SIGIRR inhibits interleukin-1 receptor- and toll-like receptor 4-mediated signaling through different mechanisms.

The Toll-interleukin-1 receptor (TIR) domain-containing orphan receptor SIGIRR (single immunoglobulin interleukin-1 receptor-related protein) acts as a negative regulator of interleukin (IL)-1 and lipopolysaccharide (LPS) signaling. Endogenous SIGIRR transiently interacted with IL-1 receptor and the receptor-proximal signaling components (MyD88, IRAK, and tumor necrosis factor receptor-associated factor 6) upon IL-1 stimulation, indicating that SIGIRR interacts with the IL-1 receptor complex in a ligand-dependent manner. Similar interaction was also observed between SIGIRR and Toll-like receptor 4 receptor complex upon LPS stimulation. To identify the domains of SIGIRR required for its interaction with the Toll-like receptor 4 and IL-1 receptor complexes, several SIGIRR deletion mutants were generated, including DeltaN (lacking the extracellular immunoglobulin (Ig) domain with deletion of amino acids 1-119), DeltaC (lacking the C-terminal domain with deletion of amino acids 313-410), and DeltaTIR (lacking the TIR domain with deletion of amino acids 161-313). Whereas both the extracellular Ig domain and the intracellular TIR domains are important for SIGIRR to inhibit IL-1 signaling, only the TIR domain is necessary for SIGIRR to inhibit LPS signaling. The extracellular Ig domain exerts its inhibitory role in IL-1 signaling by interfering with the heterodimerization of IL-1 receptor and IL-1RAcP, whereas the intracellular TIR domain inhibits both IL-1 and LPS signaling by attenuating the recruitment of receptor-proximal signaling components to the receptor. These results indicate that SIGIRR inhibits IL-1 and LPS signaling pathways through differential mechanisms.

The Toll-interleukin-1 receptor (TIR) 1 superfamily, a large family of proteins defined by the presence of an intracellular TIR domain, plays crucial roles in the immune response. This superfamily can be divided into two main subgroups, based on the extracellular domains: the immunoglobulin (Ig) domaincontaining receptors (1), and the leucine-rich repeat motifcontaining receptors (2). The Ig domain subgroup includes IL-1R1, IL-18 receptor, T1/ST2, and SIGIRR. IL-1 has been demonstrated to be a key player in the immune response and inflammatory response at both local and systemic levels by activating gene expression of such genes as MIP-2, KC, and C-reactive protein. IL-18 plays important roles in promoting Th1 cell differentiation and natural killer cell activation. T1/ ST2 (3) and SIGIRR (4), also known as TIR8 (5), have been shown to function as negative regulators for Toll-IL-1R-mediated signaling. The leucine-rich repeat motif subgroup consists of at least 11 Toll-like receptors (TLRs) (2, 6 -10). These receptors have received intense attention because different TLRs were found to be activated by specific pathogen products (8,(11)(12)(13)(14)(15)(16).
Due to the similarity in their intracellular domain, the Toll-IL-1 receptors employ related yet distinct signaling components and downstream pathways. Genetic and biochemical studies revealed that IL-1R mediates a very complex pathway involving a cascade of kinases organized by multiple adapter molecules into signaling complexes, leading to activation of the transcription factors NF-B, ATF, and AP-1 (17)(18)(19). Based on published studies (20 -23), a model of the IL-1 pathway is postulated. Upon IL-1 stimulation, adapter molecule MyD88 (24) is first recruited to the IL-1 receptor, followed by the recruitment of two serine-threonine kinases, IRAK4 (25,26) and IRAK (27,28), and the adapter TRAF6 (29), resulting in the formation of the receptor complex (Complex I). During the formation of Complex I, IRAK and IRAK4 are activated, leading to the hyperphosphorylation of IRAK. Pellino 1⅐IRAK4⅐IRAK⅐TRAF6 complex is then formed, releasing these signaling molecules from the receptor (20). The released components interact with the membrane bound pre-associated TAK1⅐TAB1⅐TAB2⅐TAB3 (21,23), resulting in the formation of Complex II (IRAK⅐TRAF6⅐TAK1⅐TAB1⅐TAB2⅐TAB3), followed by the translocation of TRAF6⅐TAK1⅐TAB1⅐TAB2⅐TAB3 (Complex III) from the membrane to the cytosol. The translocated Complex III interacts with additional factors in the cytosol, leading to TAK1 activation. It has been implicated that TRAF6 functions as part of a unique E3 complex, mediating TAK1 activation through nonclassical ubiquitination catalyzed by the ubiquitination proteins Ubc13 and Uev1A (30,31). Once activated, TAK1 can directly phosphorylate IKK␤ and mitogenactivated protein kinase kinase 6, leading to the activation of both the JNK and NF-B signaling pathways (32)(33)(34)(35). In addition to TAK1, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 1 and 3 have also been implicated in the activation of IKK and mitogen-activated protein kinase, leading to the activation of NF-B and JNK (36 -39).
