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J. Biol. Chem., Vol. 279, Issue 49, 51697-51703, December 3, 2004
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From the Department of Medicine, Wake Forest University School of Medicine, Winston Salem, North Carolina 27157
Received for publication, September 9, 2004 , and in revised form, September 27, 2004.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B activation. Yet IRAK1 knock-out mice still exhibit NFB activation upon lipopolysaccharide (LPS) challenge, suggesting that IRAK1 may play other un-characterized function. In this report, we show that IRAK1 is mainly involved in Stat3 activation and subsequent interleukin-10 (IL-10) gene expression. Splenocytes from IRAK1-deficient mice fail to exhibit LPS-induced Stat3 serine phosphorylation and IL-10 gene expression yet still maintain normal IL-1
gene expression upon LPS challenge. Mechanistically, we observe that IRAK1 modification upon LPS challenge leads to its modification, nuclear distribution, and interaction with Stat3. IRAK1 can directly use Stat3 as a substrate and cause Stat3 serine 727 phosphorylation. In addition, nuclear IRAK1 binds directly with IL-10 promoter in vivo upon LPS treatment. Atherosclerosis patients usually have elevated serum IL-10 levels. We document here that IRAK1 is constitutively modified and localized in the nucleus in the peripheral blood mononuclear cells from atherosclerosis patients. These observations reveal the mechanism for the novel role of IRAK1 in the complex Toll-like receptor signaling network and indicate that IRAK1 regulation may be intimately linked with the pathogenesis and/or resolution of atherosclerosis. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B (NFB), activator protein-1 (AP-1), interferon regulatory factors, and signal transducers and activators of transcription (Stats) are shown to be activated upon challenges with various TLR ligands (4). The mechanism for the selective activation of distinctive transcription factor is not clearly understood and may be caused by the differential recruitment of intracellular adaptor molecules such as MyD88, Mal/TIRAP, TRIF, and TRAM as well as the downstream IRAK kinases (4).
IRAK1 was the first IRAK family kinase being identified to be associated with the intracellular domain of IL-1 receptor (5). Since TLRs share the TIR domain with IL-1 receptor, it was hypothesized that IRAK1 may also participate in TLR mediated signaling. Subsequent work (6-9) including ours have confirmed that indeed various TLR ligands can activate endogenous IRAK1 kinase activation. Biochemically, we and others (7, 10, 11) have shown that IRAK1 undergoes covalent modification likely due to phosphorylation and ubiquitination upon IL-1 or LPS challenge. IRAK1 can form a complex with MyD88, as well as TRAF6 (6). Since the most apparent downstream target of LPS signaling is the activation of NFB, IRAK1 is the apparent candidate to fulfill such role. Therefore, IRAK1 has historically been linked with IL-1/LPS-mediated NFB activation. Yet the majority of published evidence supporting the role of IRAK1 in mediating IL-1/LPS-induced NFB activation has been derived from studies employing cell lines with IRAK1 overexpression (12-14). Upon overexpression, both the wild type and the kinase-dead IRAK1 (which has a point mutation in the ATP-binding pocket (K239S) or in the catalytic site (D340N)) can strongly induce NFB reporter activation (12, 14). The fact that despite being an active kinase, its kinase activity is not required for its function raises the concern that IRAK1 may perform other novel unidentified function besides activating NFB.
