The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

The Journal of Immunology, 2007, 179, 4125 -4134
Copyright © 2007 by The American Association of Immunologists, Inc.
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Liu, G.
Right arrow Articles by Abraham, E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Liu, G.
Right arrow Articles by Abraham, E.

Variant IL-1 Receptor-Associated Kinase-1 Mediates Increased NF-{kappa}B Activity1

Gang Liu, Yuko Tsuruta, Zhiqian Gao, Young-Jun Park and Edward Abraham2

Department of Medicine, University of Alabama, Birmingham, AL 35294


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
IL-1R-associated kinase (IRAK)-1 is a critical mediator of TLR/IL-1R-induced activation of the transcription factor NF-{kappa}B. We previously described that a commonly occurring IRAK-1 variant haplotype, containing amino acid changes from serine to phenylalanine at position 196 and from leucine to serine at position 532, is associated with increased activation of NF-{kappa}B in LPS-stimulated neutrophils from patients with sepsis-induced acute lung injury and also higher mortality and more severe clinical outcomes in such patients. To investigate the underlying molecular mechanisms, we examined the ability of wild-type and variant IRAK-1 to modulate NF-{kappa}B activation. We found increased NF-{kappa}B transcriptional activity and expression of NF-{kappa}B-dependent proinflammatory cytokines in IL-1beta-stimulated IRAK-1-deficient cells transfected with variant IRAK-1 as compared with IRAK-1 wild type. I{kappa}B-{alpha} degradation was faster and p65 phosphorylation more prolonged after IL-1beta stimulation in cells expressing the IRAK-1 variant. However, IL-1-induced activation of MAPKs and nuclear translocation of NF-{kappa}B are comparable in both IRAK-1 variant- and IRAK-1 wild-type-expressing cells. Autophosphorylation of the IRAK-1 variant is greater than that found with wild-type IRAK-1. Additionally, variant IRAK-1 has greater interaction with TNFR-associated factor 6 than does wild-type IRAK-1. The enhanced activity of variant IRAK-1 appeared to be due to the alteration at aa 532, with only minimal effects being associated with change at aa 196. These results demonstrate that variant IRAK-1 is associated with alterations in multiple intracellular events that are likely to contribute to increased NF-{kappa}B activation and inflammatory responses in individuals with this IRAK-1 haplotype.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Sepsis is a leading cause of death in the U.S., with an estimated 750,000 new cases each year, resulting in >200,000 deaths (1). Although sepsis is initiated by infection, most of the deaths are not due to infection itself, but rather multiple organ dysfunctions, including acute lung injury (2, 3, 4, 5). High circulating and tissue levels of proinflammatory cytokines, such as TNF-{alpha}, IL-1, and IL-8, are present in septic patients and appear to contribute to organ system dysfunction in this setting (6, 7).

The transcription factor NF-{kappa}B plays a central role in regulating inflammation and immune response by modulating the expression of cytokines and other proinflammatory mediators (8, 9, 10, 11, 12). Increased activation of NF-{kappa}B is found in PBMC, neutrophils, and alveolar macrophages from patients with sepsis (8, 9, 10, 11, 12). In addition, greater or more persistent nuclear accumulation of NF-{kappa}B is associated with higher mortality and more severe organ dysfunction in such patients (8, 9, 10, 11, 12).

Cells recognize distinct microbial products, which exist as pathogen-associated molecular patterns, through the TLRs (13, 14, 15, 16). TLRs and IL-1R share a common intracellular domain known as TLR- and IL-1R-related (TIR)3 domain (13, 16, 17). Once TLR/IL-1R is associated with ligands, the TIR domain recruits adaptor proteins, such as MyD88 (18, 19, 20). IL-1R-associated kinase (IRAK)-1 and IRAK-4 then join the receptor complex, in which IRAK-4 phosphorylates IRAK-1, which results in autophosphorylation (21, 22). In parallel, hyperphosphorylated IRAK-1 interacts with TNFR-associated factor (TRAF)6, facilitating the assembly of the TAK-1, TAB-1, and TAB-2 complex (23, 24). After phosphorylation, TAK-1 and TAK-1-binding protein (TAB)-2 enter the cytosol, where TAK-1 phosphorylates and activates IKK and MAPKs, leading to phosphorylation, ubiquitination, and degradation of I{kappa}B-{alpha}, with subsequent translocation of NF-{kappa}B into the nucleus (23, 24, 25, 26).

IRAK-1 contains an N-terminal death domain (aa 1–102), a C-terminal domain (aa 523–712), and a central kinase domain (aa 198–522). There is also a regulatory domain (aa 103–197), termed the ProST region because of unusual abundance of proline, serine, and threonine residues, between the death domain and the kinase domain (27). Previous studies demonstrated that the kinase domain is not required for IRAK-1-mediated signaling transduction (27, 28). It was also shown that IRAK-1 phosphorylation is involved in NF-{kappa}B activation in response to IL-1 stimulation (28). Because IRAK-1 is subject to autophosphorylation, it is likely that the kinase activity of IRAK-1, although not strictly necessary for NF-{kappa}B activation, still contributes to optimal activation of this transcriptional factor.

Single nucleotide polymorphisms (SNPs) within the IRAK-1 gene occur frequently in the human population (29, 30). In particular, a variant haplotype consisting of five intron SNPs and three exon SNPs that have an r (2) near 1.0, suggesting nearly complete linkage disequilibrium, has been described in ~20–25% of Caucasians and 75% of Japanese (30). In this variant IRAK-1 haplotype, two of the three SNPs within exons are nonsynonymous, resulting in the change of aa 196 from serine to phenylalanine and aa 532 from leucine to serine (29, 30). We previously found that the variant IRAK-1 haplotype is associated with increased nuclear translocation of NF-{kappa}B in LPS-stimulated neutrophils from patients with sepsis-induced acute lung injury and also higher mortality and more severe clinical outcomes in such patients (29). In addition, decreased bone density was found to be present in Japanese women with this variant IRAK-1 haplotype (30).

To investigate underlying molecular mechanisms for these variant IRAK-1-associated effects, we examined the ability of wild-type (WT) and variant IRAK-1 to modulate central intracellular events associated with TLR/IL-1R-induced NF-{kappa}B activation. These studies demonstrate that variant IRAK-1 is associated with alterations in multiple intracellular events that contribute to increased NF-{kappa}B activation and inflammatory response.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cell culture

A human embryonic kidney (HEK)-293 cell line that is deficient in IRAK-1 (HEK-293 I1A) was provided by X. Li (Cleveland Clinic, Cleveland, OH) (28). Cells were cultured in DMEM supplemented with 10% of FBS.

