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¹ Division of Molecular and Cellular Immunology, Medical Institute of Bioregulation, Kyushu University, Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
² Department of Molecular Oncology and Leukemia Program Project, Research Institute for Radiation Biology and Medicine, Hiroshima University, Kasumi, Minami-ku, Hiroshima 734-8553, Japan
³ Laboratory for Lymphocyte Differentiation, RIKEN Research Center for Allergy and Immunology, 1-7-22, Suehirocho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan

	Abstract

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In macrophages and monocytes, microbial components trigger the production of pro-inflammatory cytokine through Toll-like receptors (TLRs). Although major TLR signaling pathways are mediated by serine/threonine kinases, including TAK1, IKK and MAP kinases, tyrosine phosphorylation of intracellular proteins by TLR ligands has been suggested in a number of reports. Here, we demonstrated that peptidoglycan (PGN) of a Gram-positive bacterial cell wall component, a TLR2 ligand and lipopoysaccharide (LPS) of a Gram-positive bacterial component, a TLR4 ligand induced tyrosine phosphorylation of phospholipase C-2 (PLC2), leading to intracellular free Ca²⁺ mobilization in bone marrow-derived macrophages (BMM) and bone marrow-derived dendritic cells (BMDC). PGN- and LPS-induced Ca²⁺ mobilization was not observed in BMDC from PLC2 knockout mice. Thus, PLC2 is essential for TLR2 and TLR4-mediated Ca²⁺ flux. In PLC2-knockdown cells, PGN-induced IB- phosphorylation and p38 activation were reduced. Moreover, PLC2 was necessary for the full production of TNF- and IL-6. These data indicate that the PLC2 pathway plays an important role in bacterial ligands-induced activation of macrophages and dendritic cells.

	Introduction

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Toll-like receptors (TLRs), which recognize the structure of microorganisms, are essential for innate immune signaling (Akira 2003; Akira et al. 2006). Peptidoglycan (PGN) is a major component of the cell wall of Gram-positive bacteria, and it activates the innate immune system of the host. TLR2 has been shown to be a main receptor recognizing PGN, and its activation in response to PGN induces the production of pro-inflammatory cytokines, chemokines and adhesion molecules (Akira et al. 2006; O’Neill & Bowie 2007). For TLR2 signaling, TLR2 utilizes adaptor proteins Myeloid differentiation 88 (MyD88) and MyD88-adaptor like (Mal) to activate IL-1 receptor-associated kinase. Then, the activated IL-1 receptor-associated kinase dissociates the MyD88/Mal complex from the receptor, followed by association with tumor necrosis factor-associated factor 6 (TRAF6). This triggers the activation of the Rel family transcription factor NF-B, which is required for transactivation of gene expression (Akira et al. 2006; O’Neill et al. 2007). In addition, MAP kinases, including p38, JNK and ERK, are also activated in response to PGN, which leads to activation of AP-1 and ATF2 transcription factors (Chang et al. 2005).

In lymphocytes, Ca²⁺ is important for cellular function as a second messenger (Gallo et al. 2006). It is generally established that ligation of antigen receptors induces the generation of inositol-1,4,5-triphospate (IP₃) and diacylglycerol (DAG). IP₃ binds to IP₃R in the membrane of the endoplasmic reticulum (ER) and induces the release of Ca²⁺ into the cytoplasm, whereas DAG, together with Ca²⁺, activates protein kinase C (PKC) (Berridge 1993; Nishizuka 1995; Carpenter & Ji 1999). Recent studies suggest that the Ca²⁺ flux is also activated via TLR signaling (Chun & Prince 2006; Zhou et al. 2006). The TLR2 ligand, Pam₃Cys-Ser-Lys₄ (P3C), stimulated the release of Ca²⁺ by activating TLR2 phosphorylation by c-Src and recruited phosphatidylinositol 3-kinase (PI3K) and phospholipase C (PLC) to affect Ca²⁺ release through IP₃ in airway epithelial cells (Chun & Prince 2006). Furthermore, Ca²⁺ release has also been shown to increase upon the stimulation of TLR4 (Zhou et al. 2006). However, few studies have demonstrated a direct contribution of PLC2 to the PGN-induced Ca²⁺ mobilization. Furthermore, the importance of this pathway for pro-inflammatory cytokine production has not been elucidated.