Whereas most of the TLRs share the MyD88-dependent pathway outlined above for IL-1 signaling, several Toll-IL-1 receptors also utilize variations of the above common signaling * 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.
Whereas the positive regulation of NF-B, AP-1, and IRF3 through the Toll-IL-1R superfamily has been studied extensively, recent studies have also begun to unravel how these pathways are negatively regulated. Several negative regulators have been shown to inhibit the Toll-IL-1R signaling, including IRAKM (48), IRAK2 (49), MyD88s (50), SOCS1 (51), and Triad3A (52). Recently, two orphan receptors of the Toll-IL-1R superfamily, SIGIRR (4,53), also known as TIR8 (5), and ST2 (3), have been identified as negative regulators for the signaling pathways mediated by members of this receptor superfamily. SIGIRR represents a unique subgroup of the Toll-IL-1R superfamily because its extracellular domain consists of a single Ig domain. SIGIRR has been shown not to bind IL-1 or enhance IL-1 signaling, but it does inhibit IL-1-mediated signaling. SIGIRR is expressed at high levels in primary kidney and intestinal epithelial cells and at moderate levels in splenocytes and immature dendritic cells, but it is not expressed in primary macrophages, fibroblasts, and endothelial cells (4). LPS stimulation leads to down-regulation of SIGIRR expression in different mouse tissues, suggesting an important regulatory role of SIGIRR in the inflammatory process (4). Consistent with the expression pattern of SIGIRR, SIGIRR-deficient kidney and intestinal epithelial cells and splenocytes, but not macrophages, exhibit enhanced responsiveness to IL-1 and Toll ligands (4). Furthermore, SIGIRR-deficient dendritic cells showed increased cytokine production in response to TLR ligands, including LPS and CpG DNA (5). Moreover, SIGIRRdeficient mice show an enhanced inflammatory response to IL-1, specifically in the lung and colon, and a reduced threshold for lethal endotoxin challenge (4). Importantly, it has been shown that the SIGIRR-deficient mice are more susceptible to inflammatory bowel disease induced by dextran sulfate sodium, as compared with wild-type mice (5). Taken together, these observations indicate that SIGIRR functions as an important modulator for the Toll-IL-1R system expressed in epithelial cells and dendritic cells and is crucial for tuning inflammation in gastrointestinal tract. The detailed signaling mechanism for SIGIRR is still unclear.
To understand the molecular mechanism of the inhibitory effect of SIGIRR, we recently studied the structure-function relationship of SIGIRR. Here we show that whereas both the extracellular Ig domain and the intracellular TIR domains are important for SIGIRR to inhibit IL-1 signaling, only the TIR domain is necessary for SIGIRR to inhibit LPS signaling. The fact that different domains of SIGIRR are used to inhibit IL-1 FIG. 1. A, SIGIRR-deficient kidney cells show enhanced activation in response to LPS or IL-1. a, SIGIRR-deficient kidney cells show a more profound NF-B gel shift and JNK activation after IL-1 or LPS treatment. Primary mouse kidney cells were either left untreated or treated (times are shown above the blot) with IL-1 (10 ng/ml), LPS (5 g/ml), peptidoglycan (PGN; 10 g/ml), or TNF␣ (10 ng/ml). Nuclear extracts were prepared and analyzed by electrophoretic mobility shift assay with an NF-B-specific probe. JNK activation was determined by immunoblot of cell extracts with anti-phospho-JNK (P-JNK). b, top panel, SI-GIRR-deficient kidney cells for wild-type and SIGIRR-deficient mice were stimulated with 10 ng/ml IL-1␤, 5 g/ml LPS, 10 g/ml peptidoglycan (PGN), or 5 ng/ml TNF␣ for the indicated times. Total RNA (15 g) was extracted and subjected to Northern blot analysis for expression of gene encoding Cxcl11. b, bottom panel, ethidium bromide staining after RNA transfer to the membrane. WT, wild-type; KO, SIGIRR-deficient. B, overexpression of SIGIRR inhibits IL-1 and LPS signaling in multiple cell types. NF-B-dependent luciferase reporter construct was tran-siently co-transfected with expression vectors (for empty vector or SI-GIRR) into 293IL-1R/TLR4/MD2 cells stably transfected with IL-1R, TLR4, and MD2; HeLa 229 cells; and HepG2 cells. The transfected cells were treated with various stimuli (horizontal axes), followed by luciferase reporter assay. Data are from one of three independent experiments with similar results.
versus LPS signaling indicates that SIGIRR probably inhibits these two pathways through differential mechanisms.