Plasmids

pcDNA3-WT-IRAK-1 expressing human full-length IRAK-1 was obtained from M. Martin (Hannover Medical School, Hannover, Germany). To generate a construct that encodes S196F IRAK-1 variant, a cDNA fragment was amplified by PCR using pcDNA3-WT-IRAK-1 as a template. The forward primer was (IRAK-1-C666T-F) 5'-TCC TGC AGG GAG CCC GCC CCT TTC CGT TTT GCT GGC CCC T 3', and the reverse primer was BGH-R. The codon change of aa 196 from serine to phenylalanine was created by a nucleotide substitution (bold italic form) within the forward primer IRAK-1-C666T-F. The PCR product was cut by PstI and EcoRI and used to replace the corresponding region in pcDNA3-WT-IRAK-1. The resulting construct was designated as pcDNA3-S196F-IRAK-1. The L532S IRAK-1 variant was generated by site-directed mutagenesis using pcDNA3-WT-IRAK-1 as a template. The specific primers for site-directed mutagenesis were as follows: forward, 5'-CGG GGG TGC CCG GGC ATT CGG AGG CCG CCA GCT GCA TC and reverse, 5'-GAT GCA GCT GGC GGC CTC CGA ATG CCC GGG CAC CCC CG. The nucleotide substitution corresponding to the change of aa 532 from leucine to serine was displayed in bold italic form. The resulting plasmid was designated as pcDNA3-L532S-IRAK-1. To generate the S196F/L532S IRAK-1 variant, pcDNA3-S196F-IRAK-1 was digested with BglII and EcoRI and replaced by the corresponding region in pcDNA3-L532S-IRAK-1. The resulting plasmid was designated as pcDNA3-S196F/L532S-IRAK-1.

Transfection

HEK-293 I1A cells were plated 1 day before transfection. The empty vector pcDNA3 and constructs expressing various IRAK-1 were transfected into the cells using Lipofectamine 2000, according to the manufacturer’s instructions.

Dual luciferase assay

Cotransfection of 100 ng of the empty vector pcDNA3, pcDNA3 construct expressing WT IRAK-1, or pcDNA3 construct expression S196F/L532S IRAK-1 with 100 ng of a luciferase reporter pNF-{kappa}Bluc (BD Clontech) was performed into HEK-293 I1A cells. As an internal control, a firefly luciferase reporter that is controlled by the thymidine kinase (TK) promoter (pRL-TK) (Promega) was also cotransfected. At 24 h after transfection, cells were either left untreated or cultured with 10 ng/ml IL-1beta for 30, 60, and 120 min. The dual luciferase assay was performed, according to the manufacturer’s instructions (Promega). The fold increase of luciferase activity is the product of values of various transfected groups divided by that of pcDNA3 control group without IL-1beta stimulation.

Real-time RT-PCR

Total RNA was purified with TRI reagent (Sigma-Aldrich). cDNA was synthesized using Taqman reverse-transcription reagents (Roche). Real-time PCR was performed using a Lightcycler 480 SYBR Green I Master system (Roche), according to the manufacturer’s instructions. The primers used to amplify human IL-8 transcripts were as follows: forward, 5'-GTG CAG TTT TGC CAA GGA GT-3' and reverse, 5'-CTC TGC ACC CAG TTT TCC TT-3'. The primers for human I{kappa}B-{alpha} were as follows: forward, 5'-CCC TGT AAT GGC CGG ACT G-3' and reverse, 5'-CAG CAT CTG AAG GTT TTC TAG TG-3'. Human GAPDH transcript was used as an internal control. The primers for GAPDH were as follows: 5'-GCG AGA TCC CTC CAA AAT CAA-3' and reverse, 5'-GTT CAC ACC CAT GAC GAA CAT-3'.

Immunoblotting assay

HEK-293 I1A cells that were transfected with pcDNA3 and pcDNA3 constructs encoding IRAK-1 WT or various IRAK-1 variants were washed twice with cold PBS. Cells were then lysed in 0.1% Triton X-100 lysis buffer (50 mM sodium phosphate, 150 mM sodium chloride, 5 mM EDTA, 0.1% Triton X-100 (pH 7.4)), supplemented with 1:100 protease inhibitor mixture (Sigma-Aldrich) and 1:100 phosphatase inhibitor mixture (Pierce). Protein concentrations were measured using dendritic cell protein assay kit (Bio-Rad), according to the manufacturer’s instructions. A quantity amounting to 30 µg of total protein was resolved by SDS-PAGE gel and then transferred to a nitrocellulose membrane. The membrane was blotted with specific Abs and protein bands detected using Supersignal ECL (Pierce). Mouse anti-IRAK-1 mAb, rabbit anti-p65 polyclonal Ab, rabbit anti-TRAF6 polyclonal Ab, rabbit anti-CREB-binding protein (CBP) polyclonal Ab, rabbit anti-GAPDH polyclonal Ab, and rabbit anti-p300 polyclonal Ab were purchased from Santa Cruz Biotechnology. Rabbit anti-actin polyclonal Ab was from Sigma-Aldrich. Rabbit anti-IRAK-4 polyclonal Ab was from Abcam. Rabbit anti-I{kappa}B-{alpha}, anti-p-S536, anti-p-JNK, anti-p-p38, anti-p-ERK, anti-JNK, anti-p38, and anti-ERK Abs were purchased from Cell Signaling Technology.

Immunoprecipitation assay

Cells were lysed in 0.1% Triton X-100 lysis buffer and 300 µg of total protein used for immunoprecipitation assays. A total of 1 µg of anti-IRAK-1 or anti-TRAF Ab was added to the cell lysate and incubated at 4°C for 2 h. Protein G beads (20 µl; Upstate Biotechnology) were then added and incubated for 1 h to capture the immunocomplex. The beads were washed three times with 1 ml of 0.1% Triton X-100 lysis buffer and protein bound to the beads eluted by adding 40 µl of 2x SDS sample buffer and boiled for 10 min. Proteins in the immunocomplex were resolved by SDS-PAGE, and immunoblotting was performed to detect specific proteins.

Kinase assay

HEK-293 I1A cells were transfected with pcDNA3 and pcDNA3 constructs encoding IRAK-1 WT or various IRAK-1 variants. At 24 h after transfection, cells were stimulated without or with IL-1beta (10 ng/ml) for 15 min. Cells were washed twice with cold PBS and lysed in 0.1% Triton X-100 lysis buffer. IRAK-1 WT and various IRAK-1 variants were immunoprecipitated by anti-IRAK-1 Ab and protein G agarose. The beads were washed three times with 0.1% Triton X-100 lysis buffer, and twice with kinase buffer (20 mM HEPES (pH 7.6) and 20 mM MgCl2). The beads were then incubated at 37°C in a final volume of 50 µl of kinase buffer in the presence of myelin basic protein (Sigma-Aldrich) as a substrate (1 µg/sample), 100 µM ATP, and 2.5 µCi of [{gamma}-32P]ATP (Amersham). After SDS sample buffer was added to the protein G beads, the samples were boiled for 10 min and then subjected to SDS-PAGE analysis. The gel was transferred to a nitrocellulose membrane and exposed to film. After obtaining an radioautograph, the membrane was blotted with total anti-IRAK-1 Ab to determine that an equal amount of precipitated IRAK-1 was present in each sample.

Isolation of nuclei

HEK-293 I1A cells were collected in PBS and centrifuged. The cell pellet was resuspended with 500 µl of lysis buffer (10 mM Tris (pH 7.5), 10 mM NaCl, 3 mM MgCl2, 0.05% Nonidet P-40, 1 mM EGTA, 1:100 protease inhibitor mixture, and 1:100 phosphatase inhibitor mixture). Lysed cells were collected in microcentrifuge tubes and centrifuged at 2,700 x g for 10 min at 4°C. The supernatant containing the cytosol was further centrifuged at 20,800 x g for 15 min at 4°C to obtain the cytosolic fraction. The nuclei in the pellet were washed three times by gently resuspending the nuclei in 200 µl of wash buffer (10 mM PIPES (pH 6.8), 300 mM sucrose, 3 mM MgCl2, 1 mM EGTA, 25 mM NaCl, 1:100 protease inhibitor mixture, and 1:100 phosphatase inhibitor mixture) and centrifuging at 2,700 x g for 5 min at 4°C. For a final wash, the nuclei were resuspended in 100 µl of wash buffer, layered over a cushion of 1 ml of sucrose buffer (1 M sucrose), and centrifuged at 2,700 x g for 10 min. The sucrose buffer containing nonsedimented cellular debris was discarded, and the pellet containing nuclei was washed in 100 µl of lysis buffer and centrifuged at 2,700 x g for 5 min at 4°C to remove residual sucrose buffer.