In this study, we investigated a loss-of-function effect of PLC2 in PGN-stimulated macrophages and dendritic cells. PLC2 depletion using siRNA reduced PGN-induced p38, Akt and IB- phosphorylation in murine macrophage RAW 264.7 cells. Moreover, PGN and lipopolysaccharide (LPS) stimulation failed to activate TLR2- and TLR4-dependent Ca²⁺ mobilization in bone marrow-derived dendritic cells (BMDC) from Tie2Cre/PLC2^flox/flox mutant mice. PLC2 knockdown in RAW264.7 cells and knockout in bone marrow-derived macrophages (BMM) resulted in a reduction in the production of TNF-, IL-6 in response to PGN. Our results indicated that Ca²⁺ mobilization plays an important role in pro-inflammatory cytokine production induced by the PGN/TLR2 as well as the LPS/TLR4 pathways.

	Results

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PGN-and LPS-induced tyrosine phosphorylation of PLC2 in BMM and BMDC

Previously, we identified several tyrosine-phosphorylated proteins in LPS-activated RAW 264.7 cells using proteomic approaches. Among them, we noticed PLC2 (Aki et al. 2005). To confirm the tyrosine phosphorylation of PLC2 in the stimulation to LPS, we performed immunoblot analysis using an anti-phosphotyrosine (4G10) and PLC2 antibody in LPS-stimulated BMDC. As a result, the tyrosine phosphorylation level of PLC2 was increased after 30 min of LPS stimulation (Fig. 1A). Furthermore, we found that PGN, one of the bacterial ligands for TLR2, also induced tyrosine phosphorylation of PLC2. As shown in Fig. 1B, total cell lysates from RAW264.7 cells were isolated 0, 15, 30, 60 and 180 min after stimulation with PGN. Tyrosine-phosphorylated proteins were immunoprecipitated with 4G10 and immunoblotted with anti-PLC2 antibody or 4G10. Tyrosine phosphorylation of PLC2 increased within 30 min of PGN stimulation (Fig. 1B). To investigate whether tyrosine phosphorylation of PLC2 is induced in response to other TLR ligands, we examined the effects of zymosan (TLR2), Poly(I:C) (TLR3) and CpG-ODN (TLR9). As a result, tyrosine-phosphorylation of PLC2 was induced by zymosan as well as LPS and PGN, but Poly(I:C) and CpG-ODN which is mimic a viral infection failed to induce ligands-dependent PLC2 tyrosine-phosphorylation (Fig. 1C). This data indicated that PLC2 is induced tyrosine-phosphorylation by TLR2 and TLR4 stimulation. In addition, after pre-treatment with the tyrosine kinases inhibitor PP2 in BMM, PGN-induced PLC2 tyrosine phosphorylation was completely inhibited (Fig. 1D), suggesting that tyrosine phosphorylation of PLC2 is PTKs-dependent. Interestingly, PLC2 tyrosine phosphorylation continued for 180 min (Fig. 1B). This suggests an indirect effect of PGN on the tyrosine phosphorylation of PLC2. Thus, BMM were pre-treated with a translational inhibitor, cycloheximide for 30 min and then treated with PGN for 180 min. PGN-induced tyrosine phosphorylation of PLC2, as well as the tyrosine phosphorylation of the entire protein, was partially inhibited by cycloheximide (Fig. 1E). These data suggest that PLC2 was tyrosine-phosphorylated by direct activation of tyrosine kinases and an indirect effect, which required a new protein synthesis.