Three related IRAK genes have been later identified, namely IRAK2, IRAK-M, and IRAK4 (13, 15, 16). All IRAKs consist of a conserved N-terminal death domain and a central kinase domain. Upon overexpression, each of these IRAKs can activate the NFB reporter gene, suggesting that they may play redundant roles in activating NFB (13, 17). However, studies using transgenic mice have indicted otherwise. So far, IRAK1-/-, IRAK4-/-, and IRAK-M-/- transgenic mice have been generated. Mice with IRAK4 disruption exhibit marked reduction in NFB activation upon LPS challenge (18). Furthermore, sequence comparison with the fly IRAK counterpart pelle kinase suggests that IRAK4 is the structural orthologue of fly pelle kinase (16). These studies and analyses indicate that IRAK4 is the default kinase responsible for activating NFB. In contrast, deletion of IRAK-M was shown to lead to elevated NFB activity and pro-inflammatory gene expression such as tumor necrosis factor 
, indicating that IRAK-M may negatively regulate NFB activation (19). Intriguingly, IRAK1-deficient mice still retain LPS-induced NFB activation (20), suggesting that IRAK1 may rather fulfill other distinct yet unidentified function in LPS/TLR signaling. Besides NFB activation, TLR ligands such as LPS can also activate other transcription factors such as interferon regulatory factors and Stats (21, 22). Whether and which particular IRAK participates in the activation of interferon regulatory factors and/or Stats is not clear.
In this study, we have characterized the gene expression pattern of murine splenocytes from wild type and IRAK1-deficient mice. We have found that IL-10 induction is severely compromised in IRAK1-deficient cells upon LPS challenge. In contrast, IL-1 gene expression is induced to a similar extent. Since Stat3 has been shown to be critical for IL-10 gene expression, we have further studied the Stat3 activation and phosphorylation status. We have observed that IRAK1-deficient cells exhibit defective nuclear Stat3 serine 727 phosphorylation. Furthermore, we have shown that LPS induces IRAK1 modification and nuclear localization. In addition, nuclear IRAK1 interacts with Stat3 as well as endogenous IL-10 promoter element upon LPS treatment.
IL-10 is undetectable in normal healthy human sera or blood cells. However, under many pathological circumstances such as atherosclerosis, IL-10 message and protein can be readily detectable in the sera (23). The production of IL-10 may help alleviate excessive inflammation and therefore be beneficial for the resolution of atherosclerosis. We have examined the IRAK1 status in the peripheral blood mononuclear cells (PBMC) obtained from healthy and atherosclerosis patients. Our study shows that IRAK1 is constitutively modified and localizes inside the nucleus in atherosclerosis patient blood monocuclear cells (PBMC). Our finding reveals a novel role of IRAK1 in specifically mediating LPS-induced Stat3 activation and IL-10 expression.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mice—C57BL/6 wild type mice were purchased from the Charles River laboratory. IRAK-deficient mice were a kind gift from Dr. James Thomas from the University of Texas Southwestern Medical School. These mice were bred and maintained in the animal facility at the Wake Forest University School of Medicine with the approved Animal Care and Use Committee protocol. All mice were 7-10 weeks of age when experiments were initiated. Splenocytes were harvested as described (24).
Human Blood PBMC Isolation and Culture—Blood was drawn from healthy donors and atherosclerosis patients undergoing percutaneous coronary interventions at the Wake Forest University Medical Center with an approved protocol. PBMC were separated by Ficoll density gradient as described (25). Isolated PBMC were washed twice with phosphate-buffered saline and incubated in RPMI 1640, supplemented with 10% (v/v) fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 2 mM glutamine.
RNA Isolation and Real-time Quantitative PCR—Total cellular RNAs were extracted using the TRIzol reagent (Invitrogen) (26). Isolated total RNAs were reverse-transcribed using the QmniscriptTM RT kit (Qiagen). Quantitative real-time PCR analyses of IL-10, IL-1, as well as the control GAPDH mRNA transcripts were carried out using the assay-on-demandTM gene-specific fluorescently labeled TaqMan MGB probe in an ABI Prism 7000 sequence detection system (Applied Biosystems).
Tansient Transfection and Luciferase Reporter Activity Assay—HeLa-MAT cells stably expressing TLR4/MD2 (27) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 2 mM glutamine. A total of 2 x 105 cells were co-transfected using DMRIE-C reagent (Invitrogen) with the pIL-10Luci reporter plasmid (0.2 µg) and 1 µg of either wild type Stat3, Stat3-S727A, or Stat3-Y705A mutant plasmids (kindly provided by Dr. James Darnell, Rockefeller University). Twenty-four hours after transfection, cells were stimulated with LPS at 500 ng/ml. Four hours later cells were harvested, and the lyses were used to perform luciferase assay (26).
