1

IRAK1 serves as a novel regulator essential for LPS-induced IL-10 gene expression

Yingsu Huang, Tao Li, David C. Sane, and Liwu Li*

Department of Medicine, Wake Forest University School of Medicine, Winston Salem, NC 27157 Key words: Innate immunity, LPS, IRAK1, Stat3, IL-10, atherosclerosis * Correspondence: Dr. Liwu Li Department of Medicine Wake Forest University School of Medicine Winston Salem, NC 27157 Phone: 336-716-6040 Fax: 336-716-1214 Email: lwli@wfubmc.edu

JBC Papers in Press. Published on October 12, 2004 as Manuscript M410369200

Copyright 2004 by The American Society for Biochemistry and Molecular Biology, Inc.

by guest on April 3, 2018

http://ww

w.jbc.org/

Dow

nloaded from

2

ABSTRACT Being one of the key kinases downstream of TLR receptors, IRAK1 has initially thought to be

responsible for NFκB activation. Yet IRAK1 knockout mice still exhibit NFkB activation upon LPS

challenge, suggesting that IRAK1 may play other uncharacterized function. In this report, we show

that IRAK1 is mainly involved in Stat3 activation and subsequent 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 β 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 TLR signaling network, and indicate that IRAK1 regulation may be intimately

linked with the pathogenesis and/or resolution of atherosclerosis.

by guest on April 3, 2018

http://ww

w.jbc.org/

Dow

nloaded from

3

INTRODUCTION

Innate immunity signaling mediated by Toll-like-receptors (TLRs) leads to the expression of a wide

variety of genes (1-4). Numerous transcription factors including Nuclear factor -κB (NFκB),

Activator protein-1 (AP-1), Interferon regulatory factors (IRFs), 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 works

including ours have confirmed that indeed various TLR ligands can activate endogenous IRAK1

kinase activation (6-9). Biochemically, we and others have shown that IRAK1 undergoes covalent

modification likely due to phosphorylation and ubiquitination upon IL-1 or LPS challenge (7;10;11).

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 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 (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 NFκB reporter

activation (12;14). The fact that despite being an active kinase, its kinase activity is not required for

by guest on April 3, 2018

http://ww

w.jbc.org/

Dow

nloaded from

4

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

(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 NFκB reporter gene, suggesting that they

may play redundant roles in activating NFκB (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 NFκB 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 NFκB. In contrast, deletion of IRAK-M was shown to

lead to elevated NFκB activity and pro- inflammatory gene expression such as TNF α, indicating that

IRAK-M may negatively regulate NFκB activation (19). Intriguingly, IRAK1 deficient mice still

retain LPS induced NFκB activation (20), suggesting that IRAK1 may rather fulfill other distinct yet

un-identified function in LPS/TLR signaling. Besides NFκB activation, TLR ligands such as LPS can

also activate other transcription factors such as IRFs and Stats (21;22). Whether and which particular

IRAK participates in the activation of IRFs 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 β 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

by guest on April 3, 2018

http://ww

w.jbc.org/

Dow

nloaded from

5

activation and phosphorylation status. We have observed that IRAK1 deficient cells exhibit defective

nuclear Stat3 serine-727 phsophorylation. Furthermore, we have shown that LPS induces IRAK1

modification and nuclear localization. In addition, nuclear IRAK-1 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.

by guest on April 3, 2018

http://ww

w.jbc.org/

Dow

nloaded from

6

MATERIALS AND METOHDS Reagents. E. coli 0111:B4 LPS was obtained from Sigma (St. Louis, MO). Antibody against IRAK1

was from Upstate Biotechnology (Lake Placid, NY). Antibodies against Stat3, Phospho-Stat3(Ser727),

Phospho-Stat3(Tyr705) was from Cell Signaling (Beverly, MA). Murine and human IL-10 enzyme-

linked immunosorbent assay (ELISA) kits were from BenderMed Systems (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 ACUC protocol. All mice were 7-10 weeks of age when

experiments were initiated. Splenocytes were harvested as described (24).

