111.B4 was purchased from Sigma, dissolved in
deoxycholate, and re-extracted by phenol/chloroform as described in
Ref. 10. Poly(I-C) was purchased from Amersham Biosciences, and
MALP2 and peptidoglycan were purchased from EMC Microcollec-
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Nicole Cusson-Hermance‡, Smriti Khurana‡, Thomas H. Lee‡, Katherine A. Fitzgerald§, and Michelle A. Kelliher‡1
From the Departments of ‡Cancer Biology and §Medicine, University of Massachusetts Medical School,
Worcester, Massachusetts 01605
Rip1 Mediates TLR3 and TLR4 NF-!B Pathways
tions (Tuebingen, Germany). Sendai virus was purchased from Charles
River Laboratories (Wilmington, MA). Anti-Rip1 antibody was purchased from BD Biosciences (catalog number 601459), and anti-phospho-I"B! was from Cell Signaling (catalog number 9246s). Anti-I"B!
(catalog number sc-7183), anti-Traf6 (catalog number sc-6224), antiIKK! (catalog number sc-371), anti-extracellular signal-regulated
kinase (catalog number sc-154), anti-Tak1 (catalog number sc-7162),
and anti-ubiquitin (catalog number sc-8017) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-#-actin was from Sigma (catalog number A5441), and antibodies were used according to the manufacturer’s suggestions. The RANTES enzyme-linked immunosorbent
assay (ELISA) kit and recombinant TNF-! and IL-1# were purchased
from R & D Systems. The IRF3–5D plasmid was from John Hiscott
(Montreal, Canada). The NF-"B-luciferase, the ISG54 ISRE luciferase,
and the Trif and p65 expression constructs are described in Ref. 11.
Real Time PCR—RNA from wild type and rip1!/! MEF was isolated, and cDNA was synthesized using the Superscript First Strand
System (Invitrogen). Real time PCR was performed using the DNA
Engine-Opticon 2 PCR Machine (MJ Research). The reaction included
2# SYBR Green PCR Master Mix, forward and reverse primer (10 $M),
and the appropriate amount of cDNA. The thermal cycling parameter
was 95 °C for 15 min, 95 °C for 15 s, 58.5 °C for 30 s, 72 °C for 30 s for 45
cycles followed by 72 °C for 10 min. The primers for real time PCR were
murine IFN-# (5$-AGC TCC AAG AAA GGA CGA ACA T-3$),
murine IFN-# (3$-GCC CTG TAG GTG AG TTG ATC T-5$); murine
#-actin (5$-TTG AAC ATG GCA TTG TTA CCA A-3$); and murine
#-actin (3$-TGG CAT AGA GGT CTT TAC GGA-5$).
Transfection Assays—HEK 293 cells (1.5 # 104 cells/well) were
seeded into 96-well plates and transfected on the following day with 40
ng of luciferase reporter genes (NF-"B, ISRE, or Viperin promoter)
using Genejuice (Novagen). The Renilla luciferase reporter gene (Promega) was cotransfected for normalization. In all cases, cell lysates were
prepared, and reporter gene activity was measured using the Dual Luciferase Assay System (Promega). Data are expressed as the mean relative
stimulation % S.D. for a representative experiment from three separate
experiments, each performed in triplicate.
NOVEMBER 4, 2005 • VOLUME 280 • NUMBER 44
Coimmunoprecipitation and Western Blotting—HEK293 cells stably
transfected with FLAG-tagged human TLR3 left unstimulated or
treated with poly(I-C) (100 $g/ml) were lysed in endogenous lysis buffer
(150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS) and immunoprecipitated with anti-FLAG antibody,
and associated proteins were detected by immunoblotting with antiRip1, -Traf6, or -Tak1 antibodies. To measure NF-"B activity, wild type
or rip1!/! MEF were left untreated or stimulated with IL-1! (10
ng/ml), TNF-! (10 ng/ml), or TLR ligands poly(I-C) (100 $g/ml), purified LPS (100 ng/ml), peptidoglycan, or MALP2 or infected with Sendai
virus, and total cell lysates were examined for evidence of phosphorylated I"B! using a phosphospecific I"B! antibody (Cell Signaling). To
ensure that an equivalent amount of total protein was examined, lysates
were probed with either an IKK-! antibody (Santa Cruz) or anti-extracellular signal-regulated kinase antibody. To measure TNF- and LPSinduced NF-"B activation in the rip1!/!MyD88!/! MEF, I"B! degradation was measured by probing equivalent amounts of total protein
with an anti-I"B! antibody (Santa Cruz Biotechnology). Polyubiquitinated Rip1 was detected by stimulating the RAW264.7 macrophage or
U373 astrocytoma cell lines with poly(I-C) or TNF-! and immunoprecipitating the lysates with anti-Rip1 antibody, followed by immunoblotting with an anti-ubiquitin antibody.
