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• W2014120138 abstract "Being one of the key kinases downstream of Toll-like receptors, IRAK1 has initially thought to be responsible for NFκB activation. Yet IRAK1 knock-out mice still exhibit NFκB 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-1β 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. Being one of the key kinases downstream of Toll-like receptors, IRAK1 has initially thought to be responsible for NFκB activation. Yet IRAK1 knock-out mice still exhibit NFκB 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-1β 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. Innate immunity signaling mediated by Toll-like receptors (TLRs) 1The 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.1The 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. leads to the expression of a wide variety of genes (1Vogel S.N. Fitzgerald K.A. Fenton M.J. Mol. Interv. 2003; 3: 466-477Crossref PubMed Scopus (203) Google Scholar, 2Akira S. Adv. Immunol. 2001; 78: 1-56Crossref PubMed Scopus (290) Google Scholar, 3Aderem A. Crit. Care Med. 2001; 29: S16-S18Crossref PubMed Scopus (95) Google Scholar, 4Beutler B. Hoebe K. Du X. Ulevitch R.J. J. Leukocyte Biol. 2003; 74: 479-485Crossref PubMed Scopus (497) Google Scholar). Numerous transcription factors including nuclear factor-κB (NFκB), 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 (4Beutler B. Hoebe K. Du X. Ulevitch R.J. J. Leukocyte Biol. 2003; 74: 479-485Crossref PubMed Scopus (497) Google Scholar). 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 (4Beutler B. Hoebe K. Du X. Ulevitch R.J. J. Leukocyte Biol. 2003; 74: 479-485Crossref PubMed Scopus (497) Google Scholar).IRAK1 was the first IRAK family kinase being identified to be associated with the intracellular domain of IL-1 receptor (5Cao Z. Henzel W.J. Gao X. Science. 1996; 271: 1128-1131Crossref PubMed Scopus (766) Google Scholar). Since TLRs share the TIR domain with IL-1 receptor, it was hypothesized that IRAK1 may also participate in TLR mediated signaling. Subsequent work (6Medzhitov R. Preston-Hurlburt P. Kopp E. Stadlen A. Chen C. Ghosh S. Janeway Jr., C.A. Mol. Cell. 1998; 2: 253-258Abstract Full Text Full Text PDF PubMed Scopus (1290) Google Scholar, 7Li L. Cousart S. Hu J. McCall C.E. J. Biol. Chem. 2000; 275: 23340-23345Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 8Moors M.A. Li L. Mizel S.B. Infect. Immun. 2001; 69: 4424-4429Crossref PubMed Scopus (77) Google Scholar, 9Jacinto R. Hartung T. McCall C. Li L. J. Immunol. 2002; 168: 6136-6141Crossref PubMed Scopus (140) Google Scholar) including ours have confirmed that indeed various TLR ligands can activate endogenous IRAK1 kinase activation. Biochemically, we and others (7Li L. Cousart S. Hu J. McCall C.E. J. Biol. Chem. 2000; 275: 23340-23345Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 10Yamin T.T. Miller D.K. J. Biol. Chem. 1997; 272: 21540-21547Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar, 11Li X. Commane M. Jiang Z. Stark G.R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4461-4465Crossref PubMed Scopus (142) Google Scholar) 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 (6Medzhitov R. Preston-Hurlburt P. Kopp E. Stadlen A. Chen C. Ghosh S. Janeway Jr., C.A. Mol. Cell. 1998; 2: 253-258Abstract Full Text Full Text PDF PubMed Scopus (1290) Google Scholar). Since the most apparent downstream target of LPS signaling is the activation of NFκB, IRAK1 is the apparent candidate to fulfill such role. Therefore, IRAK1 has historically been linked with IL-1/LPS-mediated NFκB activation. Yet the majority of published evidence supporting the role of IRAK1 in mediating IL-1/LPS-induced NFκB activation has been derived from studies employing cell lines with IRAK1 overexpression (12Li X. Commane M. Burns C. Vithalani K. Cao Z. Stark G.R. Mol. Cell. Biol. 1999; 19: 4643-4652Crossref PubMed Scopus (187) Google Scholar, 13Wesche H. Gao X. Li X. Kirschning C.J. Stark G.R. Cao Z. J. Biol. Chem. 1999; 274: 19403-19410Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar, 14Maschera B. Ray K. Burns K. Volpe F. Biochem. J. 1999; 339: 227-231Crossref PubMed Scopus (79) Google Scholar). 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 NFκB reporter activation (12Li X. Commane M. Burns C. Vithalani K. Cao Z. Stark G.R. Mol. Cell. Biol. 1999; 19: 4643-4652Crossref PubMed Scopus (187) Google Scholar, 14Maschera B. Ray K. Burns K. Volpe F. Biochem. J. 1999; 339: 227-231Crossref PubMed Scopus (79) Google Scholar). 