EXPERIMENTAL PROCEDURES
Biological Reagents and Cell Culture-Recombinant human IL-1 was provided by the National Cancer Institute. Recombinant human IL-18 and mouse TNF were obtained from Peprotech. LPS from Escherichia coli serotype O11:B4 was purchased from Sigma. Poly(I:C), peptidoglycan, and flagellin were purchased from Fluka, Amersham Biosciences, and InvivoGen. The 293, HeLa 229, and HepG2 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, penicillin G (100 g/ml), and streptomycin (100 g/ml). Antibody to FLAG (anti-FLAG) was purchased from Sigma. Antibodies SIGIRR (AF990; R&D Systems), MyD88 (Stressgen), TRAF6 (sc-7221; Santa Cruz Biotechnologies), IRAK (sc-7883; Santa Cruz Biotechnologies), IL-1R (sc-993; Santa Cruz Biotechnologies), TLR4 (sc-8693; Santa Cruz Biotechnologies), phosphorylated JNK (9251; Cell Signaling Technology) and JNK1 (sc-474; Santa Cruz Biotechnologies) were used. Anti-IRAK4 polyclonal antibody was kindly provided by Dr. Holger Wesche (Tularik, South San Francisco, CA). Primary kidney cells were prepared by cutting the kidney into small pieces and incubating them in 0.5% trypsin (Invitrogen) for 30 min at 37°C. The cells from each kidney were plated on a 15-cm plate and grown to confluence for 7-10 days in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. The cells were then split 24 h prior to treatment and analyzed as described.
Luciferase Reporter Assays-293 (1 ϫ 10 6 ), HeLa 229 (1ϫ 10 6 ), or HepG2 cells (4 ϫ 10 5 ) were transiently transfected using FuGENE 6 (Roche Applied Science), following the manufacturer's protocol. Cells were transfected with the indicated expression vectors plus 100 ng of the endothelial cell leukocyte adhesion molecule-1 promoter-derived NF-B luciferase reporter plasmid and 10 ng of ␤-galactosidase plasmid for normalization, with a 1:3 ratio of DNA:FuGENE 6. Transfection of empty vector was used to ensure all samples received equal amounts of DNA. At 36 h after transfection, cells were stimulated with cytokines for 6 h. Cells were lysed, and luciferase activity was assessed using Reporter lysis buffer and Luciferase Assay Reagent (Promega). All results reported represent duplicate experiments with at least three independent transfections.
Western Blot Analysis-Cells were harvested, washed in cold PBS buffer, pelleted, and lysed in ice-cold lysis buffer (30 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, and 1 mM phenylmethylsulfonyl fluoride) for 30 min. Cell debris was pelleted by centrifugation for 10 min at 13,000 ϫ g. Supernatants were separated on 10% SDS-PAGE, transferred to supported nitrocellulose membrane (Millipore), and blocked in a 5% solution of nonfat dry milk prepared in 1ϫ PBS and 0.05% Tween 20. Blots were incubated with primary antibody diluted in PBS overnight at 4°C, washed three to four times for 10 min each with PBS, detected with horseradish peroxidase-conjugated secondary antibody diluted 1:5000 in PBS ϩ 5% nonfat milk, and developed using the enhanced chemiluminescence method (ECL Plus; Amersham Biosciences) following the manufacturer's protocol.
buffer (89 mM Tris base, 89 mM boric acid, 2 mM EDTA, pH 8.0). After electrophoresis for 2 h at 200 V (at 4°C), the gel was dried and exposed to Kodak films at Ϫ80°C for 3-24 h with intensifying screens. The radiolabeled bands were detected by autoradiography. Each gel mobility shift assay was repeated with at least two independently prepared nuclear extracts.