RESULTS
Rip1 Mediates the Trif-dependent NF-"B Pathway—Recently, the
Rip1 protein has been implicated in the innate immune response to
double-stranded RNA viruses (8, 12). Mouse embryonic fibroblasts that
lack Rip1 fail to activate NF-"B when stimulated with poly(I-C) (8);
however, the response to other Toll-like ligands has not been examined.
To test whether Rip1, like Rip2, mediates multiple Toll-like receptor
pathways, we treated wild type and rip1!/! MEF with IL-1!, poly(I-C),
purified LPS, and the TLR2 ligand peptidoglycan. To measure NF-"B
activity, we assayed cell lysates for phosphorylated I"B! by immunoblotting. As expected, we found poly(I-C)-induced NF-"B activation
impaired in rip1!/! MEF (8) (Fig. 1A) yet observed normal NF-"B
responses when rip1!/! cells were stimulated with TLR2 or TLR4
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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 phosphospecific I"B! 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
I"B! 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. Activation of NF-"B was measured by probing the cell
lysates with a phosphospecific I"B! 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.
Rip1 Mediates TLR3 and TLR4 NF-!B Pathways
ligands (Fig. 1, B and C). Thus, the NF-"B responses to Toll-like receptor 2 and 4 ligands appear unaffected by a Rip1 deficiency (Fig. 1, B and
C), indicating that, unlike Rip2, Rip1 may only mediate the Trif-dependent pathways.
The lack of TLR3 responsiveness in rip1!/! MEF could reflect
decreased TLR3 expression in Rip1-deficient cells. Thus, we examined
wild type and rip1!/! MEF for expression of TLR3 and TLR4 using
reverse transcription-PCR. We found similar TLR3 and TLR4 expression levels in wild type and rip1!/! cells (not shown), suggesting that
the TLR3 signaling defect is not due to decreased TLR3 receptor levels
in Rip1-deficient cells.
Decreased IFN-# and RANTES Production in Poly(I-C)-stimulated
rip1!/! Cells—To foster antiviral responses, TLR3 and TLR4 activate
the transcription factors NF-"B and IRF-3 and induce IFN-# expression. The transcriptional enhancer of the IFN-# promoter contains four
positive regulatory domains, which function cooperatively to induce
IFN-# expression in response to viral infection. The transcription factors that regulate IFN-# expression include NF-"B, IRF-3, and the ATF2-c-Jun heterodimer (9, 13). Thus, the initial induction of IFN-# expression requires the activation of NF-"B, yet in our previous published
studies, we observed induction of IFN-# in poly(I-C)-treated rip1!/!
cells (8). Since these studies were performed using reverse transcription-PCR (13), we reexamined IFN-# expression in poly(I-C)-stimulated rip1!/! MEF using quantitative real time PCR. At early time
36562 JOURNAL OF BIOLOGICAL CHEMISTRY
FIGURE 3. Poly(I-C) and LPS-induced cytokine production is severely impaired in
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)
(100 or 10 mg/ml), LPS (100 or 10 ng/ml), or mTNF-! (10 ng/ml). Culture supernatants
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
rip1!/!tnfr1!/! splenocytes were left untreated or stimulated with poly(I-C) (100 or
10 $g/ml) or purified LPS (100 or 10 ng/ml). Twenty-four hours later, culture supernatants were examined for RANTES production by ELISA. A minimum of three rip1!/! MEF
and splenocytes from three rip1"/"tnfr1!/! and two rip1!/!tnfr1!/! mice were
examined in triplicate. A representative experiment for each is shown. wt, wild type.