In Vitro Transcription/Translation and Phosphorylation Assay of Stat3—Wild type IRAK1 and wild type and Stat3-S727A mutant proteins were synthesized using the pflag-IRAK1, wild type Stat3, and Stat3-S727A mutant plasmids with the Promega TNT quick-coupled transcription/translation system (Promega). In vitro synthesized IRAK1 was incubated with either Stat3 or Stat3-S727A protein at 37 °C for 30 min in 50 µl of kinase buffer (20 mM HEPES, pH 7.6, 20 mM MgCl2, 20 mM -glycerophosphate, 20 mM para-nitrophenylphosphate, 1mM EDTA, 1 mM sodium orthovanadate, 1 mM benzamidine). Reaction products were separated on SDS-PAGE and transferred to polyvinylidene difluoride membranes. The phosphorylated as well as total Stat3 proteins were visualized by Western blot using antibodies specific for either Stat3-S727P or total Stat3 protein.
Isolation of Cytoplasmic and Nuclear Extracts, Immunoprecipitation, and Western Blot—Cell lysis, isolation of total, cytoplasmic, and nuclear extracts were as described previously (28). Briefly, various cells (5 x 106/ml) were washed in 10 mM HEPES, pH 7.9, and subsequently lysed on ice in the lysis buffer (10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml pepstatin). After centrifugation for 3 min at 12,000 rpm, the supernatant cytoplasmic fractions were transferred and saved. Pellets containing intact nuclei were lysed and solubilized with the high salt buffer (20 mM HEPES, pH 7.9, 1.5 mM MgCl2, 0.4 M NaCl, 0.2 mM EDTA, 0.5 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride) for 30 min and yielded the nuclear extracts. Immunoprecipitation and Western detection of corresponding proteins were performed as described (7).
Chromatin Immunoprecipitation Assay—Fresh and LPS-stimulated cells were fixed by adding formaldehyde (HCHO, from a 37% HCHO, 10% methanol stock (Calbiochem) into the medium for a final formaldehyde concentration of 1% and incubated at room temperature for 10 min with gentle shaking. Incubation with the lysis buffer was extended to 20 min at 4 °C. The chromatin was sheared by sonication using a Branson 250 sonicator with microtip at a power setting of 2 and 40% duty cycle. The samples were placed on ice for 1 min between sonication bursts. Each condition was divided into two samples, providing a preimmunoprecipitation or "input" sample that was not incubated with specific antibodies, and an immunoprecipitated "IP" sample that was incubated overnight with antibodies specific for IRAK1 (Upstate Biotechnology, Lake Placid, NY). Preimmune IgG was used as a negative control. DNA was isolated by phenol/chloroform extraction, ethanol-precipitated, and re-suspended in 20 µl of distilled H2O. 4 µl of immunoprecipitated DNA was used for each PCR. The following primers specific for the IL-10 promoter -300- to -60-bp region were used: 5' CAG CTG TTC TCC CCA GGA AA 3' and 5' AGG GAG GCC TCT TCA TTC AT 3'. PCR products were separated on 1% agarose gel. The amplified band was visualized using Bio-Rad Gel DocTM.
Data Analysis—The significance of the data was evaluated by means of one-factor analysis of variance followed by Student-Newman-Keuls test using SPSS 10.0 software. A p value <0.05 was considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B such as IL-1 and tumor necrosis factor were induced to similar levels by LPS in both wild type and IRAK1-deficient splenocytes. In contrast, we identified that IL-10 message was only induced by LPS in wild type, but not IRAK1-deficient, splenocytes.