Human blood peripheral blood mononuclear cell (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. Peripheral

blood mononuclear cells (PBMC) were separated by Ficoll density gradient as described (25). Isolated

PBMC were washed twice with PBS and incubated in RPMI 1640, supplemented with 10% (v/v) FBS,

100 U/ml penicillin, 100 µg/ml of streptomycin, and 2 mM glutamine.

RNA isolation and real-time quantitative PCR: Total cellular RNAs were extracted using the

TRIzol reagent (Invitrogen life technologies, CA)(26). Isolated total RNAs were reverse transcribed

using the Qmniscript TMRT Kit (QIAGEN). Quantitative real-time PCR analyses of IL-10, IL-1 β, 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).

by guest on April 3, 2018

http://ww

w.jbc.org/

Dow

nloaded from

7

Tansient transfection and luciferase reporter activity assay: Hela-MAT cells stably expressing

TLR4/MD2 (27) were cultured in DMEM medium supplemented with 10% FBS, 100 U/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 life technologies, CA) 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, the 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 lueiferase assay (26).

In vitro transcription/translation and phosphorylation assay of Stat3: Wild type IRAK1, wild

type and Stat3-S727A mutant proteins were synthesized using the pflag-IRAK1, wild type Stat3 and

Stat3-S727A mutant plasmid with the Promega TNT quick coupled transcription/translation system

(Promega, WI). In vitro synthesized IRAK1 was incubated with either Stat3 or Stat3-S727A protein at

37oC for 30 minutes in 50 µl kinase buffer (20 mM HEPES, pH 7.6, 20 mM MgCl2, 20 mM β-

glycerophosphate, 20 mM para-nitrophenylphosphate, 1 mM EDTA, 1 mM sodium orthovanadate, 1

mM benzamidine). Reaction products were separated on SDS-PAGE, and transferred to PVDF

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.

Isolation of cytoplasmic and nuclear extracts, immunoprecipitation, and Western blot: Cell lysis,

isolation of total, cytoplasmic, and nuclear extracts were as described (28). Briefly, various cells (5 x

106/ml) were washed in 10 mM HEPES, pH 7.9 and subsequently lyzed on ice in the lysis buffer (10

mM HEPES, pH7.9, 1.5 mM MgCl2, 10 mM KCL, 0.5 mM EDTA, 0.5mM DTT, 0.5 mM PMSF,

by guest on April 3, 2018

http://ww

w.jbc.org/

Dow

nloaded from

8

1µg/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 lyzed and

solubilized with the high salt buffer (20 mM HEPES, pH 7.9, 1.5 mM MgCl2, 0.4 M NaCl, 0.2mM

EDTA, 0.5mM DTT, 1 mM PMSF) for 30 minutes, and yielded the nuclear extracts.

Immunoprecipitation and western detection of corresponding proteins were performed as described (7).

Chromatin immunoiprecipitation 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 pre-immunoprecipitation 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). Pre-immune IgG was used as

a negative control. DNA was isolated by phenol/chloroform extraction, ethanol precipitated and re-

suspended in 20µl of dH2O. 4 µl of immunoprecipitated DNA was used for each polymerase chain

reaction (PCR). The following primers specific for the IL-10 promoter -300 to -60bp 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 .

by guest on April 3, 2018

http://ww

w.jbc.org/

Dow

nloaded from

9

Data Analysis: The significance of the data was evaluated by means of one factor ANOVA followed

by Student-Newman-Keuls test using SPSS 10.0 software. A p value <0.05 was considered statistically

significant.

by guest on April 3, 2018

http://ww

w.jbc.org/

Dow

nloaded from

10

RESULTS

IRAK1 is critical for IL-10 gene expression. To identify potential downstream gene targets of

IRAK-1, 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 genespring data

analysis software. We observed that several typical pro-inflammatory genes under the control of

NFκB such as IL-1 β and TNF 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.