points, induction of IFN-# expression was observed in poly(I-C)-stimulated, wild type MEF but not in rip1!/! MEF stimulated with
poly(I-C) (Fig. 2A). In contrast, late phase (24-h) IFN-# expression
appears less affected, although expression remained significantly
reduced in poly(I-C)-treated rip1!/! MEF (Fig. 2A). Similarly,
RANTES production was reduced when rip1!/! cells were stimulated
with either poly(I-C) or TNF-! (Fig. 2B). However, cytokine production
was unaffected when rip1!/! MEF were infected with Sendai virus, a
single-stranded RNA virus, that induces IFN-# expression via a Trifand TLR3-independent pathway (14). These data suggest that Rip1 contributes to anti-viral responses mediated by TLR3 but is not required for
NF-"B activation by the intracellular retinoic acid-inducible gene I
pathway (15).
Decreased RANTES Production When rip1!/! Splenocytes Are
Stimulated with Purified LPS—Although Rip1 has been shown to interact with the TLR3 and TLR4 adapter Trif, cytokine responses to
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FIGURE 2. Decreased IFN-$ expression and cytokine production in poly(I-C)-stimulated 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 stimulated 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 examined for RANTES production by ELISA. Three independent rip1!/! MEF lines were examined in triplicate, and one representative experiment is shown.
Rip1 Mediates TLR3 and TLR4 NF-!B Pathways
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 production 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 examine 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
reduced when neonatal rip1!/!tnfr1!/! splenocytes are stimulated
with poly(I-C) and LPS (Fig. 3). These findings suggest that Rip1 contributes to both the Trif-dependent TLR3 and TLR4 pathways and demonstrate that activation of both the Trif-Rip1 and MyD88-dependent
TLR4 pathways is required for robust cytokine production.
Rip1 Mediates the Trif-dependent TLR4 Pathway—The decreased
cytokine responses to the TLR4 ligand LPS observed in rip1!/! MEF
and splenocytes suggested that Rip1 may also mediate the Trif-dependent, TLR4-induced NF-"B pathway. To test this possibility, we mated
our rip1"/! mice with MyD88!/! mice (generously provided by
S. Akira) and then stimulated wild type, rip1"/"MyD88!/!, or
rip1"/!MyD88!/! and rip1!/!MyD88!/! MEF with TNF-! or
LPS and examined cytokine-induced I"B! degradation. TNF-!-induced I"B! degradation was observed in wild type cells and in rip1"/"
MyD88!/! 10 min following cytokine treatment (Fig. 4, A and B). As
expected, no TNF-!-induced I"B! degradation is observed in the double knockout rip1!/!MyD88!/! cells due to the absence of Rip1 (Fig.
4C). Wild type MEF exhibit I"B! degradation 15 min following LPS
treatment, whereas I"B! degradation is delayed in rip1"/
"MyD88!/! cells with evidence of I"B! degradation detected
between 45 min and 1 h poststimulation (Fig. 4, A and B). In contrast, no
LPS-induced I"B! degradation was observed in cells deficient for both
Rip1 and MyD88 even at 120 min following LPS treatment (Fig. 4C). The
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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
with a Viperin-dependent reporter construct (C), together with expression constructs
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.
double knock out cells failed to respond to TNF-!, LPS, and poly(I-C)
but remained capable of activating NF-"B in response to treatment with
phorbol 12-myristate 13-acetate/ionomycin (not shown). These studies
provide genetic evidence that Rip1 contributes to the Trif-dependent
TLR4-induced NF-"B pathway.