RESULTS
SIGIRR Inhibits IL-1 and LPS Signaling-We have previously shown that SIGIRR plays a negative regulatory role in immediate signaling events mediated by members of the Toll-IL-1R family (4). Several primary cell types from SIGIRR-null mice were examined. The SIGIRR-null cells exhibited increased activation of NF-B and JNK in response to IL-1, LPS, and CpG DNA (but not peptidoglycan, flagellin, poly(I:C), and TNF), as compared with cells from wild-type littermate control mice (4) (Fig. 1A; data not shown). We recently examined the effects of SIGIRR overexpression on Toll-IL-1R signaling. Specifically, we used 293 cells transfected with IL-1RI, TLR4, and MD2, which were responsive to IL-1 and LPS; HeLa cells, which were responsive to IL-1, flagellin, and poly(I:C); and HepG2 cells, which were responsive to IL-1. Regardless of the cell type, SIGIRR overexpression substantially reduced the IL-1-and LPS-mediated activation of NF-B (50 -70%), as measured by an NF-B-dependent luciferase reporter assay (Fig. 1B). In contrast, SIGIRR overexpression had no significant effect on TLR3-, TLR5-or TNF␣-mediated NF-B activation. Thus, SIGIRR functions as a negative regulator for the signaling pathways mediated by some members of the Toll-IL-1R superfamily.
Interactions of SIGIRR with IL-1R and TLR4 Complex-To investigate the molecular mechanism of the action of SIGIRR, we examined the interaction of SIGIRR with the known signaling components of the IL-1R-and TLR4-mediated pathways. We stably transfected IL-1RI, TLR4, and MD2 into the human embryonic kidney epithelial 293 cells. The resulting cell line, 293IL-1RI/TLR4/MD2, is highly responsive to IL-1 and LPS with very low constitutive NF-B and JNK activity (Figs. 1, 3C, and 4B). To examine the interaction of SIGIRR with IL-1R and TLR4 complex, 293IL-1RI/TLR4/MD2 cells were stimulated with IL-1, LPS, or TNF␣ for 5, 10, and 30 min, followed by immunoprecipitation with anti-SIGIRR antibody and Western analysis with antibodies against IL-1RI, TLR4, MyD88, IRAK, TRAF6, and SIGIRR. As shown in Fig. 2, endogenous SIGIRR transiently interacted with IL-1RI, MyD88, IRAK, and TRAF6 upon IL-1 stimulation, indicating that SIGIRR associates with the IL-1 receptor complex in a ligand-dependent manner. Similar interaction was also observed between SIGIRR and TLR4 receptor complex upon LPS stimulation (Figs. 2 and 4A). These data were confirmed using a reverse co-immunoprecipitation approach, in which IL-1RI was immunoprecipitated and analyzed by immunoblotting with anti-SIGIRR (Fig. 2). Furthermore, IL-1 stimulation also leads to interaction of endogenous SIGIRR with endogenous IL-1 receptor in HeLa cells (data not shown). The TIR domain is located between amino acids 161 and 313. SIGIRR also contains a C-terminal 95-amino acid cytoplasmic tail. B, domains required for association of SIGIRR with IL-1R complex. 293IL-1RI/TLR4/ MD2 cells transiently transfected with empty vector, FLAG-tagged full-length (WT), and truncated SIGIRR mutants (lacking the extracellular Ig domain (⌬N), the TIR domain (⌬TIR), or the C-terminal portion (⌬C)) were either untreated or stimulated with 10 ng/ml IL-1 for 5, 10, and 30 min, followed by immunoprecipitation with anti-FLAG or normal rabbit serum (NS) and Western analysis with antibodies against IL-1RI, MyD88, IRAK, and TRAF6. The same lysates were simultaneously blotted with anti-FLAG antibody to monitor the expression of full-length and truncated SIGIRR mutants. C, domains involved in SIGIRR-mediated inhibition of IL-1-induced NF-B and JNK activation. NF-B-dependent luciferase reporter construct was transiently co-transfected into 293IL-1RI/TLR4/MD2 cells with empty vector, fulllength SIGIRR, or SIGIRR mutants lacking the extracellular Ig domain (⌬N), the TIR domain (⌬TIR), or the C-terminal portion (⌬C). a, the transfected cells were treated with IL-1 (10 ng/ml) for 4 h, followed by luciferase reporter assay. Data are from one of three independent experiments with similar results. b, nuclear extracts were prepared from the transfected cells treated with IL-1 (10 ng/ml) for the indicated times and analyzed