Densitometric and statistical analysis

Densitometry of Western blots was performed by AlphaEaseFC 4.0 software. Student’s t test was used for comparisons between two groups. A value of p < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Variant IRAK-1 is more active than IRAK-1 WT in stimulating NF-{kappa}B transcriptional activity

We previously found that the IRAK-1 variant haplotype is not only associated with greater LPS-induced translocation of NF-{kappa}B to the nucleus in neutrophils from septic patients, but also correlates with a more severe clinical course in the setting (29). Because of these findings, we hypothesized that variant IRAK-1 may be more potent than the WT form in mediating NF-{kappa}B activation in response to clinically relevant TLR/IL-1R ligands. To explore this possibility, we generated various constructs that encode WT IRAK-1, as well as variant versions that included S196F, L532S, and both S196F and L532S IRAK-1. We first compared the ability of WT and the double-mutant S196F/L532S IRAK-1 to affect NF-{kappa}B activation in HEK-293 cells without or with IL-1beta stimulation. To achieve this aim, HEK-293 I1A cells, which lack IRAK-1, were transiently transfected with either the empty vector (pcDNA3), pcDNA3-WT-IRAK-1, or pcDNA3-S196F/L532S-IRAK-1.

WT and S196F/L532S IRAK-1 protein were expressed at an equivalent level in transfected cells without or with IL-1beta stimulation (Fig. 1, A and B). Next, we examined the expression of two well-defined NF-{kappa}B-regulated genes, IL-8 and I{kappa}B-{alpha}, in cells transfected with the various IRAK-1 constructs. We found that transfection of WT IRAK-1 significantly activates IL-8 expression, even without IL-1beta stimulation (Fig. 1C). This finding was not surprising because it has been reported that overexpressed IRAK-1 is autophosphorylated, thus contributing to activation of the NF-{kappa}B signaling pathway (24). When the WT IRAK-1-transfected HEK-293 I1A cells were cultured with IL-1beta, IL-8 expression was increased even further, indicating that IRAK-1 contributes to NF-{kappa}B activation, as expected. Of note, culture with IL-1beta induced increased IL-8 expression even in HEK I1A cells transfected with the empty vector (Fig. 1C), indicating that IRAK-1 plays a redundant role, with other kinases, such as IRAK-2, also involved in IL-1-induced NF-{kappa}B activation.


Figure 1
View larger version (57K):
[in this window]
[in a new window]

  		FIGURE 1. Variant IRAK-1 is more active than WT IRAK-1 in stimulating NF-{kappa}B transcriptional activity. A, The levels of expression of WT IRAK-1 and S196F/L532S IRAK-1 are comparable in transfected I1A cells. IRAK-1-deficient HEK-293 I1A cells were transfected with 0.5 µg of pcDNA3, pcDNA3-WT-IRAK-1, or pcDNA3-S196F/L532S-IRAK-1. At 24 h after transfection, the cells were treated without or with 10 ng/ml IL-1beta for 30 min. Cell extracts were prepared and Western blots were performed to detect IRAK-1. Actin was also examined to ensure equal loading. B, Densitometric analysis of IRAK-1 expression in A. Relative level of IRAK-1 expression was obtained by dividing the densitometric value of WT IRAK-1 or S196F/L532S IRAK-1 with that of actin. C and D, S196F/L532S IRAK-1 is more potent than WT IRAK-1 in increasing the expression of IL-8 and I{kappa}B-{alpha}. I1A cells were transfected, as described in A. At 24 h after transfection, cells were treated without and with 10 ng/ml IL-1beta for 30 or 120 min. Total RNA was purified and cDNA synthesized. Real-time PCR was performed, as described in Materials and Methods, to determine the expression of IL-8 (C) and I{kappa}B-{alpha} (D). The relative expression level of IL-8 or I{kappa}B-{alpha} in cells transfected with pcDNA3 without IL-1beta treatment was regarded as 1, and the fold increase in relative expression of IL-8 or I{kappa}B-{alpha} in cells with different treatments was calculated based on the value for the control group. #, p < 0.05 vs pcDNA control at the indicated time point. *, p < 0.05 vs WT IRAK-1 group at the indicated group. E, S196F/L532S IRAK-1 is more potent than WT IRAK-1 in activating NF-{kappa}B-dependent luciferase activity. HEK-293 I1A cells were transfected with 100 ng of pcDNA3 and pcDNA3 construct expressing WT IRAK-1 or S196F/L532S IRAK-1, respectively. The luciferase reporter, pNF-{kappa}Bluc, and the internal control, PRL-TK, were cotransfected. At 24 h after transfection, cells were left untreated or were treated with 10 ng/ml IL-1beta for 30, 60, and 120 min. #, p < 0.05 vs pcDNA control at the indicated time point. *, p < 0.05 vs WT IRAK-1 group. F, The levels of expression of WT, S196F, and L532S IRAK-1 are comparable in transfected I1A cells. I1A cells were transfected with 0.5 µg of pcDNA3, pcDNA3-WT-IRAK-1, pcDNA3-S196F-IRAK-1, or pcDNA3-L532S-IRAK-1. Twenty-four hours after transfection, cells were treated without or with 10 ng/ml IL-1beta for 60 min. Cell extracts were prepared and Western blots were performed to detect IRAK-1. G, Densitometric analysis of IRAK-1 expression in F. Relative level of IRAK-1 expression was obtained by dividing the densitometric value of IRAK-1 by that of actin. H and I, L532S IRAK-1 is more potent than WT IRAK-1 in increasing the expression of IL-8 and I{kappa}B-{alpha}. I1A cells were transfected, as described in A. Twenty-four hours after transfection, cells were treated without and with IL-1beta for 60 or 120 min. Real-time PCR was performed, as described in C, to measure the expression of IL-8 (H) and I{kappa}B-{alpha} (I). #, p < 0.05 vs pcDNA control at the indicated time point. *, p < 0.05 vs WT IRAK-1 group at the indicated group.

 
Compared with WT IRAK-1, there was increased IL-8 expression both with and without IL-1beta stimulation in cells transfected with the S196F/L532S IRAK-1 construct (Fig. 1C). Similarly, S196F/L532S IRAK-1 was more active than WT IRAK-1 in inducing the expression of another NF-{kappa}B-dependent gene, I{kappa}B-{alpha} (Fig. 1D).

To examine directly the abilities of WT and S196F/L532S IRAK-1 to mediate NF-{kappa}B activation, we used a luciferase reporter construct that is under the control of NF-{kappa}B-responsive elements. The transcription of the reporter gene is solely dependent on NF-{kappa}B activity. Although both WT and S196F/L532S IRAK-1 stimulate luciferase activity even without IL-1beta stimulation, there was significantly greater activity with the S196F/L532S construct (Fig. 1E). Luciferase activity was further increased after 2 h of culture with IL-1beta, but again, S196F/L532S IRAK-1 was significantly more potent than WT IRAK-1 in activating the luciferase reporter (Fig. 1E).