Rip1 Expression Stimulates NF-"B but Not IRF-3 Activation—The
presence of a Rip homotypic interaction domain in Trif and Rip1 suggests that Rip1 and Trif may interact to mediate both NF-"B and IRF-3
activation. Deletion of the Rip homotypic interaction motif in Trif
ablates Trif-mediated NF-"B activation but has no effect on IRF-3 activation (8). To test whether Rip1 can stimulate both NF-"B and IRF-3
activation, we transfected HEK293 cells with a NF-"B or IRF reporter
constructs (ISG54 and ISRE) with expression plasmids containing p65
(RelA), Rip1, or Trif or with a constitutively active IRF-3 expression
construct, the phosphomimetic IRF3-5D. Twenty-four hours later,
luciferase reporter gene activity was measured. RelA, Rip1, and Trif
expression all stimulated NF-"B reporter activity (Fig. 5A), yet only Trif
and IRF3-5D were capable of stimulating IRF-3-dependent reporter
activity (ISRE reporter (Fig. 5B) and Viperin reporter (Fig. 5C)). This
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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 antiI"B! 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.
Rip1 Mediates TLR3 and TLR4 NF-!B Pathways
FIGURE 6. Poly(I-C) and LPS-induced cytokine production is impaired in traf6#/#
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
$g/ml), LPS (100 ng/ml), or TNF-! (10 ng/ml) for the indicated time periods. Cell lysates
were separated on a 12% SDS-polyacrylamide gel, and NF-"B activity was assessed by
immunoblotting with an anti-phospho-I"B! antibody. B, poly(I-C) or LPS treatment fails
to stimulate cytokine production in traf6!/! MEF. Wild type and traf6!/! MEF were left
untreated or stimulated with various concentrations of poly(I-C) (100, 10, or 1 $g/ml),
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
experiment was repeated a minimum of three times.
experiment suggests that Rip1 may not participate in the Trif-dependent IRF-3 pathway and is recruited by Trif to mediate IKK-# activation.
LPS- and Poly(I-C)-induced Cytokine Production Is Diminished in
traf6!/! MEF—The MyD88-dependent NF-"B pathway is mediated
by the recruitment of IRAK kinases and Traf6 and the Tak1-Tab1-Tab2
complex. Thus, we reasoned that Traf6 may also contribute to the Trifdependent NF-"B pathway. To test this possibility, we stimulated wild
type and traf6!/! MEF with TLR ligand poly(I-C) or LPS or with
TNF-! and examined IKK activity using a phospho-I"B! antibody. In
contrast to wild type MEF, traf6!/! MEF responded only to TNF-!
and failed to phosphorylate I"B! when stimulated with TLR3 or TLR4
ligands (Fig. 6A). Culture supernatants were also harvested 24 h poststimulation, and RANTES production was measured by ELISA (Fig. 6B).
NF-"B activation and cytokine production in response to TLR2, -3, and
-4 ligands was ablated in traf6!/! MEF, whereas TNF-induced IKK
activation and cytokine production remained unaffected by a Traf6 deficiency (Fig. 6, A and B). Taken together, these studies suggest that Traf6
and Rip1 contribute to the Trif-dependent TLR3 and TLR4 NF-"B
pathways.
36564 JOURNAL OF BIOLOGICAL CHEMISTRY
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 antibody and tested whether Rip1 or Traf6 associated with TLR3. We
detected Rip1 and Traf6 recruitment to TLR3 30 min following
poly(I-C) treatment (Fig. 7, A and B). We also examined the TLR3associated proteins for evidence of Tak1 recruitment. Tak1 recruitment
to TLR3 was observed at 60 min when maximal phosphorylated I"B!
protein is also detected (Fig. 7C). Interestingly, Rip1 and Traf6 recruitment appears to precede Tak1 recruitment, suggesting that Traf6 may
modify Rip1 and thereby signal Tak1 recruitment.