View larger version (26K): [in this window] [in a new window]   Figure 1  TLR2 and TLR4 signaling induced tyrosine phosphorylation of PLC2. (A) BMDC were stimulated with 100 ng/mL LPS for 30 min. Cell extracts were immunoprecipitated with anti-PLC2 antibody and then immunoblotted with anti-phosphotyrosine antibody (4G10). (B) RAW264.7 cells were stimulated with 30 µg/mL PGN for the indicated time and then lysed. Cell extracts were immunoprecipitated with 4G10 and then immunoblotted with anti-PLC2 antibody. (C) BMM were stimulated with 1 µg/mL LPS, 20 µg/mL PGN, 20 µg/mL zymosan, 1 µM CpG-ODN or 100 µg/mL Poly(I : C) for 180 min. Cell extracts were immunoprecipitated with anti-PLC2 antibody and then immunobloted with the indicated antibodies. (D, E) BMM were pre-treated with 10 µM PP2 for 15 min (D) and 10 µM cycloheximide for 30 min (E) and then stimulated with PGN for the indicated period. Cell extracts were immunoprecipitated with anti-PLC2 antibody and immunoblotted with 4G10.

To examine whether TLR-ligands can induce intracellular Ca²⁺ mobilization, BMM were loaded with Fluo-3/AM and then stimulated with PGN and their fluorescence intensity was measured using confocal microscope. A transient increase in fluorescence intensity was induced after PGN treatment (Fig. 2A). The concentration of Ca²⁺ reached its maximum level within a few seconds and quickly returned to the baseline level (Fig. 2A). In BMDC, similar PGN-dependent intracellular Ca²⁺ mobilization to that for BMM was induced (Fig. 2B). Moreover, we also observed that LPS stimulation also induced Ca²⁺ mobilization in BMDC (Fig. 2C). These results indicated that bacterial ligands caused a transient increase in the concentration of Ca²⁺ via TLR signaling in BMM and BMDC.

View larger version (15K): [in this window] [in a new window]   Figure 2  TLR2 and TLR4 activate intracellular Ca2+ mobilization in BMM and BMDC. (A) BMM were loaded with Fluo-3/AM and stimulated with 50 µg/mL PGN. (B, C) BMDC were loaded onto Indo-1 AM and then stimulated with 50 µg/mL PGN (B) or 100 ng/mL LPS (C). Intracellular Ca2+ mobilization was evaluated as described in Experimental procedures. Sequentially, these cells were stimulated with PMA or ionomycin as positive controls. The data shown are representative of several experiments that produced similar results.

PLC2 is required for PGN- and LPS-induced Ca²⁺ signaling

The Ca²⁺ flux is essential for many cellular responses. Inositol 1,4,5-triphosphate (IP₃) generated by PLC has been shown to be responsible for Ca²⁺ mobilization from the ER (Berridge 1993; Nishizuka 1995; Carpenter & Ji 1999). To elucidate whether PLC2 is involved in Ca²⁺ mobilization in response to PGN, we first assessed the effect of a PLC inhibitor, U73122 [GenBank] , on PGN-stimulated hTLR2-expressing HEK 293 cells. U73122 [GenBank] pre-treatment for 30 min, but not DMSO, blocked PGN-induced intracellular Ca²⁺ mobilization (Fig. 3A). Next, to confirm the essential role of PLC2 in TLR-mediated intracellular Ca²⁺ mobilization, we generated Tie2-Cre PLC2^flox/flox mice. PLC2^flox/flox mice, in which an exon encoding the phosphatidylinositol-4, 5-biphosphate (PIP₂) binding site of the PLC2 gene is flanked by two loxP sites, have been described elsewhere (Hashimoto et al. 2000). The PLC2 gene was efficiently deleted in BMDC from homozygous mutant mice (Fig. 3B). We then examined the PGN-induced intracellular Ca²⁺ release of BMDC from Tie2-Cre PLC2^flox/flox mice. PGN-mediated Ca²⁺ mobilization was completely blocked in the BMDC from mutant mice (Fig. 3C). This result was consistent with a blocking effect of U73122 [GenBank] on Ca²⁺ mobilization in PGN-treated HEK293 cells stably expressing TLR2 (Fig. 3A). In addition, the LPS-induced transient Ca²⁺ increase was also significantly inhibited in BMDC from Tie2-Cre PLC2^flox/flox mutant mice (Fig. 3D). These data indicate that PLC2 is necessary for intracellular Ca²⁺ mobilization in response to TLR stimulation in BMM and BMDC.