To determine which amino acid change contributes to the increased activity of S196F/L532S IRAK-1, WT, S196F, and L532S IRAK-1 were overexpressed at equivalent levels in I1A cells (Fig. 1, F and G). We found that S196F IRAK-1 only marginally increased the induction of NF-{kappa}B transcriptional activity compared with WT IRAK-1 (Fig. 1, H and I). However, L532S IRAK-1 was significantly more active than WT IRAK-1 in its ability to contribute to NF-{kappa}B activation.

Variant IRAK-1 is more potent than WT IRAK-1 in inducing I{kappa}B-{alpha} degradation after IL-1 stimulation

To examine the mechanisms by which variant IRAK-1 mediates increased NF-{kappa}B activation, we initially determined I{kappa}B-{alpha} degradation in response to IL-1beta stimulation. IL-1beta treatment resulted in I{kappa}B-{alpha} degradation in I1A cells deficient in IRAK-1 (Fig. 2A), further suggesting that the role of IRAK-1 is redundant and that IRAK-2 and other kinases compensate for the lack of IRAK-1. However, more rapid degradation of I{kappa}B-{alpha} was present in WT IRAK-1-expressing cells than in cells transfected with empty vector (Fig. 2A). I1A cells transfected with variant IRAK-1 demonstrated a significantly greater degree of I{kappa}B-{alpha} degradation at early time points (15 and 30 min post-IL-1beta treatment) than that found in cells transfected with WT IRAK-1 (Fig. 2, A and B). These data suggest that the increased NF-{kappa}B activation mediated by variant IRAK-1 may involve more rapid and complete degradation of I{kappa}B-{alpha}, leading to greater NF-{kappa}B translocation to the nucleus.


Figure 2
View larger version (49K):
[in this window]
[in a new window]

  		FIGURE 2. Variant IRAK-1 induces a greater degree of I{kappa}B-{alpha} degradation at early time points than does WT IRAK-1 and is associated with increased p65 phosphorylation after IL-1 stimulation. A, HEK-293 I1A cells were transfected with 0.5 µg of pcDNA3, pcDNA3-WT-IRAK-1, or pcDNA3-S196F/L532S-IRAK-1. Twenty-four hours after transfection, cells were treated without or with 10 ng/ml IL-1beta for 15, 30, or 60 min. Cell extracts were prepared and Western blots were performed to detect IRAK-1 and I{kappa}B-{alpha}. p300 was used as a loading control. A representative experiment is shown. Two additional experiments provided similar results. B, Densitometric analysis of I{kappa}B-{alpha} expression. After normalization of I{kappa}B-{alpha} with that of the loading control, the value of pcDNA group at 15 or 30 min post-IL-1beta stimulation was regarded as 1, and the percentage of pcDNA3 control was obtained by dividing the value of WT IRAK-1 or S196F/L532S IRAK-1 with that of the pcDNA3 control. #, p < 0.05 vs the pcDNA control at the indicated time point. *, p < 0.05 vs WT IRAK-1 group at the indicated group. C, HEK-293 I1A cells were transfected with 0.5 µg of pcDNA3, pcDNA3-WT-IRAK-1, or pcDNA3-S196F/L532S-IRAK-1. Twenty-four hours after transfection, cells were treated without or with 10 ng/ml IL-1beta for 15, 30, or 60 min. Cell extracts were prepared and Western blots were performed to detect IRAK-1, p65, p-S536-p65, and I{kappa}B-{alpha}. Actin was used as a loading control. A representative experiment is shown. Two additional experiments provided similar results. D, Densitometric analysis of p-S536-p65 expression. After normalization of p-S536-p65 with that of the loading control, the value of pcDNA group at 15, 30, or 60 min post-IL-1beta stimulation was regarded as 1, and the relative fold of pcDNA3 control was obtained by dividing the value of WT IRAK-1 or S196F/L532S IRAK-1 with that of the pcDNA3 control.

 
Variant IRAK-1 is associated with increased p65 phosphorylation after IL-1 stimulation

After translocation to the nucleus, the p65 subunit of NF-{kappa}B undergoes multiple modifications, including phosphorylation and acetylation (31, 32, 33). Such events affect p65 DNA-binding activity as well as association with coactivators and corepressors that subsequently modulate p65 transcriptional activity (34, 35). To this end, we examined p65 phosphorylation on serine 536, a modification known to enhance its transcriptional activity (33).

At early time points (15 min) after cellular exposure to IL-1beta, p65 is phosphorylated in IRAK-1-deficient I1A cells to a degree comparable with that observed in WT IRAK-1-expressing cells (Fig. 2, C and D). However, p65 phosphorylation persisted longer when WT IRAK-1 was expressed (Fig. 2, C and D). Phosphorylation of p65 was even greater and sustained for a longer period in I1A cells transfected with variant IRAK-1 (Fig. 2, C and D).

Variant and WT IRAK-1 mediate similar levels of MAPK activation upon IL-1 stimulation

IRAK-1 mediates phosphorylation and activation of IKKs and MAPKs after engagement of TLR/IL-1R by cytokines and microbial products (23, 25, 26). MAPKs participate in modulating NF-{kappa}B activity (36). To investigate whether variant IRAK-1 mediates a differential activation of MAPKs, we examined phosphorylation of MAPKs in I1A HEK cells transfected with empty vector or constructs expressing WT IRAK-1 or S196F/L532S IRAK-1, both before and after IL-1beta stimulation.

JNK, ERK, and p38 were only marginally phosphorylated in response to IL-1beta in IRAK-1-deficient cells (Fig. 3), suggesting that IL-1-induced activation of MAPKs requires IRAK-1. However, transfection of WT IRAK-1 or S196F/L532S IRAK-1 into I1A cells resulted in equivalent phosphorylation of JNK, ERK, and p38 after IL-1 stimulation (Fig. 3). Thus, it is unlikely that modulation of MAPK activation is involved in the increased NF-{kappa}B activity mediated by variant IRAK-1.


Figure 3
View larger version (32K):
[in this window]
[in a new window]

  		FIGURE 3. Variant and WT IRAK-1 mediate similar levels of MAPK activation upon IL-1 stimulation. HEK-293 I1A cells were transfected with 0.5 µg of pcDNA3, pcDNA3-WT-IRAK-1, or pcDNA3-S196F/L532S-IRAK-1. Twenty-four hours after transfection, cells were treated without or with 10 ng/ml IL-1beta for 30 min. Cell extracts were prepared and Western blots were performed to detect IRAK-1, phosphorylated JNK, and total JNK (A), phosphorylated ERK and total ERK (C), and phosphorylated p38 and total p38 (E). B, D, and F, Densitometric analysis of phosphorylated JNK (B), ERK (D), and p38 (F). The level of phosphorylated JNK, ERK, or p38 was normalized by that of total JNK, ERK, or p38. The pcDNA3 group after IL-1beta stimulation was regarded as 1.

 
Variant IRAK-1 is more sensitive to autophosphorylation than is WT IRAK-1

IRAK-1 is phosphorylated after engagement of TLR/IL-1R by IL-1 or microbial products. Such modification of IRAK-1 appears to participate in pathways leading to NF-{kappa}B activation (27). To this end, we examined IL-1beta-induced phosphorylation of WT and variant IRAK-1.