by electrophoretic mobility shift assay. JNK activation was also determined by immunoblot of cell extracts from the transfected cells treated with IL-1 (10 ng/ml) for the indicated times with anti-phospho-JNK (P-JNK). The NF-B and JNK activation levels have now been analyzed by Scion Image 1.62C alias and are presented as relative fold of induction of the untreated samples. interaction with the receptor complexes, several SIGIRR deletion mutants were generated (Fig. 3A), including ⌬N (lacking the extracellular Ig domain with deletion of amino acids 1-119), ⌬C (lacking the C-terminal domain with deletion of amino acids 313-410), and ⌬TIR (lacking the TIR domain with deletion of amino acids 161-313). The 293IL-1RI/TLR4/MD2 cells transiently transfected with the FLAG-tagged wild-type or deletion mutants of SIGIRR were either untreated or treated with IL-1 for the indicated times, followed by immunoprecipitation with anti-FLAG antibody and Western analysis with antibodies against IL-1RI, MyD88, IRAK, and TRAF6 (Fig.  3B). Whereas wild-type SIGIRR interacted with IL-1R, MyD88, IRAK, and TRAF6 upon IL-1 stimulation, the deletion of the extracellular Ig domain (⌬N) completely abolished the ability of SIGIRR to interact with the IL-1 receptor complex. The deletion of the intracellular TIR domain (⌬TIR) also reduced the ability of SIGIRR to interact with the IL-1 receptor complex, whereas the deletion of the C terminus (⌬C) had little effect. These results indicate that both the extracellular Ig domain and the intracellular TIR domain of SIGIRR participate in its association with IL-1 receptor complex.

Both Extracellular Ig and Intracellular TIR Domain of SI-GIRR Are Required for Its Inhibition on IL-1R-mediated Signaling-To identify the domains of SIGIRR required for its
The SIGIRR deletion mutants were then examined for their ability to inhibit IL-1 signaling. Whereas overexpression of full-length SIGIRR significantly inhibited IL-1-induced NF-B and JNK activation (Fig. 3C, a and b), mutant ⌬N completely lost the ability to inhibit IL-1 signaling. Mutant ⌬TIR had reduced capacity for this inhibition, whereas mutant ⌬C retained the full ability to inhibit IL-1 signaling as compared with the wild-type SIGIRR. Taken together, these results strongly suggest that both the extracellular Ig domain and the intracellular TIR domain not only participate in the interaction of SIGIRR with IL-1 receptor complex but also are important for its inhibition of IL-1 signaling. These results implicate that SIGIRR probably exerts its inhibitory effect on IL-1 signaling through its interaction with the receptor complex.
Intracellular TIR Domain of SIGIRR Is Required for Its Inhibition on TLR4-mediated Signaling-We then examined the structure-function relationship of SIGIRR in TLR4-mediated signaling. 293IL-1RI/TLR4/MD2 cells transfected with FLAG-tagged full-length or truncated SIGIRR mutants were untreated or stimulated with LPS, followed by immunoprecipitation with anti-FLAG antibody and Western analysis with antibodies against TLR4, MyD88, IRAK, and TRAF6. The deletion of the intracellular TIR domain (⌬TIR) abolished the ability of SIGIRR to interact with the TLR4 MyD88, IRAK, and TRAF6 upon LPS stimulation, whereas the deletion of the extracellular Ig domain (⌬N) or the C terminus (⌬C) did not affect the interaction of SIGIRR with the TLR4 complex (Fig.  4A). Consistent with the inability of mutant ⌬TIR to associate with TLR4 complex, this mutant also failed to inhibit LPSinduced NF-B and JNK activation (Fig. 4B). These results indicate that the TIR domain of SIGIRR is necessary for both its association with TLR4 and its inhibition of LPS-induced NF-B and JNK activation.
Taken together, the above results show that whereas both the extracellular Ig domain and the intracellular TIR domain are important for SIGIRR to inhibit IL-1 signaling, only the TIR domain is necessary for SIGIRR to inhibit LPS signaling. The fact that different domains of SIGIRR are used to inhibit IL-1 versus LPS signaling indicates that SIGIRR probably inhibits these two pathways through differential mechanisms.