To confirm the microarray data, we performed real-time PCR analysis of the induced IL-10 as well as IL-1 messages. Total RNAs isolated from wild type and IRAK1-deficient mice with or without LPS treatment were reverse-transcribed into cDNAs and subsequently subjected to real-time PCR analysis using the assay-on-demand IL-10, IL-1, and the control GAPDH primer sets purchased from Applied Biosystems. GAPDH message levels remain steady in all the samples assayed. IL-10 message levels were undetectable after 40 cycles of amplifications in resting splenocytes from wild type as well as IRAK1-deficient mice. Upon LPS challenge, there was significant induction of IL-10 message in the wild type splenocytes (Fig. 1a). Based on the comparative Ct method, the induction of IL-10 by LPS was greater than at least 30-fold. In contrast, there was no statistically significant difference between IL-10 message levels in resting and LPS-treated splenocytes from IRAK1-deficient mice (Fig. 1a). On the other hand, we observed that IL-1 message levels were induced to a similar extent in wild type and IRAK1-deficient splenocytes by LPS (Fig. 1b). The amplified IL-10, IL-1, and GAPDH mRNAs were also resolved and visualized on agarose gel (Fig. 1c). Subsequently, we measured IL-10 protein levels by ELISA. Wild type instead of IRAK1-deficient splenocytes produced a significant amount of IL-10 protein upon 16-h LPS treatment (Fig. 1d).
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IRAK1 and Stat3 Form a Novel Complex in the Nucleus—IRAK1 is known to undergo modification such as phosphorylation and ubiquitination upon IL-1 and/or LPS challenge. Intriguingly, it has been noted that IL-1 treatment may induce IRAK1 to localize inside the nucleus (30). To further determine the molecular mechanism for IRAK-mediated Stat3 activation, we analyzed IRAK1 protein status upon LPS challenge. Wild type splenocytes treated with or without LPS were harvested and used to prepare whole cell extract, cytoplasmic extract, as well as nuclear extract as described under "Materials and Methods." The purities of prepared extracts were confirmed by probing for the presence of cytoplasmic and nuclear specific proteins. As shown in Fig. 4a, GAPDH was only detectable in the cytoplasmic extract, while Lamin-B was only visible in the nuclear extract. -Actin was visualized to show equal loading of corresponding extracts. Equal amounts of isolated cellular extracts were subsequently used to immunoprecipitate IRAK1 protein. Immunoprecipitated IRAK1 was subjected to SDS-PAGE and Western blotted with anti-IRAK1 antibody. As shown in Fig. 4b, LPS challenge caused a major shift of IRAK1 migration from 
85 to 100 kDa, corresponding to the covalent IRAK1 modification likely due to ubiquitination and phosphorylation as reported by others (12). We then examined the subcellular distribution of IRAK1 upon LPS challenge. Strikingly, as shown in Fig. 4b, unmodified IRAK1 (85 kDa) was only detectable in the cytoplasmic extract and completely absent in the nuclear extract. In contrast, modified IRAK1 was primarily present in the nuclear extract (Fig. 4b).
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Modified IRAK1 Resides in the Nuclear Fraction of the Human Peripheral Blood Mononuclear Cells as Well as the Monocytic THP-1 Cells—We subsequently examined the IRAK1 status in human primary blood mononuclear cells (PBMC) as well as the THP-1 cells. As shown in Fig. 5, IRAK1 underwent modification upon LPS treatment in THP-1 cells (Fig. 5a) as well as in human primary PBMC (Fig. 5b). Furthermore, modified IRAK1 primarily localized in the nuclear fraction. Similar to the finding observed in mice splenocytes, modified IRAK1 formed a complex with the endogenous Stat3 in the nucleus upon LPS challenge (Fig. 5, a and b).