Phosphorylated WT IRAK-1, as demonstrated by a band of slower migration on SDS-PAGE gels, was found in I1A cells even without IL-1beta stimulation (Fig. 4, A and B). This finding is consistent with previous reports that overexpressed IRAK-1 undergoes autophosphorylation under basal conditions (21). Additional WT IRAK-1 phosphorylation was present after IL-1beta treatment (Fig. 4A). There were greater amounts of phosphorylated S196F/L532S IRAK-1 than of WT IRAK-1 in both unstimulated and IL-1beta-stimulated I1A cells (Fig. 4, A and B).


Figure 4
View larger version (48K):
[in this window]
[in a new window]

  		FIGURE 4. Variant IRAK-1 is more sensitive to autophosphorylation than is WT IRAK-1. A, S196F/L532S IRAK-1 demonstrates greater phosphorylation than does WT IRAK-1. I1A cells were transfected with 0.5 µg of pcDNA3, pcDNA3-WT-IRAK-1, or pcDNA3-S196F/L532S-IRAK-1. Twenty-four hours after transfection, cells were treated without or with 10 ng/ml IL-1 for 15, 30, or 60 min. Cell extracts were prepared and Western blots were performed to detect IRAK-1. Actin was used as a loading control. B, Densitometric analysis of phosphorylated IRAK-1 in A. The level of phosphorylated WT IRAK-1 or S196F IRAK-1 (upper band) was normalized to that of the unphosphorylated form. The value of WT IRAK-1 group at each time point after IL-1beta stimulation was regarded as 1. C, L532S IRAK-1 demonstrates greater phosphorylation than does WT IRAK-1 and S196F IRAK-1. I1A cells were transfected with 0.5 µg of pcDNA3, pcDNA3-WT-IRAK-1, or pcDNA3-S196F, and pcDNA3–532S-IRAK-1. Cells were treated as described in A. Western blots were performed to detect IRAK-1. D, Densitometric analysis of phosphorylated IRAK-1 in C. Densitometric analysis was performed, as described in B. E, L532S IRAK-1 and S196F/L532S IRAK-1 are more sensitive to autophosphorylation than IRAK-1 WT. I1A cells were transfected with 0.5 µg of pcDNA3, pcDNA3-WT-IRAK-1, pcDNA3-S196F-IRAK-1, pcDNA3-L532S-IRAK-1, or pcDNA3-S196F/L532S-IRAK-1. Twenty-four hours after transfection, cells were treated without or with 10 ng/ml IL-1 for 15 min. IRAK-1 WT and various IRAK-1 variants were immunoprecipitated, and kinase assays were performed, as described in Materials and Methods.

 
To determine which amino acid substitution is responsible for the greater phosphorylation of S196F/L532S IRAK-1, we transfected I1A cells with S196F IRAK-1 or L532S IRAK-1 and then examined their phosphorylation. The amounts of phosphorylated S196F IRAK-1 in unstimulated and IL-1beta-stimulated cells were similar to those found in cells transfected with WT IRAK-1 (Fig. 4, C and D). However, phosphorylation of L532S IRAK-1 was substantially greater than that of WT IRAK-1 under basal and IL-1beta-stimulated conditions (Fig. 4, C and D).

To examine autophosphorylation of WT and IRAK-1 variants under in vitro conditions, WT and variant IRAK-1 was immunoprecipitated for kinase assays using myelin basic protein as substrate. As shown in Fig. 4E, increased amounts of phosphorylated S196F/L532S IRAK-1 and of L532S IRAK-1 as compared with WT IRAK-1 or S196F IRAK-1 were present in unstimulated or IL-1beta-stimulated I1A cells, consistent with the results found in the experiments reported in Fig. 4, A and B. In addition, we found that L532S IRAK-1 and S196F/L532S IRAK-1 have slightly higher kinase activity than the WT IRAK-1 (Fig. 4E). Altogether, our data indicate that greater phosphorylation of variant IRAK-1, and particularly of the L532S form, may contribute to its increased ability to mediate NF-{kappa}B activation.

Interaction between variant IRAK-1 and TRAF6 is increased, but association between variant IRAK-1 and IRAK-4 is decreased compared with that found with WT IRAK-1

IRAK-1 forms a transient complex with IRAK-4 after TLR/IL-1R engagement, in which IRAK-4 contributes to IRAK-1 phosphorylation. Phosphorylated IRAK-1 then associates with TRAF6 and the IRAK-1/TRAF6 complex recruits TAK-1, TAB-1, and TAB-2 (23, 25, 26). It has been shown that TRAF6 is indispensable to IL-1R- and TLR-mediated NF-{kappa}B activation (37, 38). Thus, we investigated the interaction of variant and WT IRAK-1 with TRAF6.

In initial experiments, TRAF6 was immunoprecipitated and associated IRAK-1 was detected using anti-IRAK-1 Abs. As shown in Fig. 5, A and B, there was more S196F/L532S IRAK-1 than WT IRAK-1 associated with TRAF6 in IL-1-stimulated cells. Reverse immunoprecipitation was also performed by pull-down of IRAK-1 and determination of associated TRAF6. After normalization of the level of TRAF6 by that of coprecipitated IRAK-1, we found that L532S IRAK-1 and S196F/L532S IRAK-1 have more interaction with TRAF6 than does WT IRAK-1 or S196F IRAK-1 (Fig. 5, C and D). We then examined the interaction between IRAK-1 and IRAK-4 in cells transfected with variant or WT IRAK-1. As shown in Fig. 5, E and F, in both unstimulated and IL-1-stimulated cells, there was less IRAK-4 associated with S196F/L532S IRAK-1 than with WT IRAK-1.


Figure 5
View larger version (35K):
[in this window]
[in a new window]

  		FIGURE 5. Interaction between variant IRAK-1 and TRAF6 is increased, but association between variant IRAK-1 and IRAK-4 decreased compared with that found with WT IRAK-1. A and C, L532S IRAK-1 and S196F/L532S IRAK-1 have greater interaction with TRAF6 than WT IRAK-1. I1A cells were transfected with 0.5 µg of pcDNA3, pcDNA3-WT-IRAK-1, pcDNA3-S196F-IRAK-1, pcDNA3-L532S-IRAK-1, or pcDNA3-S196F/L532S-IRAK-1. Twenty-four hours after transfection, cells were treated without or with 10 ng/ml IL-1beta for 15 min. Cell lysates were prepared and TRAF6 or IRAK-1 precipitated with anti-TRAF6 (A) or anti-IRAK-1 (C) Abs. TRAF6 and IRAK-1 were detected by anti-TRAF6 or anti-IRAK-1 Abs. B and D, Densitometric analysis of the levels of IRAK-1 and TRAF6 in A and C. After normalization of IRAK-1 (B) or TRAF6 (D) level with that of TRAF6 or IRAK-1, the value of WT IRAK-1 was considered as 1. The percentage of the WT IRAK-1 group was obtained by dividing the value of S196F/L532S IRAK-1 (B), S196F IRAK-1, L532S IRAK-1, or S196F/L532S IRAK-1 (D) with that of the WT IRAK-1. E, S196F/L532S IRAK-1 has greater interaction with IRAK-4 than WT IRAK-1. I1A cells were transfected with 0.5 µg of pcDNA3, pcDNA3-WT-IRAK-1, or pcDNA3-S196F/L532S-IRAK-1. Twenty-four hours after transfection, cells were treated without or with 10 ng/ml IL-1beta for 15 min. Cell lysates were prepared and immunoprecipitation assays were performed with anti-IRAK-1 Abs. IRAK-1 and IRAK-4 were detected using anti-IRAK-1 or anti-IRAK-4 Abs. F, Densitometric analysis of the level of IRAK-4 in E. After normalization of the level of IRAK-4 to that of IRAK-1, the value of WT IRAK-1 was regarded as 1. The percentage of WT IRAK-1 group was obtained by dividing the value of S196F/L532S IRAK-1 with that of WT IRAK-1.