SIGIRR Inhibits Heterodimerization between IL-1RI and IL-1RAcP through Its Extracellular Ig Domain-Because the extracellular Ig domain is essential for SIGIRR-mediated inhibition of IL-1 responsiveness, we speculated that this region might be crucial for inhibiting the assembly of IL-1RI and IL-1RAcP into a heterodimer. To test this hypothesis, 293IL-1RI/TLR4/MD2 cells were transiently transfected with empty vector as well as expression constructs encoding full-length and truncated forms of SIGIRR. The cell extracts from the transfectants untreated or stimulated with IL-1 were immunoprecipitated with anti-IL-1RAcP, followed by Western blotting with anti-IL-1R. As shown in Fig. 5, whereas wild-type SIGIRR efficiently inhibited the IL-1-induced heterodimerization between IL-1R and IL-1RAcP, the deletion of the extracellular Ig   FIG. 4. A, domains required for association of SIGIRR with LPS receptor complex. 293IL-1RI/TLR4/MD2 cells transiently transfected with empty vector, FLAG-tagged full-length, and truncated SIGIRR mutants (lacking the extracellular Ig domain (⌬N), the TIR domain (⌬TIR), or the C-terminal portion (⌬C)) were either untreated or stimulated with 10 g/ml LPS for 5, 10, and 30 min, followed by immunoprecipitation with anti-FLAG and Western analysis with antibodies against TLR4, MyD88, IRAK, and TRAF6. The same lysates were simultaneously blotted with anti-FLAG antibody to monitor the expression of full-length and truncated SIGIRR mutants. B, domains involved in SIGIRR-mediated inhibition of LPS-induced NF-B and JNK activation. NF-B-dependent luciferase reporter construct was transiently co-transfected into 293IL-1RI/TLR4/MD2 cells with empty vector, fulllength SIGIRR, or SIGIRR mutants lacking the extracellular Ig domain (⌬N), the TIR domain (⌬TIR), or the C-terminal portion (⌬C). a, the transfected cells were treated with LPS (10 g/ml) for 4 h, followed by luciferase reporter assay. Data are from one of three independent experiments with similar results. b, nuclear extracts were prepared from the transfected cells treated with LPS (10 g/ml) for the indicated times and analyzed by electrophoretic mobility shift assay. JNK activation was also determined by immunoblot of cell extracts from the transfected cells treated with LPS (10 g/ml) for the indicated times with anti-phospho-JNK (P-JNK). The NF-B and JNK activation levels have now been analyzed by Scion Image 1.62C alias and are presented as relative fold of induction of the untreated samples.
domain completely abolished such inhibition. These results indicate that at least one of the mechanisms for SIGIRR to inhibit IL-1 signaling is to block the heterodimerization between IL-1RI and IL-1RAcP through its extracellular domain.

SIGIRR Interferes with Recruitment of the Receptor-proximal Signaling Components through Its Intracellular TIR Domain-
Whereas the TIR domain is not required for SIGIRR to block IL-1R dimerization (Fig. 5), the deletion of the TIR domain did reduce the interaction of SIGIRR with the IL-1 receptor complex and decreased the subsequent inhibition of IL-1-induced NF-B and JNK activation (Fig. 3C). These results suggest that SIGIRR may also inhibit IL-1 signaling by attenuating the recruitment of TIR domain-containing adapter protein MyD88 to the TIR domain in the IL-1R and the subsequent recruitment of other receptor-proximal signaling components. To test this idea, full-length SIGIRR as well as empty vector was transiently transfected into 293IL-1RI/TLR4/MD2 cells. The receptor complex was immunoprecipitated with anti-IL-1RI antibody from the transfected cells untreated or stimulated with IL-1, followed by Western analysis with antibodies against IL-1R, MyD88, IRAK4, IRAK, and TRAF6. Compared with the amounts of MyD88, IRAK4, IRAK, and TRAF6 in the receptor complex in cells transfected with an empty vector, reduced amounts of MyD88, IRAK4, IRAK, and TRAF6 were detected in the receptor complex from cells expressing the full-length SIGIRR protein (Fig. 6A). As expected, the deletion of the extracellular Ig domain of SIGIRR (⌬N) lost its ability to inhibit the recruitment of these receptor-proximal signaling components to the IL-1 receptor because this mutant completely failed to interact with the IL-1 receptor complex (Fig.  6A). Importantly, whereas deletion of the TIR domain did not affect the ability of SIGIRR to inhibit receptor dimerization, it did reduce the inhibition of SIGIRR on the recruitment of receptor-proximal signaling components (Fig. 6A). This result suggests that SIGIRR perturbs not only receptor dimerization but also recruitment of these receptor-proximal signaling components to the IL-1 receptor through its TIR domain.