In order to confirm the microarray data, we performed real time PCR analysis of the induced IL-10 as

well as IL-1 β 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 β, and the control GAPDH primer sets

purchased from Applied BiosystemsTM. 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 (figure 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 (figure 1a). On the other hand, we observed that IL-1

β message levels were induced to the similar extent in wild type and IRAK1 deficient splenocytes by

LPS (figure 1b). The amplified IL-10, IL-1 β and GAPDH mRNAs were also resolved and visualized

on agarose gel (figure 1c). Subsequently, we measured IL-10 protein levels by ELISA. Wild type,

by guest on April 3, 2018

http://ww

w.jbc.org/

Dow

nloaded from

11

instead of IRAK1 deficient splenocytes produced significant amount of IL-10 protein upon 16 hour

LPS treatment (figure 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 (22).

Stat3 activation is achieved through phosphorylation at the serine 727 and tyrosine 705 residues (29).

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 (27) with the IL-10 promoter

reporter plasmid pIL-10Luci (22) together with various Stat3 expression constructs. As shown in

figure 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

Stat3 S727A or Y705A mutant, confirming that serine 727 as well as tyrosine 705 phosphorylation is

essential for Stat3 mediated IL-10 gene transcription.

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 figure 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 (figure 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 (figure 3b). LPS induced

dramatic increase of nuclear Stat3 serine 727 phsophorylation in the wild type splenocytes.

by guest on April 3, 2018

http://ww

w.jbc.org/

Dow

nloaded from

12

Strikingly, serine-727-phosphorylated Stat3 was completely absent in the nuclear fraction from

IRAK1 deficient splenocytes (figure 3b).

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 in the 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 37C.

Reaction products were resolved by SDS-PAGE. Total and S727 phosphorylated Stat3 proteins were

detected through Western blot using anti-Stat3 and anti-Stat3-S727 antibodies. As shown in figure 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 (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 in the

methods. The purities of prepared extracts were confirmed by probing for the presence of

cytoplasmic and nuclear specific proteins. As shown in figure 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 figure 4b, LPS challenge

caused a major shift of IRAK1 migration from ~85KD to ~100KD, corresponding to the covalent

by guest on April 3, 2018

http://ww

w.jbc.org/

Dow

nloaded from

13

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

figure 4b, un-modified IRAK1 (~85KD) 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 (figure 4b).

We next asked whether IRAK1 could form a complex with endogenous Stat3. Immunoprecipitated

IRAK1 complex from the cytoplasmic as well as nuclear extracts were separated on SDS-PAGE,

transferred to PVDF membrane, and probed with anti-Stat3 antibody. As shown in figure 4c, Stat3

was only detected in the IRAK1 immunoprecipitated complex obtained from the nuclear extract.

Furthermore, the intensity of co-immunoprecipitated Stat3 in the nucleus increased dramatically from

the samples treated with LPS. We further performed immunoprecipitation of Stat3 using the

cytoplasmic and nuclear extracts. As shown in figure 4c, Stat3 existed in both the nuclear and

cytoplasmic fractions. The total Stat3 levels remained steady in resting as well as LPS treated

splenocytes. Consistently, the modified IRAK1 (100KD) was present in the immunoprecipitated Stat3

complex in the nuclear fraction upon LPS treatment (figure 4c).

Modified IRAK-1 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 figure 5, IRAK1

underwent modification upon LPS treatment in THP-1 cells (figure 5a) as well as in human primary

PBMC (figure 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 (figure 5a, b).

by guest on April 3, 2018

http://ww

w.jbc.org/

Dow

nloaded from

14

IRAK1 binds directly to the endogenous IL-10 promoter region. Since IRAK1 is distributed into

the nucleus and interacts with Stat3 upon LPS challenge, and its presence is critical for IL-10 gene

expression, we hypothesized and tested that IRAK1 may interact with the IL-10 promoter element.