 
Variant IRAK-1 does not affect TLR/IL-1R-induced nuclear translocation of NF-{kappa}B

Because our experiments demonstrated more rapid degradation of I{kappa}B-{alpha} in I1A cells transfected with S196F/L532S IRAK-1 as compared with cells expressing WT IRAK-1, we expected to find increased nuclear translocation of NF-{kappa}B following TLR/IL-1R engagement. However, as shown in Fig. 6, there were no differences in the amounts of NF-{kappa}B accumulating in the nucleus after IL-1beta stimulation in cells transfected with S196F/L532S IRAK-1 as compared with those transfected with WT IRAK-1.


Figure 6
View larger version (26K):
[in this window]
[in a new window]

  		FIGURE 6. Variant IRAK-1 does not affect TIR-induced nuclear translocation of NF-{kappa}B. A, I1A cells were transfected with 2 µg of pcDNA3, pcDNA3-WT-IRAK-1, or pcDNA3-S196F/L532S-IRAK-1. Twenty-four hours after transfection, cells were treated without or with 10 ng/ml IL-1beta for 30 min. Cytosolic and nuclear fractions were prepared, as described in Materials and Methods. p65 nuclear translocation was determined using anti-p65 Abs. CBP was used as a nuclear marker, and GAPDH was used as a cytosolic marker. B, Densitometric analysis of the level of nuclear-translocated p65. The level of nuclear p65 was normalized to that of CBP. The value of the pcDNA3 group before IL-1beta stimulation was regarded as 1.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
IRAK-1 is an indispensable mediator of NF-{kappa}B activation induced through the interaction of proinflammatory mediators, including cytokines, such as IL-1, and microbial products, with TLRs and IL-1Rs (27, 28). In the setting of sepsis, a dysregulated inflammatory response to infection that involves increased nuclear localization of NF-{kappa}B and enhanced transcription of NF-{kappa}B-dependent proinflammatory genes, including TNF-{alpha}, is associated with a higher mortality rate and more severe organ system dysfunction (6, 7). In a previous study, we found that a common variant haplotype of IRAK-1 was not only associated with greater LPS-induced translocation of NF-{kappa}B to the nucleus in neutrophils from septic patients, but also with an increased mortality rate as well as a worse profile for important indices of severity of illness, including prolonged requirement of mechanical ventilation (29).

The variant IRAK-1 haplotype that is associated with increased NF-{kappa}B activation and worsened clinical outcomes from sepsis is characterized by two amino acid changes, S->F and L->S at positions 196 and 532, respectively, which are in complete linkage disequilibrium (29, 30). In the present study, we used site-directed mutagenesis to create IRAK-1 mutants that expressed the S196F, L532S, or the combined S196F/L532S polymorphisms and then transfected IRAK-1 WT and variants into IRAK-1-deficient cells. We found that variant IRAK-1 containing the dual amino acid changes found in the haplotype associated with a higher mortality rate from sepsis has greater ability to induce NF-{kappa}B transcriptional activity, as shown by increased expression of the NF-{kappa}B-dependent genes, IL-8 and I{kappa}B-{alpha}, and greater activation of NF-{kappa}B-dependent luciferase reporter, after stimulation with IL-1. Moreover, variant IRAK-1 mediates more rapid IL-1-induced I{kappa}B-{alpha} degradation and more sustained p65 phosphorylation. We found that variant IRAK-1 has greater autophosphorylation and more interaction with TRAF6 than does WT IRAK-1. However, WT and variant IRAK-1 induce comparable activation of MAPKs in response to IL-1.

Although IRAK-1 is a kinase, its kinase activity appears to be unnecessary for the ability of IRAK-1 to contribute to NF-{kappa}B activation (28, 39). Phosphorylation of IRAK-1 appears to be involved in TLR/IL-1R-associated induction of NF-{kappa}B activity (27). IRAK-1 phosphorylation is mediated by IRAK-4 and also by IRAK-1 itself (21, 22). Additionally, overexpressed IRAK-1 is susceptible to autophosphorylation. We found that variant IRAK-1 has a higher level of phosphorylation in both unstimulated and IL-1-stimulated cells. These differences in IRAK-1 phosphorylation may contribute to the increased association of the variant isoform with TRAF6 and greater NF-{kappa}B activation in cells expressing variant IRAK-1.

Considering that IRAK-4 phosphorylates IRAK-1, it may appear surprising to find that variant IRAK-1 has less interaction with IRAK-4 than does WT IRAK-1. Nevertheless, after phosphorylation by IRAK-4, IRAK-1 and TRAF6 leave IRAK-4 to initiate recruitment of the TAK-1 complex (23, 25, 26). Therefore, less interaction with IRAK-4 is consistent with the greater degree of IRAK-1 phosphorylation found with the variant IRAK-1 isoform, resulting in separation of variant IRAK-1 and TRAF6 from IRAK-4, more rapid recruitment of TAK-1 complex, and eventually a greater activation of NF-{kappa}B.

No differences in nuclear translocation of NF-{kappa}B were found in cells expressing variant as compared with WT IRAK-1 despite the alterations in I{kappa}B-{alpha} degradation present in variant IRAK-1-transfected cells. These findings suggest that the mechanism induced by variant IRAK-1 and responsible for increased expression of NF-{kappa}B-dependent genes in variant IRAK-1-expressing cells does not involve modulation of nuclear accumulation of NF-{kappa}B, but rather other events that affect the transcriptional ability of NF-{kappa}B. A potential mechanism for the ability of variant IRAK-1 to increase NF-{kappa}B-dependent transcription is through enhancing the phosphorylation of the p65 subunit of NF-{kappa}B. Previous studies have demonstrated that phosphorylation of serine 536 in p65 is necessary for optimal transcriptional activity of NF-{kappa}B (34, 35). In particular, p65 phosphorylation at serine 536 appears to increase p65 transcriptional activity by promoting interaction with coactivators, such as CBP, and enhancing DNA-binding activity (34, 35). In the present studies, we found that levels of serine 536-phosphorylated p65 were greater when variant IRAK-1 isoforms, and particularly the L532S isoform, were present, as compared with WT IRAK-1. Activated IKK can phosphorylate p65, so signaling events upstream of IKK phosphorylation and activation that are modulated by variant IRAK-1 could contribute to the enhanced phosphorylation of p65 found in these studies. In particular, autophosphorylation of IRAK-1 and association of IRAK-1 with TRAF6, events required for activating IKK after TLR/IL-1R engagement, were increased in IL-1-stimulated cells expressing variant IRAK-1 as compared with WT IRAK-1.