Rip1 Is Phosphorylated and Polyubiquitinated in TLR3-stimulated
Cells—We have shown that the kinase activity of Rip1 is responsive to
TNFR1 activation (6), suggesting that the kinase activity of Rip1 is
responsive to specific ligands or stresses. Studies in cells expressing
kinase-inactive Rip1 have revealed that Rip1 autophosphorylation is
induced in TNF-!-stimulated cells (6) (Fig. 8, A and B). Similarly, the
related Rip2 kinase and IRAK kinases are responsive to TLR and IL-1
receptor activation (16). To provide additional evidence that Rip1
responds to TLR3/4 activation, we stimulated wild type cells with
TNF-! or with the TLR ligand LPS or poly(I-C) and examined cell
lysates for phosphorylated Rip1. As expected, phosphorylated Rip1 was
detected as early as 5 min following TNF-! treatment. We find Rip1
autophosphorylation also rapidly induced upon poly(I-C) treatment or
LPS treatment (Fig. 8, A and B). These studies reveal that the Rip1 kinase
activity responds to TNFR1, TLR3, and TLR4 activation and further
implicates Rip1 in a TLR3 and TLR4 Trif-dependent pathway.
In addition to phosphorylation, TNF-! stimulates the conjugation of
K63-linked polyubiquitin chains on Rip1 (6, 17–19). K63-linked polyubiquitin chains on Rip1, Traf6, or IKK-% (NEMO) are recognized by
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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 immunoblotting 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 Mediates TLR3 and TLR4 NF-!B Pathways
DISCUSSION
the ubiquitin receptor protein Tab2, a component of the ubiquitinactivated Tak1 complex (7). Thus, Rip1 may use similar mechanisms to
mediate IKK-# activation in TNF-!- and TLR3- or TLR4-stimulated
cells. To test this possibility, we stimulated the macrophage cell line
RAW264.7 and the U373 astrocytoma line with TNF-! for 10 min or
treated the cells with poly(I-C) for 30, 60, or 120 min. We immunoprecipitated Rip1 from the untreated and treated cells and examined the
lysates for polyubiquitinated Rip1 protein by immunoblotting with an
anti-ubiquitin antibody. We observed evidence of Rip1 polyubiquitination in both the poly(I-C) and TNF-!-treated cell lysates (Fig. 8C), suggesting that polyubiquitinated Rip1 may contribute to IKK-# activation
in both the TNFR1 and TLR3 pathway. It remains unclear whether Rip1
is modified by K63-linked and/or K48-linked polyubiquitin chains in the
TLR3-stimulated cells; however, Rip1 degradation does not appear
induced in the poly(I-C)-treated cells, suggesting that Rip1 may be modified by K63-linked polyubiquitin chains. Moreover, the recruitment of
Traf6 and Tak1 would support the idea that K63-linked polyubiquitinated Rip1 stimulates IKK-# activation in TLR3/4-stimulated cells.
These studies provide genetic and biochemical evidence that Rip1 is an
essential mediator of the TLR3 and TLR4 NF-"B responses and suggest
that the Trif-dependent NF-"B pathway may be mediated by polyubiquitinated Rip1 and the ubiquitin-activated Tak1 complex.
NOVEMBER 4, 2005 • VOLUME 280 • NUMBER 44
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G. Wen and M. A. Kelliher, unpublished data.
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FIGURE 8. Treatment with TLR3 or TLR4 ligands stimulates Rip1 phosphorylation
and polyubiquitination. A, the kinase activity of Rip1 is induced when cells are treated
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
separated on an 8% gel, and Rip1 protein was detected by immunoblotting with an
anti-Rip1 antibody. B, the TLR4 ligand LPS stimulates Rip1 phosphorylation. Wild type
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
immunoblotting with an anti-Rip1 antibody. C, TNF-! and poly(I-C) stimulation induces
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-!.
Cell lysates were immunoprecipitated with anti-Rip1 antibody and separated on a 3– 8%
gradient gel. Polyubiquitinated Rip1 was detected by immunoblotting with an antiubiquitin antibody.
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 activation 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-dependent 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 published studies on Trif-deficient and MyD88-deficient cells (20, 21), we
find poly(I-C)- and LPS-induced cytokine production impaired in Rip1deficient MEF and splenocytes. Taken together, these studies support
the model that TLR4-induced cytokine responses to bacterial pathogens require activation of both the Trif-dependent and MyD88-dependent pathways.