To confirm that SIGIRR negatively regulates the IL-1 pathway by interfering with the recruitment of the receptor-proximal signaling components, we examined the interaction of endogenous IL-1RI, IRAK, and TRAF6 in wild-type and SIGIRR-deficient kidney cells before and after IL-1 stimulation at different time points by immunoprecipitation with anti-IRAK antibody. Compared with the amounts of IL-1RI and TRAF6 in the receptor complex in wild-type cells, enhanced amounts of IL-1RI and TRAF6 were detected in the receptor complex from SIGIRR-deficient cells (Fig. 6B).
We further analyzed the mechanistic basis for the inhibitory effect of SIGIRR on LPS signaling. 293IL-1RI/TLR4/MD2 cells transfected with FLAG-tagged full-length or truncated mutants were untreated or stimulated with LPS, followed by immunoprecipitation with anti-TLR4 antibody and Western analysis with anti-MyD88, anti-IRAK, and anti-TLR4 antibodies. As shown in Fig. 7, overexpression of ⌬N, ⌬C, as well as full-length SIGIRR reduced the recruitment of MyD88 and IRAK to TLR4. In contrast, ⌬TIR lost the ability to prevent the recruitment of MyD88 and IRAK to the receptor complex, consistent with the inability of this mutant to associate with TLR4 and inhibit LPS-induced NF-B and JNK activation. These results indicate that SIGIRR inhibits LPS signaling mainly by attenuating the efficient recruitment of receptor-proximal signaling components to TLR4. Such inhibition is likely achieved through the TIR-TIR domain interaction between SIGIRR and TLR4. DISCUSSION IL-1 or LPS treatment promoted the association of SIGIRR with IL-1RI or TLR4, respectively, and also led to the formation of a complex between SIGIRR, MyD88, IRAK4, IRAK, and TRAF6. Deletion studies of SIGIRR show that whereas both the extracellular Ig domain and the intracellular TIR domains FIG. 5. SIGIRR inhibits the assembly of IL-1RI and IL-1RAcP into a heterodimer through its extracellular domain. a, 293IL-1RI/ TLR4/MD2 cells were transiently transfected with empty vector as well as expression constructs encoding FLAG-tagged full-length and truncated forms of SIGIRR (lacking the extracellular Ig domain (⌬N), the TIR domain (⌬TIR), or the C-terminal portion (⌬C)). Cell extracts from the transfectants untreated or stimulated with IL-1 were immunoprecipitated with anti-IL-1RAcP, followed by Western analysis with anti-IL-1RI. The same lysates were simultaneously blotted with anti-FLAG antibody to monitor the expression of full-length and truncated SIGIRR mutants. b, 293IL-1RI/TLR4/MD2 cells were transiently transfected with 1.0, 2.5, and 5.0 g of expression plasmids for FLAG-tagged full-length and truncated forms of SIGIRR. The total amount of plasmids DNA was kept at 5.0 g by using an empty vector. Cell extracts from the transfectants untreated or stimulated with IL-1 were immunoprecipitated with anti-IL-1RAcP or normal rabbit serum (NS), followed by Western analysis with anti-IL-1RI. The same lysates were simultaneously blotted with anti-FLAG antibody to monitor the expression of full-length and truncated SIGIRR mutants. are important for SIGIRR to inhibit IL-1 signaling, only the TIR domain is necessary for SIGIRR to inhibit LPS signaling. The fact that different domains of SIGIRR are used to inhibit IL-1 versus LPS signaling indicates that SIGIRR probably inhibits these two pathways through differential mechanisms. The extracellular Ig domain exerts its inhibitory role in IL-1 signaling by interfering with the heterodimerization of IL-1R and IL-1RAcP, whereas the intracellular TIR domain interferes with both IL-1 and LPS signaling by attenuating the recruitment of receptor-proximal signaling components to the receptors. The TIR domain of SIGIRR might function through its interaction with the TIR domains of target receptors, suggesting the possibility to search for inhibitors to interfere with the TIR domain function. Development of low molecular weight inhibitors of the TIR domain interactions to modulate Toll-IL-1R signaling may yield an entirely new generation of potential anti-inflammatory compounds to modulate the innate immune system.
The spectrum of action of SIGIRR is restricted to several members of the Toll-IL-1R superfamily, including IL-1R, IL-18 receptor, TLR4, and TLR9. The specificity of SIGIRR might be determined by its ability to interact with the TIR domain of the target receptors. Whereas it is clear that SIGIRR has an inhibitory effect on LPS signaling, it still needs to be clarified whether SIGIRR impacts both TLR4-mediated MyD88-dependent and -independent pathways (44, 45, 54, 55).