In our previous study (29), the variant IRAK-1 haplotype was found to be associated with increased LPS-induced NF-{kappa}B nuclear translocation in neutrophils obtained from patients with severe sepsis and acute lung injury, whereas in the present experiments, there were no differences found in IL-1beta-induced nuclear accumulation of NF-{kappa}B in cells expressing variant vs WT IRAK-1. The systemic proinflammatory milieu that accompanied the life-threatening clinical conditions in our previous study may contribute to the apparent differences in nuclear translocation of NF-{kappa}B in the two studies. In particular, patients with severe sepsis often have circulating LPS as well as high levels of proinflammatory cytokines that are capable of priming neutrophils to respond with increased activation after a second exposure to LPS, and resulting in higher NF-{kappa}B nuclear translocation (40, 41). An increased proinflammatory environment, as a result of variant IRAK-1-induced proinflammatory cytokine production, may directly contribute to such neutrophil priming. In addition, although the IRAK-1-deficient HEK-293 cell line is a widely used model to study TLR/IL-1R signaling, there may be differences in intensity and regulation of NF-{kappa}B-related signaling compared with that in myeloid cells, such as neutrophils.

The variant IRAK-1 haplotype contains five intron and three exon polymorphisms in complete linkage disequilibrium (29). In this study, we only examined the activity of variant IRAK-1 arising from the two nonsynonymous polymorphisms within exons that lead to amino acid changes. Although it is likely that the alterations in protein structure produced by these two nonsynonymous SNPs are the major factor responsible for the greater ability of the IRAK-1 variant to stimulate NF-{kappa}B activity and presumably to contribute to a greater inflammatory response associated with sepsis-induced mortality and organ failure, it is possible that SNPs within introns may still play a role in modulating IRAK-1 activity by affecting mRNA splicing. Thus, it may be worthwhile in the future to investigate whether there are additional IRAK-1 isoforms associated with the variant IRAK-1 haplotype that are even more active in mediating TLR/IL-1R-induced NF-{kappa}B activation in human populations.


   Acknowledgments
 
We thank Dr. Xiaoxia Li for the HEK-293 I1A cell line, and Michael Martin for his critical reading of this manuscript and multiple useful suggestions.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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 This work was supported by National Institutes of Health Grants HL62221 and HL068743. Back

2 Address correspondence and reprint requests to Dr. Edward Abraham, Department of Medicine, University of Alabama, School of Medicine, 420 Boshell Building, 1808 7th Avenue South, Birmingham, AL 35294. E-mail address: eabraham{at}uab.edu Back

3 Abbreviations used in this paper: TIR, TLR- and IL-1R-related; CBP, CREB-binding protein; HEK, human embryonic kidney; IKK, I{kappa}B kinase; IRAK, IL-1R-associated kinase; SNP, single nucleotide polymorphism; TAB, TAK1-binding protein; TAK, TGF-beta-activated kinase; TK, thymidine kinase; TRAF, TNFR-associated factor; WT, wild type. Back