The involvement of Rip1 in the Trif-dependent TLR3 NF-"B pathway 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
pathway, we generated rip1!/!MyD88!/! cells and stimulated them
with either TNF-! or LPS. As expected, NF-"B activation is delayed in
LPS-treated rip1"/"MyD88!/! cells; however, in the absence of both
MyD88 and Rip1, LPS-induced NF-"B activation is not observed. Taken
together, these studies demonstrate that Rip1 is responsive to TLR4
activation and demonstrates that Rip1 mediates both the Trif-dependent TLR3 and TLR4 NF-"B pathways. Interestingly, the dsRNA-activated protein kinase PKR has also been implicated in poly(I-C)-induced
NF-"B activation and in apoptotic responses to poly(I-C), TNF-!, and
LPS (22, 23), raising the possibility that dsRNA-induced NF-"B activation may involve PKR and Rip1. Consistent with this model, Li and
co-workers (24) find PKR recruited to Trif upon poly(I-C) stimulation.
However, unlike PKR-deficient cells that are resistant to poly(I-C)- and
TNF-!-induced apoptosis, Rip1-deficient cells are sensitive to TNF-!and poly(I-C)-induced cell death, suggesting that Rip1 and PKR have
opposing functions in the TNF-!- and dsRNA-activated pathways.3
We demonstrate that, like TNFR1 activation, TLR3 activation stimulates Rip1 recruitment and Rip1 polyubiquitination. In TNF-!-treated
cells, ubiquitin chain conjugation is mediated by the Ubc13-Uev1a complex and facilitated by Traf2, the E3 ubiquitin ligase recruited to the
TNFR1 that also interacts with Rip1 (6, 19). We find TLR3 and TLR4
responses diminished in traf6!/! cells and observe ligand-dependent
recruitment of Rip1, Traf6, and Tak1 to TLR3 (Fig. 7). Our studies
suggest that Traf6 also contributes to TLR3/4-induced antimicrobial
responses. Traf6 has been shown by others to be recruited to TLR3 and
bind Trif (24, 25) and has been implicated in TLR signaling with
decreased cytokine responses observed when traf6!/! macrophages
are stimulated with TLR2, -4, -7, and -9 ligands (26).
Rip1 polyubiquitination is induced upon TNF-! or poly(I-C) treatment of cells, suggesting that Rip1 may use similar mechanisms to activate NF-"B in the TNFR1- and Trif-dependent TLR pathways. Thus,
polyubiquitinated Rip1 may be recognized by the Tab2 protein (7), a
Rip1 Mediates TLR3 and TLR4 NF-!B Pathways
Acknowledgments—We thank Annette Schoenmeyer for the real time PCR
assay for mouse IFN-#, Neal Silverman for critical reading of the manuscript,
and members of the University of Massachusetts Medical School Innate
Immunity Data Club for helpful discussion.
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component of the Tak1 complex, and thereby, the ubiquitin-activated
kinase Tak1 may be recruited to the TLR3. Consistently, we find Traf6
and Tak1 recruited to TLR3, and we find TLR3/4-induced cytokine
production ablated in traf6!/! cells. Additionally, TLR3 and TLR4
NF-"B and IRF-3 responses have been shown to be negatively regulated
by the ubiquitin-modifying enzyme A20 (27–29).
Parallels can also be made to Drosophila melanogaster, where the Imd
protein, the mammalian counterpart to Rip1, mediates the antimicrobial immune response to Gram-negative bacteria. Imd is required for
activation of Drosophila Tak1 and acts through a pathway that includes
the Drosophila I"B kinase complex (30). Recent studies suggest that Imd
signaling requires the fly homologues of the ubiquitin-conjugating
enzymes Ubc13/Uev1A4 and a Drosophila homologue of the human
Tab2 protein. A Drosophila Tab2 mutant that is defective in activating
the Imd pathway contains a nonsense mutation in the C-terminal zinc
finger domain that recognizes K63-linked polyubiquitin chains.5 Thus,
the antimicrobial response and mechanism of IKK activation in flies and
mammals appear highly conserved and may be dependent on the ubiquitin modification of Rip1 and potentially Imd.