View larger version (17K): [in this window] [in a new window]   Figure 3  PLC2 is essential for TLR-dependent Ca2+ signaling. (A) HEK293 stably expressing hTLR2 cell lines were stimulated with 50 µg/mL PGN after pre-treatment with DMSO or 10 µM U73122. Cells were imaged using confocal microscope. (B) PCR analysis of genomic DNA from BMDC. The PCR product of wild-type PLC2 locus is 554 bp. In Tie2-Cre PLC2fl/fl mice, a band of 386 bp, (KO band), indicates the Cre-mediated deletion of PLC2. (C, D) BMDC from wild-type and mutant mice stimulated with 30 µg/mL PGN (C) or 100 ng/mL LPS (D) were analyzed by flow cytometric analysis as described in Experimental procedures.

TLR2-PLC2 signaling pathway is involved in PGN-induced p38, Akt, IB- and pro-inflammatory cytokine production

To explore the physiological role of PLC2 in TLR signaling, we generated RAW264.7 cells that were transfected with a vector expressing PLC2 siRNA. Immunoblot analysis indicated the silencing of PLC2 expression in RAW264.7 cells by PLC2 siRNA. The reduction of PLC2 levels by siRNA was greater in #2 than in #1 (Fig. 4A).

View larger version (20K): [in this window] [in a new window]   Figure 4  PLC2 regulates TLR signaling and pro-inflammatory cytokine production. Two individual RAW 264.7 transformants, each containing vector alone as control or PLC2 siRNA expression construct, were analyzed. (A) cell extracts were immunoblotted with the indicated antibodies. (B) Transformants were stimulated with 30 µg/mL PGN. The levels of IB- and phospho-IB- were determined 15 min after stimulation. Those of p38, Akt and Erk were measured 30 min after stimulation. (C–E) For RT-PCR analysis, cells were stimulated with PGN for 1 h and then total RNA was extracted from the cells (C). For ELISA, cells were stimulated with 30 µg/mL PGN (C, D) or 100 ng/mL LPS (E) for 6 h, and IL-6 and TNF- in the cell culture supernatants were then analyzed.

Finally, we found that, upon stimulation of PLC2^flox/flox BMM with PGN, IL-6 and TNF-a production were suppressed to 50% and 37% of the control levels, respectively (Fig. 4D). Furthermore, LPS stimulation of PLC2^flox/flox BMM inhibited cytokine production to the same degree as PGN stimulation did (Fig. 4E). These data suggest that PLC2 is required for pro-inflammatory cytokine production mediated by PGN-and LPS-dependent Ca²⁺ flux in BMM and BMDC.

	Discussion

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In agreement with our present study, several recent studies have demonstrated that TLRs induced rapid changes in the intracellular Ca²⁺ concentration (Supajatura et al. 2002; Kim et al. 2004; Chun & Prince 2006; Zhou et al. 2006). Ca²⁺ signaling regulates downstream signaling molecules, such as nuclear factor of activated T cells transcriptional complexes (NF-ATc) and calcineurin, followed by many cellular responses through the change of gene expression (Gallo et al. 2006). It has been shown that Ca²⁺-dependent signaling pathway results in the gene expression of inducible nitric-oxide synthase as well as the production of TNF- upon LPS stimulation of rat peritoneal macrophages (Zhou et al. 2006). Furthermore, it has been suggested that TLR-dependent PLC2 activation participated in this event (Shinji et al. 1997; Chun & Prince 2006; Zhou et al. 2006). However, there has been little direct evidence for the involvement of PLC2 in Ca²⁺ mobilization in response to TLR ligands. In the present study, we found that PGN and LPS stimulation induced PLC2 phosphorylation and intracellular Ca²⁺ mobilization in BMM and BMDC. We clearly demonstrated that this pathway played an important role in the activation of macrophages using PLC2 knockdown cells and BMM from PLC2-disrupted mice. Thus, it is now clear that PLC2 plays an important role in TLR2 and TLR4-mediated Ca²⁺ mobilization and signal transduction.