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 NFκB.Three related IRAK genes have been later identified, namely IRAK2, IRAK-M, and IRAK4 (13Wesche H. Gao X. Li X. Kirschning C.J. Stark G.R. Cao Z. J. Biol. Chem. 1999; 274: 19403-19410Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar, 15Muzio M. Ni J. Feng P. Dixit V.M. Science. 1997; 278: 1612-1615Crossref PubMed Scopus (974) Google Scholar, 16Li S. Strelow A. Fontana E.J. Wesche H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5567-5572Crossref PubMed Scopus (530) Google Scholar). All IRAKs consist of a conserved N-terminal death domain and a central kinase domain. Upon overexpression, each of these IRAKs can activate the NFκB reporter gene, suggesting that they may play redundant roles in activating NFκB (13Wesche H. Gao X. Li X. Kirschning C.J. Stark G.R. Cao Z. J. Biol. Chem. 1999; 274: 19403-19410Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar, 17Qin J. Jiang Z. Qian Y. Casanova J.L. Li X. J. Biol. Chem. 2004; 279: 26748-26753Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). 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 NFκB activation upon LPS challenge (18Suzuki N. Suzuki S. Duncan G.S. Millar D.G. Wada T. Mirtsos C. Takada H. Wakeham A. Itie A. Li S. Penninger J.M. Wesche H. Ohashi P.S. Mak T.W. Yeh W.C. Nature. 2002; 416: 750-756Crossref PubMed Scopus (653) Google Scholar). Furthermore, sequence comparison with the fly IRAK counterpart pelle kinase suggests that IRAK4 is the structural orthologue of fly pelle kinase (16Li S. Strelow A. Fontana E.J. Wesche H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5567-5572Crossref PubMed Scopus (530) Google Scholar). These studies and analyses indicate that IRAK4 is the default kinase responsible for activating NFκB. In contrast, deletion of IRAK-M was shown to lead to elevated NFκB activity and pro-inflammatory gene expression such as tumor necrosis factor α, indicating that IRAK-M may negatively regulate NFκB activation (19Kobayashi K. Hernandez L.D. Galan J.E. Janeway Jr., C.A. Medzhitov R. Flavell R.A. Cell. 2002; 110: 191-202Abstract Full Text Full Text PDF PubMed Scopus (1136) Google Scholar). Intriguingly, IRAK1-deficient mice still retain LPS-induced NFκB activation (20Swantek J.L. Tsen M.F. Cobb M.H. Thomas J.A. J. Immunol. 2000; 164: 4301-4306Crossref PubMed Scopus (233) Google Scholar), suggesting that IRAK1 may rather fulfill other distinct yet unidentified function in LPS/TLR signaling. Besides NFκB activation, TLR ligands such as LPS can also activate other transcription factors such as interferon regulatory factors and Stats (21Fitzgerald K.A. Rowe D.C. Barnes B.J. Caffrey D.R. Visintin A. Latz E. Monks B. Pitha P.M. Golenbock D.T. J. Exp. Med. 2003; 198: 1043-1055Crossref PubMed Scopus (919) Google Scholar, 22Benkhart E.M. Siedlar M. Wedel A. Werner T. Ziegler-Heitbrock H.W. J. Immunol. 2000; 165: 1612-1617Crossref PubMed Scopus (216) Google Scholar). 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-1β 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 (23Mallat Z. Heymes C. Ohan J. Faggin E. Leseche G. Tedgui A. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 611-616Crossref PubMed Scopus (251) Google Scholar). 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.MATERIALS AND METHODSReagents—Escherichia coli 0111:B4 LPS was obtained from Sigma. Antibody against IRAK1 was from Upstate Biotechnology (Lake Placid, NY). Antibodies against Stat3, phospho-Stat3(Ser727), and phospho-Stat3(Tyr705) were from Cell Signaling (Beverly, MA). Murine and human IL-10 enzyme-linked immunosorbent assay (ELISA) kits were from BenderMed Systems (Vienna, Austria).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 (24Feldmann K. Sebald W. Knaus P. Eur. J. Immunol. 2002; 32: 1393-1402Crossref PubMed Scopus (3) Google Scholar).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 (25Mueller L.P. Yoza B.K. Neuhaus K. Loeser C.S. Cousart S. Chang M.C. Meredith J.W. Li L. McCall C.E. Shock. 2001; 16: 430-437Crossref PubMed Scopus (19) Google Scholar). 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) (26Li T. Hu J. Li L. Mol. Immunol. 