IL-10 promoter region responsive to LPS challenge has been previously mapped to the -300 to -60bp

region (22). We therefore performed chromatin immunoprecipitation assays (Chip) to test whether

IRAK1 can bind with this region in vivo. THP-1 cell treated with and without LPS were fixed with

1% formaldehyde for 10 minutes and subjected to Chip assays as described in the methods. As shown

in figure 6, there was no binding of IRAK1 to the IL-10 promoter element in the resting cells.

Strikingly, LPS induced binding of IRAK1 to the endogenous IL-10 promoter region based on Chip

assay.

Blood cells from atherosclerosis patients have elevated IRAK1 modification and constitutive IL-

10 expression. Inflammation has been well noticed to be intimately linked with the pathogenesis of

atherosclerosis (31). Elevated IL-10 levels have been detected in human sera from atherosclerosis

patients (23). The presence of IL-10 may be a self-protective mechanism to prevent excessive

inflammation. The mechanism for elevated IL-10 expression in human atherosclerosis patients is not

clear. We indeed found that the sera from 11 atherosclerosis patients all exhibited detectable IL-10

protein based on ELISA (~5pg/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 figure 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 figure 7b, IRAK1 primarily exists as the modified 100KD form in the patient

by guest on April 3, 2018

http://ww

w.jbc.org/

Dow

nloaded from

15

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 figure 7c, the modified IRAK1 protein was constitutively

migrated as the modified 100 KD form and resided exclusively in the nuclear fraction from the patient

PBMC.

by guest on April 3, 2018

http://ww

w.jbc.org/

Dow

nloaded from

16

DISCUSSION

Our current study has presented evidence indicating that IRAK1 plays a novel and critical role in Stat3

mediated IL-10 gene expression. IRAK1 is essential for Stat3 serine 727 phosphorylation. IRAK1

forms a complex with Stat3 as well as the IL-10 promoter element in the nucleus upon LPS challenge.

Furthermore, atherosclerosis patients exhibit constitutive nuclear distribution of modified IRAK1 in

their peripheral blood mononuclear cells, in concert with constitutively elevated blood IL-10 levels.

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 85KD form, and the other being modified (phosphorylated and/or ubiquitinated)

100KD form. In 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 β stimulation may lead to

IRAK1 nuclear localization (30). Yet this report has been largely ignored by the field. In this study,

we further analyzed the distribution of IRAK1 in the fractionated cellular and nuclear extracts.

Strikingly, the majority of the 85KD 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

reporting IRAK1 nuclear localization that the apparent molecular weight of nuclear IRAK1 is

~100KD, 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

by guest on April 3, 2018

http://ww

w.jbc.org/

Dow

nloaded from

17

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

have 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 NFκB

reporter activation, studies using IRAK1 deficient mice have indicated that LPS can still induce NFκB

activation in IRAK1 deficient cells (20). Therefore, IRAK1 may perform other unidentified role

besides NFκB activation. Our data presented herein unveils 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 Y705 and S727 are 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 (figure 2). Janus kinase 3 (JAK3) has been shown to be the main kinase responsible for Stat3

tyrosine phosphorylation (33). Till to date, the mechanism for LPS induced Stat3 serine 727

phosphorylation is not clear. In consistent with previous reports, we have documented that LPS can

induce Stat3 S727 and Y705 phosphorylation in wild type murine splenocytes. Strikingly, we have

observed that although LPS-induced Y727 phosphorylation is normal, LPS-induced Stat3 S727

phosphorylation is greatly compromised in IRAK1 deficient splenocytes (figure 3). Furthermore, we

by guest on April 3, 2018

http://ww

w.jbc.org/

Dow

nloaded from

18

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

phsophorylation. This is one of 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

dramatic increase of nuclear Stat3 serine 727 phsophorylation 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 IL-10 promoter element in vivo upon LPS

by guest on April 3, 2018

http://ww

w.jbc.org/

Dow

nloaded from

19

challenge (figure 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 NFκB. Furthermore, constitutive IRAK1 modification and

nuclear localization may be intimately linked with either the progression or resolution of

atherosclerosis.

Acknowledgement

We thank Cynthia Hickman for help with the chromatin immunoprecipitation assay, Abbie Connoy for

help with the mice splenocyte isolation, and Jean Hu for her excellent technical assistance. This work

is supported in part by the National Institute of Health Grant AI50089 to L.L.

by guest on April 3, 2018

http://ww

w.jbc.org/

Dow

nloaded from

20

REFERENCES 1. Vogel, S. N., Fitzgerald, K. A., and Fenton, M. J. (2003) Mol.Interv. 3, 466-477

2. Akira, S. (2001) Adv.Immunol. 78, 1-56

3. Aderem, A. (2001) Crit Care Med. 29, S16-S18

4. Beutler, B., Hoebe, K., Du, X., and Ulevitch, R. J. (2003) J.Leukoc.Biol. 74, 479-485

5. Cao, Z., Henzel, W. J., and Gao, X. (1996) Science 271, 1128-1131

6. Medzhitov, R., Preston-Hurlburt, P., Kopp, E., Stadlen, A., Chen, C., Ghosh, S., and Janeway, C. A., Jr. (1998) Mol.Cell 2, 253-258

7. Li, L., Cousart, S., Hu, J., and McCall, C. E. (2000) J.Biol.Chem. 275, 23340-23345

8. Moors, M. A., Li, L., and Mizel, S. B. (2001) Infect.Immun. 69, 4424-4429

9. Jacinto, R., Hartung, T., McCall, C., and Li, L. (2002) J.Immunol. 168, 6136-6141

10. Yamin, T. T. and Miller, D. K. (1997) J.Biol.Chem. 272, 21540-21547

11. Li, X., Commane, M., Jiang, Z., and Stark, G. R. (2001) Proc.Natl.Acad.Sci.U.S.A 98, 4461-4465

12. Li, X., Commane, M., Burns, C., Vithalani, K., Cao, Z., and Stark, G. R. (1999) Mol.Cell Biol. 19, 4643-4652

13. Wesche, H., Gao, X., Li, X., Kirschning, C. J., Stark, G. R., and Cao, Z. (1999) J.Biol.Chem. 274, 19403-19410

14. Maschera, B., Ray, K., Burns, K., and Volpe, F. (1999) Biochem.J. 339 ( Pt 2), 227-231

15. Muzio, M., Ni, J., Feng, P., and Dixit, V. M. (1997) Science 278, 1612-1615

16. Li, S., Strelow, A., Fontana, E. J., and Wesche, H. (2002) Proc.Natl.Acad.Sci.U.S.A 99, 5567-5572

17. Qin, J., Jiang, Z., Qian, Y., Casanova, J. L., and Li, X. (2004) J.Biol.Chem. 279, 26748-26753

18. Suzuki, 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., and Yeh, W. C. (2002) Nature 416, 750-756

19. Kobayashi, K., Hernandez, L. D., Galan, J. E., Janeway, C. A., Jr., Medzhitov, R., and Flavell, R. A. (2002) Cell 110, 191-202

20. Swantek, J. L., Tsen, M. F., Cobb, M. H., and Thomas, J. A. (2000) J.Immunol. 164, 4301-4306

by guest on April 3, 2018

http://ww

w.jbc.org/

Dow

nloaded from

21

21. Fitzgerald, K. A., Rowe, D. C., Barnes, B. J., Caffrey, D. R., Visintin, A., Latz, E., Monks, B., Pitha, P. M., and Golenbock, D. T. (2003) J.Exp.Med. 198, 1043-1055