As discussed above, SIGIRR probably directly interferes with the formation of the receptor complex, including receptor dimerization, appropriate recruitment, and activation of the receptor-proximal signaling components, inhibiting the activation of downstream signaling events. In addition to imposing direct steric inhibition on the receptor complexes, SIGIRR may actively recruit intracellular inhibitory molecules to the receptors, thereby exerting its inhibitory effects.
The other known endogenous inhibitors for TLR signaling are IRAKM (48), IRAK2 (49), MyD88s (50), SOCS1 (51), and Triad3A (52). IRAKM expression is induced upon TLR stimulation and negatively regulates TLR signaling. IRAKM-deficient cells exhibit increased cytokine production upon Toll-IL-1 stimulation and bacterial challenge, and IRAKM-deficient mice show increased inflammatory responses to bacterial infection. At the molecular level, IRAKM was shown to prevent the dissociation of IRAK and IRAK4 from MyD88 and the formation of IRAK⅐TRAF6 complexes. The detailed molecular mechanism for how IRAKM negatively regulates Toll-IL-1R-mediated signaling is still unclear. The IRAK family consists of two active kinases, IRAK and IRAK4, and two inactive kinases, IRAK2 and IRAKM. Recent studies showed that two specific splicing variants of mouse IRAK2, IRAK2c and IRAK2d, also have inhibitory effects on signaling pathway-mediated members of the Toll-IL-1R superfamily (49). In addition, the splicing variant of MyD88, MyD88s, has been demonstrated to act as a FIG. 6. A, SIGIRR acts as a competitor for the association of MyD88 with IL-1RI. 293IL-1RI/TLR4/MD2 cells transiently transfected with empty vector, FLAGtagged full-length SIGIRR, and truncated SIGIRR mutants were either untreated or stimulated with 10 ng/ml IL-1 for 5, 10, and 30 min, followed by immunoprecipitation with anti-IL-1RI and Western analysis with antibodies against MyD88, IRAK4, IRAK, and TRAF6. The same lysates were simultaneously blotted with anti-FLAG antibody to monitor the expression of full-length and truncated SI-GIRR mutants. Western analysis of the same blot with antiserum to IL-1RI indicates that similar amounts of IL-1RI were immunoprecipitated (IL-1RI IgG H ). B, the recruitment of IRAK and TRAF6 to IL-1RI upon IL-1 stimulation in SIGIRR-deficient kidney cells. Cell extracts prepared from wild-type and SIGIRR-deficient kidney cells untreated or stimulated with IL-1 (times are shown above the lanes) were immunoprecipitated with anti-IRAK, followed by Western analysis with anti-IL-1RI and anti-TRAF6. The same cell lysates were subjected to direct Western analysis with anti-SIGIRR. negative regulator of Toll/IL-1 signaling by blocking the recruitment of IRAK-4 (50,56). Furthermore, it has been reported recently that a Ring finger protein, Triad3A, acts as an E3 ubiquitin-protein ligase and enhances ubiquitination and proteolytic degradation of some TLRs, thereby regulating the intensity and duration of TLR signaling (52). The mechanism of action of SOCS1 is not known. Mice deficient in IRAKM or SOCS1 have a very similar phenotype to that of SIGIRR-deficient mice in terms of LPS hyper-responsiveness, and both IRAKM and SOCS1 are important in LPS tolerance, in which cells become unresponsive to LPS. Taken together, these findings suggest that multiple regulatory mechanisms are probably involved in the down-regulation of TLR signaling. It is conceivable that the inhibitory orphan receptors T1/ST2 and SIGIRR may activate an inhibitory signaling pathway(s) by employing some of these intracellular inhibitory molecules. Elucidation of the detailed molecular mechanism of action of SIGIRR in the future is critical for understanding the regulatory role of SI-GIRR in inflammatory and innate immune responses and essential for evaluating the therapeutic potential of SIGIRR. FIG. 7. SIGIRR acts as a competitor for the association of MyD88 with TLR4. 293IL-1RI/TLR4/MD2 cells transiently transfected with empty vector, FLAG-tagged full-length SIGIRR, and truncated SIGIRR mutants were either untreated or stimulated with 10 g/ml LPS for 5, 10, and 30 min, followed by immunoprecipitation with anti-TLR4 and Western analysis with antibodies against MyD88, IRAK, TRAF6, and TLR4. The same lysates were simultaneously blotted with anti-FLAG antibody to monitor the expression of full-length and truncated SIGIRR mutants. Western analysis of the same blot with antiserum to TLR4 indicates that similar amounts of TLR4 were immunoprecipitated (TLR4).