Received for publication January 9, 2007. Accepted for publication June 29, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Hotchkiss, R. S., I. E. Karl. 2003. The pathophysiology and treatment of sepsis. N. Engl. J. Med. 348: 138-150. [Free Full Text]
  2. Dunn, D. L.. 1994. Gram-negative bacterial sepsis and sepsis syndrome. Surg. Clin. North Am. 74: 621-635. [Medline]
  3. Guillou, P. J.. 1993. Biological variation in the development of sepsis after surgery or trauma. Lancet 342: 217-220. [Medline]
  4. Natanson, C., W. D. Hoffman, A. F. Suffredini, P. Q. Eichacker, R. L. Danner. 1994. Selected treatment strategies for septic shock based on proposed mechanisms of pathogenesis. Ann. Intern. Med. 120: 771-783. [Abstract/Free Full Text]
  5. Parrillo, J. E.. 1993. Pathogenetic mechanisms of septic shock. N. Engl. J. Med. 328: 1471-1477. [Free Full Text]
  6. Liu, S. F., A. B. Malik. 2006. NF-{kappa}B activation as a pathological mechanism of septic shock and inflammation. Am. J. Physiol. 290: L622-L645.
  7. Perl, M., C. S. Chung, M. Garber, X. Huang, A. Ayala. 2006. Contribution of anti-inflammatory/immune suppressive processes to the pathology of sepsis. Front. Biosci. 11: 272-299. [Medline]
  8. Arnalich, F., E. Garcia-Palomero, J. Lopez, M. Jimenez, R. Madero, J. Renart, J. J. Vazquez, C. Montiel. 2000. Predictive value of nuclear factor {kappa}B activity and plasma cytokine levels in patients with sepsis. Infect. Immun. 68: 1942-1945. [Abstract/Free Full Text]
  9. Bohrer, H., F. Qiu, T. Zimmermann, Y. Zhang, T. Jllmer, D. Mannel, B. W. Bottiger, D. M. Stern, R. Waldherr, H. D. Saeger, et al 1997. Role of NF{kappa}B in the mortality of sepsis. J. Clin. Invest. 100: 972-985. [Medline]
  10. Moine, P., R. McIntyre, M. D. Schwartz, D. Kaneko, R. Shenkar, Y. Le Tulzo, E. E. Moore, E. Abraham. 2000. NF-{kappa}B regulatory mechanisms in alveolar macrophages from patients with acute respiratory distress syndrome. Shock 13: 85-91. [Medline]
  11. Schwartz, M. D., E. E. Moore, F. A. Moore, R. Shenkar, P. Moine, J. B. Haenel, E. Abraham. 1996. Nuclear factor-{kappa}B is activated in alveolar macrophages from patients with acute respiratory distress syndrome. Crit. Care Med. 24: 1285-1292. [Medline]
  12. Yang, K. Y., J. J. Arcaroli, E. Abraham. 2003. Early alterations in neutrophil activation are associated with outcome in acute lung injury. Am. J. Respir. Crit. Care Med. 167: 1567-1574. [Abstract/Free Full Text]
  13. Akira, S., S. Uematsu, O. Takeuchi. 2006. Pathogen recognition and innate immunity. Cell 124: 783-801. [Medline]
  14. Barton, G. M., R. Medzhitov. 2003. Toll-like receptor signaling pathways. Science 300: 1524-1525. [Abstract/Free Full Text]
  15. O’Neill, L. A.. 2000. The interleukin-1 receptor/Toll-like receptor superfamily: signal transduction during inflammation and host defense. Sci. STKE 2000: RE1
  16. Yamamoto, M., K. Takeda, S. Akira. 2004. TIR domain-containing adaptors define the specificity of TLR signaling. Mol. Immunol. 40: 861-868. [Medline]
  17. Janssens, S., R. Beyaert. 2003. Functional diversity and regulation of different interleukin-1 receptor-associated kinase (IRAK) family members. Mol. Cell 11: 293-302. [Medline]
  18. Burns, K., J. Clatworthy, L. Martin, F. Martinon, C. Plumpton, B. Maschera, A. Lewis, K. Ray, J. Tschopp, F. Volpe. 2000. Tollip, a new component of the IL-1RI pathway, links IRAK to the IL-1 receptor. Nat. Cell Biol. 2: 346-351. [Medline]
  19. Burns, K., F. Martinon, C. Esslinger, H. Pahl, P. Schneider, J. L. Bodmer, F. Di Marco, L. French, J. Tschopp. 1998. MyD88, an adapter protein involved in interleukin-1 signaling. J. Biol. Chem. 273: 12203-12209. [Abstract/Free Full Text]
  20. Wesche, H., W. J. Henzel, W. Shillinglaw, S. Li, Z. Cao. 1997. MyD88: an adapter that recruits IRAK to the IL-1 receptor complex. Immunity 7: 837-847. [Medline]
  21. Kollewe, C., A. C. Mackensen, D. Neumann, J. Knop, P. Cao, S. Li, H. Wesche, M. U. Martin. 2004. Sequential autophosphorylation steps in the interleukin-1 receptor-associated kinase-1 regulate its availability as an adapter in interleukin-1 signaling. J. Biol. Chem. 279: 5227-5236. [Abstract/Free Full Text]
  22. Lye, E., C. Mirtsos, N. Suzuki, S. Suzuki, W. C. Yeh. 2004. The role of interleukin 1 receptor-associated kinase-4 (IRAK-4) kinase activity in IRAK-4-mediated signaling. J. Biol. Chem. 279: 40653-40658. [Abstract/Free Full Text]
  23. Qian, Y., M. Commane, J. Ninomiya-Tsuji, K. Matsumoto, X. Li. 2001. IRAK-mediated translocation of TRAF6 and TAB2 in the interleukin-1-induced activation of NF{kappa}B. J. Biol. Chem. 276: 41661-41667. [Abstract/Free Full Text]
  24. Takaesu, G., J. Ninomiya-Tsuji, S. Kishida, X. Li, G. R. Stark, K. Matsumoto. 2001. Interleukin-1 (IL-1) receptor-associated kinase leads to activation of TAK1 by inducing TAB2 translocation in the IL-1 signaling pathway. Mol. Cell. Biol. 21: 2475-2484. [Abstract/Free Full Text]
  25. Jefferies, C., A. Bowie, G. Brady, E. L. Cooke, X. Li, L. A. O’Neill. 2001. Transactivation by the p65 subunit of NF-{kappa}B in response to interleukin-1 (IL-1) involves MyD88, IL-1 receptor-associated kinase 1, TRAF-6, and Rac1. Mol. Cell. Biol. 21: 4544-4552. [Abstract/Free Full Text]
  26. Jiang, Z., J. Ninomiya-Tsuji, Y. Qian, K. Matsumoto, X. Li. 2002. Interleukin-1 (IL-1) receptor-associated kinase-dependent IL-1-induced signaling complexes phosphorylate TAK1 and TAB2 at the plasma membrane and activate TAK1 in the cytosol. Mol. Cell. Biol. 22: 7158-7167. [Abstract/Free Full Text]
  27. Li, X., M. Commane, Z. Jiang, G. R. Stark. 2001. IL-1-induced NF{kappa}B and c-Jun N-terminal kinase (JNK) activation diverge at IL-1 receptor-associated kinase (IRAK). Proc. Natl. Acad. Sci. USA 98: 4461-4465. [Abstract/Free Full Text]
  28. Li, X., M. Commane, C. Burns, K. Vithalani, Z. Cao, G. R. Stark. 1999. Mutant cells that do not respond to interleukin-1 (IL-1) reveal a novel role for IL-1 receptor-associated kinase. Mol. Cell. Biol. 19: 4643-4652. [Abstract/Free Full Text]
  29. Arcaroli, J., E. Silva, J. P. Maloney, Q. He, D. Svetkauskaite, J. R. Murphy, E. Abraham. 2006. Variant IRAK-1 haplotype is associated with increased nuclear factor-{kappa}B activation and worse outcomes in sepsis. Am. J. Respir. Crit. Care Med. 173: 1335-1341. [Abstract/Free Full Text]
  30. Ishida, R., M. Emi, Y. Ezura, H. Iwasaki, H. Yoshida, T. Suzuki, T. Hosoi, S. Inoue, M. Shiraki, H. Ito, H. Orimo. 2003. Association of a haplotype (196Phe/532Ser) in the interleukin-1-receptor-associated kinase (IRAK1) gene with low radial bone mineral density in two independent populations. J. Bone Miner. Res. 18: 419-423. [Medline]
  31. Furia, B., L. Deng, K. Wu, S. Baylor, K. Kehn, H. Li, R. Donnelly, T. Coleman, F. Kashanchi. 2002. Enhancement of nuclear factor-{kappa}B acetylation by coactivator p300 and HIV-1 Tat proteins. J. Biol. Chem. 277: 4973-4980. [Abstract/Free Full Text]
  32. Kiernan, R., V. Bres, R. W. Ng, M. P. Coudart, S. El Messaoudi, C. Sardet, D. Y. Jin, S. Emiliani, M. Benkirane. 2003. Post-activation turn-off of NF-{kappa}B-dependent transcription is regulated by acetylation of p65. J. Biol. Chem. 278: 2758-2766. [Abstract/Free Full Text]
  33. Viatour, P., M. P. Merville, V. Bours, A. Chariot. 2005. Phosphorylation of NF-{kappa}B and I{kappa}B proteins: implications in cancer and inflammation. Trends Biochem. Sci. 30: 43-52. [Medline]
  34. Zhong, H., M. J. May, E. Jimi, S. Ghosh. 2002. The phosphorylation status of nuclear NF-{kappa}B determines its association with CBP/p300 or HDAC-1. Mol. Cell 9: 625-636. [Medline]
  35. Zhong, H., R. E. Voll, S. Ghosh. 1998. Phosphorylation of NF-{kappa}B p65 by PKA stimulates transcriptional activity by promoting a novel bivalent interaction with the coactivator CBP/p300. Mol. Cell 1: 661-671. [Medline]
  36. Abraham, E.. 2003. Nuclear factor-{kappa}B and its role in sepsis-associated organ failure. J. Infect. Dis. 187: (Suppl. 2):S364-S369. [Medline]
  37. Akira, S.. 2003. Toll-like receptor signaling. J. Biol. Chem. 278: 38105-38108. [Free Full Text]
  38. Cao, Z., J. Xiong, M. Takeuchi, T. Kurama, D. V. Goeddel. 1996. TRAF6 is a signal transducer for interleukin-1. Nature 383: 443-446. [Medline]
  39. Knop, J., M. U. Martin. 1999. Effects of IL-1 receptor-associated kinase (IRAK) expression on IL-1 signaling are independent of its kinase activity. FEBS Lett. 448: 81-85. [Medline]
  40. Marshall, J. C., D. Foster, J. L. Vincent, D. J. Cook, J. Cohen, R. P. Dellinger, S. Opal, E. Abraham, S. J. Brett, T. Smith, et al 2004. Diagnostic and prognostic implications of endotoxemia in critical illness: results of the MEDIC study. J. Infect. Dis. 190: 527-534. [Medline]
  41. Vincent, J. L., E. Abraham. 2006. The last 100 years of sepsis. Am. J. Respir. Crit. Care Med. 173: 256-263. [Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Am. J. Respir. Cell Mol. Bio.Home page
E. Lorne, X. Zhao, J. W. Zmijewski, G. Liu, Y.-J. Park, Y. Tsuruta, and E. Abraham
Participation of Mammalian Target of Rapamycin Complex 1 in Toll-Like Receptor 2- and 4-Induced Neutrophil Activation and Acute Lung Injury
Am. J. Respir. Cell Mol. Biol., August 1, 2009; 41(2): 237 - 245.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
W. Song, G. Liu, C. A. Bosworth, J. R. Walker, G. A. Megaw, A. Lazrak, E. Abraham, W. M. Sullender, and S. Matalon
Respiratory Syncytial Virus Inhibits Lung Epithelial Na+ Channels by Up-regulating Inducible Nitric-oxide Synthase
J. Biol. Chem., March 13, 2009; 284(11): 7294 - 7306.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Liu, G.
Right arrow Articles by Abraham, E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Liu, G.
Right arrow Articles by Abraham, E.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS