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Originally published online as doi:10.1189/jlb.0504277 on July 16, 2004

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(Journal of Leukocyte Biology. 2004;76:904-908.)
© 2004 by Society for Leukocyte Biology

Prolonged Toll-like receptor stimulation leads to down-regulation of IRAK-4 protein

Fumihiko Hatao*,{dagger}, Masashi Muroi*, Naoki Hiki{dagger}, Toshihisa Ogawa{dagger}, Yoshikazu Mimura{ddagger}, Michio Kaminishi{dagger} and Ken-ichi Tanamoto*,1

* Division of Microbiology, National Institute of Health Sciences, Tokyo, Japan; and
{dagger} Department of Metabolic Care and Gastrointestinal Surgery, Graduate School of Medicine, and
{ddagger} Surgical Center, The University of Tokyo, Japan

1 Correspondence: Division of Microbiology, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya, Tokyo 158-8501, Japan. E-mail: tanamoto{at}nihs.go.jp


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ABSTRACT
 
Interleukin-1 receptor-associated kinase (IRAK)-4 is a key mediator in the Toll-like receptor (TLR) signaling. We found that stimulation of TLR2, TLR4, or TLR9, but not TLR3, caused a decrease in IRAK-4 protein without affecting its mRNA level in a mouse macrophage cell line, RAW 264. The decrease in IRAK-4 was accompanied by the appearance of a smaller molecular weight protein (32 kD), which was recognized by an anti-IRAK-4 antibody raised against the C-terminal region. The decrease in IRAK-4 and the appearance of the 32-kD protein occurred with slower kinetics than the activation of IRAK-1 and were suppressed by inhibitors of the proteasome, inducible inhibitor of {kappa}B{alpha} phosphorylation or protein synthesis, but not by caspase inhibitors. These results indicate that prolonged stimulation of TLR2, TLR4, or TLR9 causes a down-regulation of IRAK-4 protein, which may be mediated through cleavage of IRAK-4 by a protease induced by the activation of nuclear factor-{kappa}B.

Key Words: monocytes/macrophages • bacterial lipoprotein • CpG-DNA • lipopolysaccharide • protein kinases


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INTRODUCTION
 
Toll-like receptors (TLRs), which recognize pathogen-associated molecular patterns, play important roles in innate immunity of mammals [1 ]. Recently, 10 members of the TLR family have been identified, and several ligands recognized by TLRs have been reported [2 ]. Triacyl bacterial lipopeptides are recognized by the cooperation of TLR2 with TLR1 [3 ]. TLR3 recognizes double-stranded RNA produced by most viruses during replication [4 ]. TLR4 is an essential receptor that transduces the signals of lipopolysaccharide (LPS) [5 ]. TLR9 recognizes unmethylated CpG DNA found in bacteria [6 ].

TLRs and interleukin 1 receptor (IL-1R) share homologies in their cytoplasmic domains, the "Toll/IL-1R (TIR)" domain [7 ]. Stimulation of TIR family members causes a TIR domain-containing cytosolic adaptor protein, such as MyD88, or a TIR domain-containing adaptor inducing interferon-ß (TRIF) to be recruited to the receptor complex via homotopic TIR–TIR interaction. Signaling of TLR2 and TLR9 depends on the MyD88-dependent pathway [8 , 9 ], and TLR3 signaling depends mainly on the TRIF-dependent pathway [10 ]. LPS stimulates the MyD88- and the TRIF-dependent pathways [11 , 12 ]. MyD88 recruits serine-threonine kinases, IL-1R-associated kinase 4 (IRAK-4), and IRAK-1 to the receptor complex. As a result, IRAK-4 phosphorylates IRAK-1 [13 , 14 ], and phosphorylated IRAK-1 associates with tumor necrosis factor receptor-associated factor 6, leading to the activation of nuclear factor (NF)-{kappa}B and p38 mitogen-activated protein kinase (MAPK) pathways [14 , 15 ].

Although IRAK-1-deficient mice show attenuated responses to TLR stimulation, residual activation of NF-{kappa}B and p38 is still observed [16 ]. In contrast, no activation was observed in IRAK-4-deficient mice in response to IL-1, LPS, or other bacterial components [17 ]. Furthermore, it has been reported that children with inherited IRAK-4 deficiency failed to respond to IL-1, IL-18, or stimulations of TLR2, TLR3, TLR4, TLR5, and TLR9 [18 ]. Thus, IRAK-4 is considered to be a key mediator in the TLR/IL-1R signaling pathway. Despite the important role of IRAK-4, the regulation of IRAK-4 protein, including its activation and inactivation processes, is still poorly understood. We report here that prolonged TLR stimulation leads to down-regulation of the IRAK-4 protein.


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MATERIALS AND METHODS
 
Reagents and antibodies
LPS from Escherichia coli O111:B4, cycloheximide, actinomycin D, E-64, and CA-074 methyl ester were purchased from Sigma-Aldrich (St. Louis, MO). Bacterial lipopeptide Pam3CSK4 was from Bachem (Bubendorf, Switzerland). Peptidoglycans from Bacillus subtilis (PGBS), Staphylococcus aureus (PGSA), and Streptomyces species (PGST) were purchased from Fluka (Buchs, Switzerland). Macrophage-activating lipopeptide (MALP)-2 and Bay 117082 were purchased from Alexis (Lausen, Switzerland). Poly I:C was from Amersham Pharmacia Biotech (Buckinghamshire, UK). Phosphorothioate oligodeoxynucleotides were synthesized at Qiagen (Valencia CA), and the sequences were 5'-TCCATGACGTTCTTGACGTT-3' (CpG-DNA: ODN5001 [19 ]) and 5'-TCCATGAGCTTCTTGAGCTT-3' (non-CpG-DNA). Z-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK), Z-Asp-CH2-DCB, MG132, and SP600125 were purchased from Biomol (Plymouth Meeting, PA). Lactacystin was from Boston Biochem (Cambridge, MA). PD-98059 was from Calbiochem (San Diego, CA). An antibody specific for IRAK-1 was from Santa Cruz Biotechnology (Santa Cruz, CA). Three different anti-IRAK-4 antibodies were used. IRAK-4 (C14), raised against amino acids 436–459 of IRAK-4, was purchased from Upstate Biotechnology (Lake Placid, NY). IRAK-4 (C12), raised against amino acids at the carboxy terminus, was from ProSci (Poway, CA). IRAK-4 (NM), raised against amino acids 38–54 and 120–136, was from Imgenex (San Diego, CA). An anti-inhibitor of {kappa}B{alpha} (I{kappa}B{alpha}) antibody was a generous gift from Nancy Rice (NCI-Frederick Cancer Research and Development Center, MD).

Cell culture
A mouse macrophage-like cell line, RAW 264 (obtained from the Riken Cell Bank, Tsukuba, Japan), was grown in Dulbecco’s modified Eagle’s medium (Gibco-BRL, Rockville, MD), supplemented with 10% (vol/vol) heat-incubated fetal calf serum (Gibco-BRL), penicillin (100 U/ml), and streptomycin (100 µg/ml).

Electrophoresis and Western blotting
RAW 264 cells (2x106), seeded in six-well plates, were stimulated for the indicated times and were washed with ice-cold phosphate-buffered saline. Then, cellular extracts were prepared by adding a lysis buffer (10 mM HEPES-KOH, pH 7.9, 10 mM KOH, 5 mM EDTA, 40 mM ß-glycerophosphate, 0.5% Nonidet P-40, 30 mM NaF, 1 mM Na3VO4, 1 mM dithiothreitol, 100 nM okadaic acid) containing a protease inhibitor cocktail (Roche Diagnostics GmbH Mannheim, Germany). The protein concentrations were determined by the Bradford method, and the same amount of protein was loaded onto each lane of a discontinuous sodium dodecyl sulfate-10% polyacrylamide gel (acrylamide/bisacrylamide ratio, 29:1), according to the method of Laemmli [20 ]. Proteins were transferred to a polyvinylidene difluoride membrane (Immobilon-P, Millipore Corp., Bedford, MA) and were subjected to Western blotting with the indicated antibodies. The signals were visualized by using an enhanced chemiluminescence system (Amersham Biosciences, Piscataway, NJ).

Reverse transcriptase-polymerase chain reaction (RT-PCR)
RAW 264 cells (2x106) were plated onto 6 cm dishes and stimulated for the indicated times. After washing, total RNA was prepared using the RNeasy mini kit (Qiagen) following the manufacturer’s instructions. RNA (0.5 µg) was subjected to RT-PCR using the Super ScriptTM one-step RT-PCR system with platinum Taq DNA polymerase (Invitrogen, Carlsbad, CA). Real-time RT-PCR was performed using the Brilliant SYBR green single-step QRT-PCR master mix (Stratagene, La Jolla, CA) on a MX4000 multiplex quantitative PCR system (Stratagene). The following primer pairs were used: IRAK-4, 5'-GTC ATG ACC AGC CGA ATC GTG-3' (sense) and 5'-CAG ACA CTG GTC AGC AGC AGA-3' (antisense); glyceraldehyde 3-phosphate dehydrogenase, 5'-ATC ACT GCC ACC CAG AAG ACT-3' (sense) and 5'-TCC ACC ACC CTG TTG CTG TAG-3' (antisense). To avoid artifacts as a result of DNA contamination, an intron-spanning primer pair was selected for IRAK-4.


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RESULTS
 
TLR stimulation leads to down-regulation of IRAK-4 protein
To know whether the IRAK-4 protein was modified by TLR stimulation, RAW 264 cells were stimulated with LPS, CpG-DNA, or Pam3CSK4, and IRAK-4 protein was then detected by Western blotting (Fig. 1 ). A protein band, which corresponds to the size of IRAK-4, was decreased in response to these stimulations (Fig. 1a , anti-IRAK-4 antibody C14). The decrease was more marked in CpG-DNA and Pam3CSK4 stimulations than in LPS stimulation. To know whether this protein band was IRAK-4, two other anti-IRAK-4 antibodies (C12 and NM), which recognize different epitope sequences, were used for Western blotting. These antibodies also showed a decrease in a protein with the size of IRAK-4 in response to these stimulations (Fig. 1 , b upper and c). Thus, we concluded that the protein that was decreased by these stimulations was IRAK-4. It is interesting that the IRAK-4 antibody (C12) detected a smaller protein (~32 kD), which appeared to be a cleavage product of IRAK-4, upon the decrease in IRAK-4 (Fig. 1b , lower). Similar concentrations of LPS, CpG-DNA, and Pam3CSK4 also caused a decrease in IRAK-1 protein (Fig. 1d) .



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  		Figure 1. TLR stimulation leads to a decrease in IRAK-4 and IRAK-1 protein levels. RAW 264 cells were stimulated with the indicated concentrations of LPS, CpG-DNA (CpG), or Pam3CSK4 (CSK4) for 6 h. Cellular extracts were analyzed for IRAK-4 (a–c) and IRAK-1 (d) proteins by Western blotting. IRAK-4 was analyzed by three different antibodies (see Materials and Methods). Results are representatives of three independent experiments.

To confirm that the decrease in IRAK-4 was caused by TLR stimulation, we used other TLR ligands, and IRAK-4 protein was detected by Western blotting (Fig. 2 ). Peptidoglycans from three different bacterial species and a diacylated bacterial lipopeptide, MALP-2, also caused a decrease in IRAK-4 (Fig. 2a) . In contrast, stimulation with poly I:C, a TLR3 ligand, did not cause any significant change in the IRAK-4 protein level, although it caused a decrease in I{kappa}B{alpha} (Fig. 2b) . A non-CpG DNA, which carries no CpG motifs, also caused no significant changes in IRAK-4 protein levels, although 1 µg/ml CpG-DNA clearly decreased the protein level in the same experiment (Fig. 2c) . These results indicate that the decrease in IRAK-4 in response to Pam3CSK4 and CpG-DNA was mediated through TLR2 and TLR9, respectively.



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  		Figure 2. Ligands for TLR2 and TLR9, but not for TLR3, cause a decrease in the IRAK-4 protein level. RAW 264 cells were stimulated with the indicated concentrations of peptidoglycans (PGBS, PGSA, and PGST; see Materials and Methods) and MALP-2 (a), poly I:C (b), and CpG- as well as non-CpG-DNA (c) for 6 h. Cellular extracts were analyzed for IRAK-4 (a, b upper, c) and I{kappa}B{alpha} (b, lower). Results are representatives of three independent experiments.

IRAK-1 activation precedes the decrease in IRAK-4
We compared the time-course of the changes in IRAK-1 and IRAK-4 levels following stimulation (Fig. 3 ). The IRAK-4 level started to decrease 3 h after stimulation, and the level reached a minimum 8 h after stimulation. The level partially recovered at 16 h, probably because of resynthesis. No significant changes in the molecular weight were observed during the time-course of the experiment. In contrast, a marked molecular weight shift as a result of phosphorylation [21 ] was observed in the IRAK-1 protein within 10 min, and the level started to decrease within 1 h of the stimulation. These results showed that the activation of IRAK-1 preceded the decrease in the IRAK-4 level, and relatively longer stimulation was required for the decrease in IRAK-4.



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  		Figure 3. The decrease in IRAK-4 occurs more slowly than IRAK-1 activation. RAW 264 cells were stimulated with 100 ng/ml LPS, 1 µg/ml CpG-DNA (CpG), or 100 ng/ml Pam3CSK4 (CSK) for the indicated time periods. Cellular extracts were analyzed for IRAK-4 (a, antibody C14) and IRAK-1 (b) by Western blotting. Results are representatives of three independent experiments.

The mechanism of the decrease in IRAK-4 protein
To investigate the mechanism of the decrease in IRAK-4 protein level, we examined the level of IRAK-4 mRNA expression. RAW 264 cells were stimulated with 100 ng/ml Pam3CSK4, and total RNA was prepared from cells at 1, 2, 4, and 8 h following stimulation. The IRAK-4 mRNA level was detected by RT-PCR or quantitative RT-PCR. The level of IRAK-4 mRNA was not significantly affected by Pam3CSK4 stimulation (data not shown), indicating that the decrease in IRAK-4 protein in response to Pam3CSK4 stimulation was not caused by a decrease in IRAK-4 expression.

We next studied the effects of various protease inhibitors. As TLR stimulation is known to activate caspases [22 ], their involvement was examined first. RAW 264 cells were treated with Z-VAD-FMK (50–200 µM) or Z-Asp-CH2-DCB (50–200 µM) broad spectrum caspase inhibitors and were then stimulated with CpG-DNA or Pam3CSK4. Neither caspase inhibitor inhibited the decrease in the IRAK-4 level (data not shown). E-64 (100 µM), a cell-permeable serine protease inhibitor, and CA-074 methyl ester (10 µM), a cathepsin B inhibitor, also did not inhibit the decrease in IRAK-4.

As IRAK-1 is known to be degraded by the proteasome in response to IL-1 stimulation [23 ], the effect of the proteasome inhibitors (MG-132 and lactacystin) were examined. Both inhibitors suppressed the decrease in IRAK-4 protein in response to Pam3CSK4 (Fig. 4a ) and CpG-DNA (Fig. 4b) . It is interesting that the smaller molecular weight protein, which appeared upon stimulation, was not observed in the presence of proteasome inhibitors (Fig. 4 , a and b lower). This result supports the notion that the smaller molecular weight protein was a cleavage product of IRAK-4.



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  		Figure 4. Effects of various inhibitors on the decrease in IRAK-4. RAW 264 cells were stimulated with the indicated concentrations of Pam3CSK4 (CSK4; a, c, e) or CpG-DNA (CpG; b, d, f) for 6 h in the absence or presence of proteasome inhibitors (20 µM MG132 and 20 µM lactacystin, a, b), protein synthesis inhibitors [5 µg/ml actinomycin D (Act D) and 50 µg/ml cycloheximide (CHX), c, d], or an inducible I{kappa}B{alpha} phosphorylation inhibitor (10 µg/ml Bay 117082, e, f). Cellular extracts were analyzed for IRAK-4 by Western blotting with antibody C14 (top panels) and C12 (middle and bottom panels). A broad spectrum caspase inhibitor (100 µM Z-Asp-CH2-DCB) was included throughout the experiments to prevent apoptosis (c, d). Results are representatives of three independent experiments.

The decrease in IRAK-4 protein in response to TLR4 stimulations occurred relatively slowly. This raised the possibility of involvement of a protease induced by TLR stimulations. To examine this possibility, RAW 264 cells were treated with actinomycin D or cycloheximide, both of which inhibit protein synthesis through different mechanisms, and then were stimulated with Pam3CSK4 or CpG-DNA. Unfortunately, most of the cells died after stimulation, probably as a result of apoptosis. Therefore, 100 µM Z-Asp-CH2-DCB, a caspase inhibitor, was included throughout the experiment [24 ]. Actinomycin D and cycloheximide inhibited the decrease in IRAK-4 protein and the appearance of the smaller molecular weight protein in response to Pam3CSK4 (Fig. 4c) or CpG-DNA (Fig. 4d) . This result supports the involvement of an induced protease in the decrease in IRAK-4 protein. To further examine whether the decrease in IRAK-4 protein depends on protein synthesis, we examined the effect of inhibiting NF-{kappa}B and activated protein-1, as TLR stimulation leads to activation of these transcription factors through I{kappa}B kinase and MAPK pathways. For this purpose, we used Bay 117082, SP600125, and PD-98059, which inhibit inducible I{kappa}B{alpha} phosphorylation, Jun-N-terminal kinase, and MAPK kinase, respectively. Bay 117082 inhibited the decrease in IRAK-4 protein and the appearance of the smaller molecular weight protein in response to Pam3CSK4 (Fig. 4e) and CpG-DNA (Fig. 4f) . In contrast, SP600125 (20 µM) and PD-98059 (50 µM) had no effects (data not shown). It is therefore likely that NF-{kappa}B is involved in the decrease in IRAK-4 protein.


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DISCUSSION
 
In this study, we have reported that TLR stimulation induces a decrease in IRAK-4 protein levels. The decrease was observed with Pam3CSK4, CpG-DNA, and LPS, as well as with peptidoglycans and MALP-2, but not with non-CpG-DNA or poly I:C. These results indicate that the stimulation of TLR2, TLR9, and TLR4, but not TLR3, leads to a decrease in IRAK-4 protein and suggests that the MyD88-dependent pathway is involved in the decrease. This is because TLR2, TLR4, and TLR9, but not TLR3, stimulation leads to the activation of the MyD88-dependent pathway [1 ].

The IRAK-4 mRNA level was not affected by Pam3CSK4 stimulation, and the decrease in IRAK-4 protein in response to TLR stimulation was accompanied by the appearance of a smaller molecular weight protein (32 kD), which was recognized by an anti-IRAK-4 antibody directed against its C-terminal sequence. In addition, various inhibitors that suppressed the decrease in IRAK-4 protein level also inhibited the appearance of the smaller molecular weight protein. It is, therefore, likely that TLR stimulation induces cleavage of the IRAK-4 protein, leading to the generation of an N-terminal-truncated IRAK-4 protein. The IRAK-4 protein (459 amino acids) consists of an N-terminal death domain (amino acids 20–104), which is necessary for the interaction with MyD88, and a C-terminal kinase domain (amino acids 195–390) [21 ]. According to their sizes, the smaller molecular weight protein probably lacks the death domain. Therefore, the cleavage of IRAK-4 observed in our study would cause the disruption of the MyD88-dependent signal transduction pathway.

The activation of IRAK-1 is considered to be a downstream event of IRAK-4 in the MyD88-dependent signaling pathway [14 ]. However, our study indicates that the decrease in IRAK-4 protein in response to TLR stimulation occurred following activation of IRAK-1. Thus, we examined the possibility that the decrease in IRAK-4 protein was a feedback event induced by the activation of IRAK-1, and we found that protein synthesis inhibitors and an inducible I{kappa}B{alpha} phosphorylation inhibitor suppressed the decrease in IRAK-4 protein. It is, therefore, likely that protein synthesis and the activation of NF-{kappa}B, both of which lie downstream of the activation of IRAK-1, are involved in the decrease in IRAK-4 protein. Proteasome inhibitors also inhibited the decrease in IRAK-4 protein. It is, however, unlikely that the proteasome is involved in the decrease in IRAK-4, as the proteasome generally degrades proteins into peptides with several amino acids, and the decrease in IRAK-4 protein was accompanied by the production of a smaller molecular weight protein, which appears to be a cleavage product of IRAK-4. The proteasome is also known to be involved in the activation of NF-{kappa}B [25 ], and therefore, the inhibitory effect of proteasome inhibitors on the decrease in IRAK-4 is probably explained by the inhibition of NF-{kappa}B activation. All of these results clearly support the notion that IRAK-4 is cleaved at a site between its death and kinase domains by a protease induced by the activation of NF-{kappa}B upon TLR stimulation.

IRAK-4 is essential for TLR signaling. Overexpression of dominant-negative IRAK-4 [21 ] and IRAK-4 knockout [17 ] totally abrogated all TLR/IL-1R responses that were studied. Thus, the signal-dependent cleavage of the IRAK-4 protein at a site between its death and kinase domains would disrupt TLR/IL-1R signaling, as it is expected that cleaved IRAK-4 has lost the ability to interact with upstream adaptor molecules. It is known that prolonged stimulations of TLR2, TLR4, and TLR9 cause homo- and heterotolerance to subsequent stimulation of these TLRs [26 , 27 ]. The activation of IRAK-4 is common to these signaling events. The cleavage of IRAK-4 protein observed in our study may be involved in the induction of tolerance.


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ACKNOWLEDGEMENTS
 
This work was supported in part by grants from the Japan Health Sciences Foundation and the Ministry of the Environment. We thank Yoshihiro Sugiyama for technical assistance.

Received May 6, 2004; accepted June 19, 2004.


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