There are several important questions still to be solved. First, the manner in which TLR2 and TLR4 signaling is activated is not exactly clear. TLR2-dependent PLC2 tyrosine phosphorylation was clearly detected at 30 min after PGN stimulation, and tyrosine phosphorylation seemed to be amplified at 180 min. Tyrosine phosphorylation of PLC2 at this point was not fully inhibited by cycloheximide pre-treatment; thus, there seem to be both direct and indirect pathways for PLC2 tyrosine phosphorylation by PGN stimulation. When PP2 was used, the phosphorylation of PLC2 induced by PGN was completely inhibited. This result suggested that Src family tyrosine kinases are directly involved in TLR2 signaling, as previously reported (Chun & Prince 2006). Other tyrosine kinases were required for the activation of TLR2 signaling. In particular, Syk kinase was considered to be involved in TLR2 signaling. Syk-deficient dendritic cells were unable to produce IL-2 and IL-10. In addition, Syk binds to the phosphorylation YxxL motif of the β-glucan receptor, Dectin-1, upon ligand binding. It is, therefore, suggested that Syk is recruited to the Dectin-1 phosphorylation site and participates in TLR2 signaling events (Rogers et al. 2005). Moreover, recent studies have demonstrated that a member of Tec family tyrosine kinase, Btk has important implications for TLR signal transduction. It has been demonstrated that Btk tyrosine phosphorylation was TLR2-dependent and the lack of Btk resulted in lower amounts of TNF- and IL-1β production (Jefferies et al. 2003; Horwood et al. 2006). However, the question of how these tyrosine kinases are activated by PGN is unanswered. In addition, the question of how these kinases phosphorylate PLC2 is still not very clear.

Second, an important question is how downstream PLC2 is involved in TLR signaling. Apparently, the activation of PLC2 results in an increase of intracellular Ca²⁺ and DAG. Ca²⁺ activates NF-AT transcription factors, while DAG activates PKC. PKC/β has been shown to participate in NF-B activation upon LPS stimulation of macrophages (Asehnoune et al. 2005; Zhou et al. 2006). Cyclosporine, an inhibitor of Ca²⁺-dependent NF-AT activation, has been shown to block cytokine production (Duperrier et al. 2002; Chen et al. 2004). Thus, both the Ca²⁺-NFAT and the DAG-PKC pathways seem to be important for the downstream of PLC2 activated by TLRs. However, the precise role of NF-AT and PKC in cytokine production remains to be clarified. Furthermore, it has been shown that another PKC isoform, PKC, is also involved in LPS signaling (Shapira et al. 1997; Valledor et al. 2000; Castrillo et al. 2001; McGettrick et al. 2006). However, it has not been clarified which PKCs isoform was activated by TLR2-PLC2 pathway.

The knockdown of PLC2 is effective enough to inhibit Akt activation in particular. The pathway for Akt activation in response to TLR ligands has not been clarified. We found that Akt activation was reduced in not only PLC2-knockdown cells, but also U73122 [GenBank] treated RAW264.7 cells (data not shown). Thus, our data raise an interesting possibility that PLC2 is an upstream of PI3kinase-Akt. On the other side, the generation of PIP₃ by PI3K also activates PLC2 (Bae et al. 1998). A recent study demonstrated that phosphorylated PLC1 interacted with Akt in response to EGF stimulation (Wang et al. 2006). Thus, Akt could be a downstream target of PLC2. We have shown that the suppression of PLC2 resulted in the reduction of IB-, p38 phosphorylation as well as Akt. The mechanism underlying these suppressions has not been clarified. It has been demonstrated that the PKC-CARD pathway is critical for NF-B activation in response to T cell receptor ligation (Hara et al. 2004). Several reports indicate that PKC is important for TLR signaling and cytokine production in macrophages (Chen et al. 1998; Castrillo et al. 2001; Zhou et al. 2006). However, the contribution of this pathway has not been clarified because NF-B is strongly activated by the TRAF6-TAK1-IKK pathways. Furthermore, it is not known which pathway, Ca²⁺ or PKC, directly regulates p38 or Akt. Further study is necessary to define the link between PLC2 and these pathways.