22. Benkhart, E. M., Siedlar, M., Wedel, A., Werner, T., and Ziegler-Heitbrock, H. W. (2000) J.Immunol. 165, 1612-1617

23. Mallat, Z., Heymes, C., Ohan, J., Faggin, E., Leseche, G., and Tedgui, A. (1999) Arterioscler.Thromb.Vasc.Biol. 19, 611-616

24. Feldmann, K., Sebald, W., and Knaus, P. (2002) Eur.J.Immunol. 32, 1393-1402

25. Mueller, L. P., Yoza, B. K., Neuhaus, K., Loeser, C. S., Cousart, S., Chang, M. C., Meredith, J. W., Li, L., and McCall, C. E. (2001) Shock 16, 430-437

26. Li, T., Hu, J., and Li, L. (2004) Mol.Immunol. 41, 85-92

27. Re, F. and Strominger, J. L. (2002) J.Biol.Chem. 277, 23427-23432

28. Gomez-Angelats, M. and Cidlowski, J. A. (2003) Cell Death.Differ. 10, 791-797

29. Wen, Z., Zhong, Z., and Darnell, J. E., Jr. (1995) Cell 82, 241-250

30. Bol, G., Kreuzer, O. J., and Brigelius-Flohe, R. (2000) FEBS Lett. 477, 73-78

31. Ross, R. (1999) Am.Heart J. 138, S419-S420

32. Pickart, C. M. (2001) Mol.Cell 8, 499-504

33. Levy, D. E. and Darnell, J. E., Jr. (2002) Nat.Rev.Mol.Cell Biol. 3, 651-662

34. Schindler, C., Shuai, K., Prezioso, V. R., and Darnell, J. E., Jr. (1992) Science 257, 809-813

by guest on April 3, 2018

http://ww

w.jbc.org/

Dow

nloaded from

22

FIGURE LEGENDS Fig.1. Selective suppression of IL-10, not IL-1 β expression in IRAK1 deficient splenocytes. (a) LPS only induced IL-10 mRNA expression in splenocytes from wild type, not IRAK1 deficient mice. Primary splenocytes were either left untreated or treated with LPS for 4 hours. Cells were collected to examine the expression of IL-10 as well as GAPDH mRNAs by real-time PCR. (b) IL-1 β mRNA was induced to the similar levels by LPS in both wild type and IRAK1 deficient splenocytes. Primary splenocytes were treated as described in a. The expression of IL-1β and GAPDH mRNAs was analyzed. (c) The amplified IL-10, IL-1 β and GAPDH mRNAs were visualized on agarose gel. (d) LPS-induced IL-10 protein production from splenocytes was suppressed in IRAK1 deficient mice. Splenocytes from wild type and IRAK1 deficient mice were treated with LPS for 16 hours or left untreated. Supernatants were collected and IL-10 protein levels were quantified by ELISA. *P<0.05 vs WT, #P<0.05 vs IRAK1-/-. All points represent the mean and standard deviation from three different experiments. Fig.2. Stat3 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, Stat3 Y705F mutant plasmid. Cells were then stimulated with LPS for 4 hours. Results were presented as fold induction compared with cells without LPS treatment. *P<0.05 vs Stat3. All points represent the mean and SE from three different experiments. Fig.3. Stat-3 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 hours. 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 phsophorylation in the wild type but not IRAK1 deficient splenocytes. Primary splenocytes were treated as described in a. Cytoplasmic and nuclear 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 Serine727. 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 37oC for 30 minutes in 50 µl kinase buffer. Reaction products were separated on SDS-PAGE, and transferred to PVDF 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. Fig.4. LPS 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, cytoplasmic as well as nuclear 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, nuclear entry in murine splenocytes. Splenocytes from wild type mice were left untreated or treated with LPS for 4 hours. Whole cell, cytoplasmic as well as nuclear fractions were collected and used to perform immunoprecipitation with anti-IRAK1 antibody. Immunoblot analysis was performed with anti-IRAK1. (c) LPS induces IRAK1 interaction with Stat3 in the nuclear fraction. Immunoprecipitated IRAK1 complex from the cytoplasmic and nuclear extracts were