2004; 41: 85-92Crossref PubMed Scopus (82) Google Scholar). Isolated total RNAs were reverse-transcribed using the Qmniscript™ RT kit (Qiagen). Quantitative real-time PCR analyses of IL-10, IL-1β, as well as the control GAPDH mRNA transcripts were carried out using the assay-on-demand™ 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 (27Re F. Strominger J.L. J. Biol. Chem. 2002; 277: 23427-23432Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar) 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 × 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 (26Li T. Hu J. Li L. Mol. Immunol. 2004; 41: 85-92Crossref PubMed Scopus (82) Google Scholar).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 β-glycerophosphate, 20 mmpara-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 (28Gomez-Angelats M. Cidlowski J.A. Cell Death Differ. 2003; 10: 791-797Crossref PubMed Scopus (79) Google Scholar). Briefly, various cells (5 × 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 (7Li L. Cousart S. Hu J. McCall C.E. J. Biol. Chem. 2000; 275: 23340-23345Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar).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 Doc™.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.RESULTSIRAK1 Is Critical for IL-10 Gene Expression—To identify potential downstream gene targets of IRAK1, we prepared total RNAs from wild type and IRAK1-deficient splenocytes treated with or without LPS. Isolated RNAs were then used to perform cDNA microarray analysis using the Affymetrix mouse chip U74v2 and the data analyzed using the Affymetrix 4 and gene-spring data analysis software. We observed that several typical pro-inflammatory genes under the control of NFκB such as IL-1β and tumor necrosis factor α 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-1β 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-1β, 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-1β message levels were induced to a similar extent in wild type and IRAK1-deficient splenocytes by LPS (Fig. 1b). The amplified IL-10, IL-1β, 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).Stat3 Serine Phosphorylation Necessary for IL-10 Gene Expression Requires the Presence of IRAK1—LPS-induced IL-10 gene expression has been shown to be mediated largely by Stat3 (22Benkhart E.M. Siedlar M. Wedel A. Werner T. Ziegler-Heitbrock H.W. J. Immunol. 2000; 165: 1612-1617Crossref PubMed Scopus (216) Google Scholar). Stat3 activation is achieved through phosphorylation at the serine 727 and tyrosine 705 residues (29Wen Z. Zhong Z. Darnell Jr., J.E. Cell. 1995; 82: 241-250Abstract Full Text PDF PubMed Scopus (1729) Google Scholar). To clarify that Stat3 phosphorylation indeed is critical for LPS-induced IL-10 gene transcription, we co-transfected HeLa-MAT cells stably carrying the TLR-4/MD-2 gene (27Re F. Strominger J.L. J. Biol. Chem. 2002; 277: 23427-23432Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar) with the IL-10 promoter reporter plasmid pIL-10Luci (22Benkhart E.M. Siedlar M. Wedel A. Werner T. Ziegler-Heitbrock H.W. J. Immunol. 2000; 165: 1612-1617Crossref PubMed Scopus (216) Google Scholar) together with various Stat3 expression constructs. As shown in Fig. 2, LPS induced IL-10 reporter gene activation in cells co-transfected with wild type Stat3 construct. In contrast, there was no induction of IL-10 reporter activity in cells co-transfected with the Stat3 S727A or Y705A mutant, confirming that serine 727 as well as tyrosine 705 phosphorylation is essential for Stat3-mediated IL-10 gene transcription.Fig. 2Stat3 serine phosphorylation is essential for LPS-induced IL-10 gene transcription. HeLa-MAT cells were co-transfected with an IL-10-luciferase promoter-reporter plasmid (pIL-10Luci) and Stat3 wild type or Stat3 S727A and Stat3 Y705F mutant plasmids. Cells were then stimulated with LPS for 4 h. Results were presented as fold induction compared with cells without LPS treatment. *, p < 0.05 versus Stat3. All points represent the mean and S.E. from three different experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We then examined the Stat3 phosphorylation status in wild type and IRAK1-deficient splenocytes. Splenocytes from wild type and IRAK1-deficient mice were treated with LPS. Total protein extracts were harvested and separated on SDS-PAGE. Total Stat3 as well as phosphorylated Stat3 were monitored by Western blot using anti-Stat3 and anti-phospho-Stat3 antibodies. As shown in Fig. 3, LPS induced Stat3 phosphorylation at both serine 727 and tyrosine 