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5 pg/ml). In contrast, there was no detectable IL-10 in healthy human sera. We further isolated PBMC from healthy as well as patient blood. The expression levels of IL-10 protein were determined by ELISA. As shown in Fig. 7a, PBMC from patients constitutively secreted IL-10 protein. Based on our finding that IRAK1 plays a critical role in the induction of IL-10, we therefore compared the IRAK1 status in the PBMC from healthy donors and atherosclerosis patients. As shown in Fig. 7b, IRAK1 primarily exists as the modified 100-kDa form in the patient PBMC. We further obtained the cytoplasmic and nuclear extracts from patient PBMC and analyzed the distribution of IRAK1. IRAK1 protein was immunoprecipitated from the prepared extracts and separated on SDS-PAGE. The presence of IRAK1 was subsequently monitored through Western blot using anti-IRAK1 antibody. As shown in Fig. 7c, the modified IRAK1 protein was constitutively migrated as the modified 100-kDa form and resided exclusively in the nuclear fraction from the patient PBMC.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This report has revealed some novel biochemical aspects of IRAK1 protein regulation. It is known that IRAK1 undergoes covalent modification such as phosphorylation and ubiquitination upon IL-1 and/or LPS challenge (7, 10, 12). Its modification may eventually lead to its subsequent degradation (11). Using human THP-1 cells, primary blood mononuclear cells, as well as mice splenocytes, we have confirmed numerous previous studies that there are indeed two signature forms of IRAK1, one being the unmodified 85-kDa form and the other being the modified (phosphorylated and/or ubiquitinated) 100-kDa form. Consistent with previous studies, upon LPS challenge, we have observed that the level of the modified IRAK1 form increases, while the unmodified form decreases. With regard to its subcellular distribution, there is only one published report showing that IL-1 stimulation may lead to IRAK1 nuclear localization (30). Yet this report has been largely ignored in the innate immunity field. In this study, we further analyzed the distribution of IRAK1 in the fractionated cellular and nuclear extracts. Strikingly, the majority of the 85-kDa form exists in the cytoplasm fraction. In sharp contrast, the modified IRAK1 is mainly present in the nucleus. We have also noticed from the previous study (30) reporting IRAK1 nuclear localization that the apparent molecular mass of nuclear IRAK1 is 100 kDa, consistent with our present finding. However, the authors of that report did not elaborate in their publication the different migration patterns of nuclear and cytoplasmic IRAK1 (30). The fact that the modified IRAK1 primarily appears inside the nucleus suggests that LPS-induced IRAK1 modification such as phosphorylation and/or ubiquitination may be critical for its trafficking into the nucleus and its subsequent function in activating IL-10 gene expression. Indeed increasing evidence has shown that protein modification such as ubiquitination and/or sumoylation are critical for subcellular trafficking as well as signal transduction besides proteasome-mediated degradation (32). Further biochemical work is needed to elucidate the exact mechanism for LPS-induced IRAK1 modification and its subsequent function.
Functionally, our findings serve to clarify the elusive role of IRAK1 in LPS-mediated innate immunity signaling. As described in the Introduction, although overexpression of IRAK1 may lead to NFB reporter activation, studies using IRAK1-deficient mice have indicated that LPS can still induce NFB activation in IRAK1-deficient cells (20). Therefore, IRAK1 may perform other unidentified roles besides NFB activation. Our data presented herein unveil that IRAK1 is critically involved in the nuclear Stat3 serine phosphorylation and subsequent IL-10 gene expression. It has been documented that Stat3 is responsible for increased IL-10 gene expression upon LPS challenge (22). LPS can induce Stat3 phosphorylation at both serine 727 and tyrosine 705 residues (22), and Stat3 phosphorylation at both Tyr705 and Ser727 is critical for its maximum transcriptional activity (29). Our observation herein concurs with these studies. Overexpression of Stat3 with serine 727 to alanine mutation or tyrosine 705 to phenylalanine mutation fails to mediate LPS-induced IL-10 gene reporter activity (Fig. 2). Janus kinase 3 (JAK3) has been shown to be the main kinase responsible for Stat3 tyrosine phosphorylation (33). To date, the mechanism for LPS-induced Stat3 serine 727 phosphorylation is not clear. Consistent with previous reports, we have documented that LPS can induce Stat3 Ser727 and Tyr705 phosphorylation in wild type murine splenocytes. Strikingly, we have observed that although LPS-induced Tyr727 phosphorylation is normal, LPS-induced Stat3 Ser727 phosphorylation is greatly compromised in IRAK1-deficient splenocytes (Fig. 3). Furthermore, we have observed that nuclear Stat3 serine phosphorylation is completely absent in IRAK1-deficient splenocytes. In contrast, there is an increase in nuclear Stat3 serine phosphorylation in wild type splenocytes upon LPS challenge. The increased Stat3 serine phosphorylation inside the nucleus upon LPS challenge correlates well with our observation that LPS facilitates IRAK1 and Stat3 interaction inside the nucleus. We have documented in this report such interaction using mice splenocytes, human THP-1 cells, as well as human peripheral blood mononuclear cells. Furthermore, our in vitro analyses indicate that IRAK1 can directly use Stat3 as its substrate and induce Stat3 serine 727 phosphorylation. This is the first evidence revealing the biological substrate of IRAK1. Taken together, our data indicate that IRAK1 is essential for Stat3 serine phosphorylation inside the nucleus.