by guest on April 3, 2018

http://ww

w.jbc.org/

Dow

nloaded from

23

subjected to immunoblot analysis with anti-Stat3 antibody. Subsequently, immunoprecipitated Stat3 complex were probed with anti-Stat3 as well as IRAK1. Results are representative of three independent experiments. Fig.5. IRAK1 interacts with Stat-3 in the nucleus upon LPS challenge in human THP-1 cells as well as peripheral blood mononuclear cells. (a) IRAK1 interacts with stat-3 in the nucleus upon LPS challenge in human THP-1 cells. THP-1 cells were treated with LPS for 1 hour or left untreated. Whole cell, cytoplasmic as well as nuclear fractions were immunoprecipitated with anti-IRAK1 and Western blotted with anti-IRAK1. Subsequently, immunoprecipitated IRAK1 complex from the cytoplasmic and nuclear fractions were used to perform immunoblot using anti-Stat3 antibody. (b) IRAK1 interacts with stat-3 in the nucleus upon LPS challenge in human peripheral blood mononuclear cells. The cells were left untreated or treated with LPS for 1 hour. Whole cell, cytoplasmic as well as nuclear fractions were immunoprecipitated with anti-IRAK1 and Western blot with anti-IRAK1 or Stat3. Subsequently, immunoprecipitated IRAK1 or Stat3 complex were immunoblotted with either anti-Stat3 or anti-IRAK1 as indicated. Results are representative of three independent experiments. Fig.6. IRAK1 binds with endogenous IL-10 promoter element. THP-1 cells were treated for 2 hours with 500ng/ml LPS. The cell extracts were analyzed for the IRAK1 binding to the IL-10 promoter element by Chip assay using anti-IRAK1. The presence of amplified IL-10 promoter sequence was determined by PCR. One of three experiments is shown. Fig.7. Elevated IL-10 levels and IRAK1 modification in blood cells from atherosclerosis patients. (a) IL-10 level is constitutively high in atherosclerosis patients. PBMC from healthy donors or atherosclerosis patients were incubated in culture medium for 16 hours. Supernatants were collected and IL-10 productions from PBMCs were quantified by ELISA. *P<0.05 vs healthy. All points represent the mean and S.E. from six different experiments. (b) IRAK1 primarily exists as the modified 100KD form in the patient PBMC. Whole cell lysates from healthy or atherosclerosis patients PBMCs were collected. Immunoblot analysis was performed with anti-IRAK1 antibody. (c) Cytoplasmic and nuclear fractions extracted from atherosclerosis patients PBMCs were immunoprecipitated with anti-IRAK1 and Western blotted with anti-IRAK1 or anti-Stat3. Results are representative of three independent experiments.

by guest on April 3, 2018

http://ww

w.jbc.org/

Dow

nloaded from

by guest on April 3, 2018

http://ww

w.jbc.org/

Dow

nloaded from

by guest on April 3, 2018

http://ww

w.jbc.org/

Dow

nloaded from

by guest on April 3, 2018

http://ww

w.jbc.org/

Dow

nloaded from

by guest on April 3, 2018

http://ww

w.jbc.org/

Dow

nloaded from

by guest on April 3, 2018

http://ww

w.jbc.org/

Dow

nloaded from

by guest on April 3, 2018

http://ww

w.jbc.org/

Dow

nloaded from

by guest on April 3, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Yingsu Huang, Tao Li, David C. Sane and Liwu LiIRAK1 serves as a novel regulator essential for LPS-induced IL-10 gene expression

published online October 1, 2004 originally published online October 1, 2004J. Biol. Chem. 

  10.1074/jbc.M410369200Access the most updated version of this article at doi:

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

by guest on April 3, 2018

http://ww

w.jbc.org/

Dow

nloaded from