705 residues in wild type splenocytes. In contrast, LPS only induced Stat3 tyrosine phosphorylation, but failed to induce serine 727 phosphorylation, in IRAK1-deficient splenocytes (Fig. 3a). We further fractionated the cell extracts into cytoplasmic and nuclear fractions. Total Stat3 was present in both the cytoplasmic and nuclear fractions of splenocytes from wild type and IRAK1-deficient mice (Fig. 3b). LPS induced a dramatic increase of nuclear Stat3 serine 727 phosphorylation in the wild type splenocytes. Strikingly, serine 727-phosphorylated Stat3 was completely absent in the nuclear fraction from IRAK1-deficient splenocytes (Fig. 3b).Fig. 3Stat3 serine phosphorylation is compromised in IRAK1-deficient splenocytes. a, LPS fails to induce serine 727 phosphorylation in IRAK1-deficient splenocytes. Splenocytes from wild type and IRAK1-deficient mice were treated with LPS for 4 h. Whole cell lysates were collected, and immunoblot analyses were performed with anti-Stat3, phospho-Stat3(Ser727), and phospho-Stat3(Tyr705) antibodies b, LPS induces nuclear Stat3 serine 727 phosphorylation in the wild type but not IRAK1-deficient splenocytes. Primary splenocytes were treated as described for a. Cytoplasmic (C) and nuclear (N) fractions were prepared and used to immunoprecipitate Stat3. Immunoprecipitates were analyzed for Stat3 serine 727 and total Stat3. The results are representative of three independent experiments. c, IRAK1 can directly phosphorylate Stat3 serine 727. IRAK1, wild type Stat3, and Stat3-S727A mutant proteins were synthesized using the Promega TNT Quick-coupled transcription/translation system. 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. Reaction products were separated on SDS-PAGE and transferred to polyvinylidene difluoride membrane. The phosphorylated as well as total Stat3 proteins were visualized by Western blot using antibodies specific for either Stat3-S727P or total Stat3 protein. KD, kDa.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We also examined whether IRAK1 could directly phosphorylate Stat3 serine 727. Wild type Stat3, Stat3-S727A mutant, as well as IRAK1 proteins were synthesized via in vitro transcription/translation as described under “Materials and Methods.” In vitro kinase assays were performed by mixing IRAK1 protein with either wild type Stat3 or Stat3-S727A mutant protein in the kinase buffer at 37 °C. Reaction products were resolved by SDS-PAGE. Total and Ser727-phosphorylated Stat3 proteins were detected through Western blot using anti-Stat3 and anti-Stat3-Ser727 antibodies. As shown in Fig. 3c, IRAK1 can directly cause Stat3 serine 727 phosphorylation.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-1β treatment may induce IRAK1 to localize inside the nucleus (30Bol G. Kreuzer O.J. Brigelius-Flohe R. FEBS Lett. 2000; 477: 73-78Crossref PubMed Scopus (27) Google Scholar). 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. β-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 (12Li X. Commane M. Burns C. Vithalani K. Cao Z. Stark G.R. Mol. Cell. Biol. 1999; 19: 4643-4652Crossref PubMed Scopus (187) Google Scholar). 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).Fig. 4LPS induces IRAK1 modification, nuclear entry, and interaction with Stat3 in murine splenocytes. a, the prepared cytoplasmic and nuclear extracts are devoid of cross-contamination. Whole cell (W), cytoplasmic (C), as well as nuclear (N) fractions were subjected to Western blot using anti-GAPDH, anti-Lamin-B, and β-actin. GAPDH and Lamin-B are cytoplasmic and nuclear markers, respectively. β-Actin is used as a marker of total amount of proteins per lane. b, LPS induces IRAK1 modification and nuclear entry in murine splenocytes. Splenocytes from wild type mice were left untreated or treated with LPS for 4 h. Whole cell (W), cytoplasmic (C), as well as nuclear (N) fractions were collected and used to perfo" @default.
• W2014120138 created "2016-06-24" @default.
• W2014120138 creator A5015612883 @default.
• W2014120138 creator A5027075620 @default.
• W2014120138 creator A5086326013 @default.
• W2014120138 creator A5089952959 @default.
• W2014120138 date "2004-12-01" @default.
• W2014120138 modified "2023-10-06" @default.
• W2014120138 title "IRAK1 Serves as a Novel Regulator Essential for Lipopolysaccharide-induced Interleukin-10 Gene Expression" @default.
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