Although our study agrees with others that Stat3 phosphorylation is critical for IL-10 gene expression, there is another novel aspect of our present finding. It was thought that upon stimulation with various ligands, Stat3 is phosphorylated and then enters the nucleus (34). Our data consistently indicate that Stat3 is constitutively localized in the cytoplasm as well as the nucleus. LPS stimulation induced a dramatic increase of nuclear Stat3 serine 727 phosphorylation in the wild type splenocytes without affecting the total Stat3 protein levels in the cytoplasm and nucleus. Therefore, the function of Stat3 serine phosphorylation may not be involved in the Stat3 nuclear entry but rather is critical for LPS-induced IL-10 transcriptional regulation inside the nucleus.
Besides activating Stat3, which is critical for IL-10 gene expression, our study also reveals the intriguing phenomenon that IRAK1 may directly serve as a transcriptional regulator for IL-10 gene transcription. Using the Chip assay, our present study presents compelling evidence showing that endogenous nuclear IRAK1 can specifically bind with the IL-10 promoter element in vivo upon LPS challenge (Fig. 6). Further detailed studies are warranted to decipher the mechanism for IRAK1 binding with the IL-10 promoter element and subsequent regulation of IL-10 gene expression.
IL-10 expression is completely absent in healthy human blood cells. However, it has long been noticed that IL-10 levels are elevated in the blood sera of atherosclerosis patients. Elevated IL-10 levels may be a self-protective mechanism preventing excessive inflammation and limiting the progression of atherosclerosis. The mechanism for the increased IL-10 gene expression is not clear. Our data indicate that IRAK1 protein in the atherosclerosis patient blood samples is constitutively modified and localized in the nucleus. Elevated IRAK1 modification and nuclear localization may lead to elevated IL-10 gene expression.
Taken together, the data presented in this study provide a novel role for the IRAK1 molecule in activating Stat3 and contributing to IL-10 gene expression and dispel the notion that IRAK1 primarily serves a redundant role in activating NFB. Furthermore, constitutive IRAK1 modification and nuclear localization may be intimately linked with either the progression or resolution of atherosclerosis.
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FOOTNOTES |
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This article was selected as a Paper of the Week.
To whom correspondence should be addressed: Dept. of Medicine, Wake Forest University School of Medicine, Winston Salem, NC 27157. Tel.: 336-716-6040; Fax: 336-716-1214; E-mail: lwli{at}wfubmc.edu.
1 The abbreviations used are: TLR, Toll-like receptor; Stat, signal transducer and activator of transcription; IL, interleukin; IRAK, IL-1 receptor-associated kinase; LPS, lipopolysaccharide; PBMC, peripheral blood mononuclear cells; ELISA, enzyme-linked immunosorbent assay; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Chip, chromatin immunoprecipitation assay.
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ACKNOWLEDGMENTS |
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REFERENCES |
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