	Experimental procedures

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Antibodies and reagent

Peptidoglycan (PGN) from Staphylococcus aureus was purchased from Fluka (Bachs, Sweden). PP2, cyclohexamide and wortmannin were from Calbiochem (San Diego, CA). LPS, CpG-ODN and Poly(I : C) were from Sigma-Aldrich (Seelze, Germany). Zymosan was obtained from Invitrogen (Carlsbad, CA). Antibodies against PLC2 (Q-20), IB- (C-21) and ERK2 (C-14) were from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-actin rabbit polyclonal antibody was purchased from Sigma (St. Louis, MO). Antibodies against Akt, p38, phospho-IB, phospho-p40/p42 MAP kinase, phospho-p38, phospho-Akt and 4G10 have been described (Aki et al. 2005).

Mice

PLC2^flox/flox mice and Tie2-cre transgenic mice have been previously established (Hashimoto et al. 2000; Kimura et al. 2004; Matsumura et al. 2007). Tie2-cre transgenic mice were bred with PLC2^flox/flox mice to generate mice in which PLC2 was deleted in hemopoietic cells. All experiments were approved by the Animal Ethics Committee of Kyushu University.

Cell culture

TLR2 stably expressing HEK293 cells were prepared by transfection of hTLR2 cDNA and selected in the presence of G418. TLR2-HEK293 cells and the murine macrophage cell line RAW264.7 cells were cultured in DMEM supplemented with 10% fetal calf serum. To prepare BMM, bone marrow cells were obtained from femora and tibia of 6–8-week-old C57BL6/J mice and then cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum; the culture medium was conditioned by the L929 cells containing a macrophage colony-stimulating factor (M-CSF). After 7 days, the cells were used as BMM for the experiments. BMDC were prepared as described elsewhere (Matsumura et al. 2007).

Immunoprecipitation and immunoblotting analysis

After stimulation, cells were washed with PBS and solubilized for 30 min at 4 °C in lysis buffer (Aki et al. 2005). Cell extracts were then centrifuged, and supernatants were used for immunoprecipitation with specific antibodies. After separation by SDS-PAGE and transfer to polyvinyliden difluoride membranes, proteins were analyzed by immunoblotting with the indicated antibodies.

Measurement of cytokine

The cells were plated on 6-well plates on the day before PGN stimulation. For ELISA, the cells were stimulated with PGN for 6 h. Then, culture supernatants were collected and analyzed for TNF- and IL-6 (BD Biosciences PharMingen, San Diego, CA). For the analysis of mRNA expression, the cells were stimulated with PGN for 1 h. TNF- and IL-6 RT-PCR were carried out as described (Aki et al. 2005; Chinen et al. 2006).

RNA interference

The small interfering RNA sequence that targets mouse PLC2 was at positions 1017–1037 (5'-GTCCTCCACGGAAGCGTATAT-3'). The annealed oligonucleotides were inserted into psiRNA-hH1neo expression vector (InvivoGen, San Diego, CA). Stable transformants were selected in 1 mg/mL G418.

Ca²⁺ flux analysis

For imaging, cells were grown in coverglass chamberslides and loaded for 30 min at 37 °C with 5 µM Fluo3/AM (DOJINDO) in the dark. Cells were washed with HANKS buffer. Fluorescence imaging was obtained using Carl Zeiss LSM 510 META scanning confocal microscope and analyzed using LSM Image Browse. For flow cytometric analysis, 1.0 x 10⁷ BMDC cells were suspended in 10 mL of RPMI medium supplemented and loaded with 3 µM Indo-1 AM (Molecular Probes, Eugene, OR) at 37 °C for 30 min. After washing, 1.0 x 10⁶ cells were used for analysis. The filter set-up on the BD LSR for Indo-1 was FL-5 424/444 nm BF filter and unbound Indo-1 FL-4 530/530 nm BF filter. The Ca²⁺ flux was measured as the ratio between the calcium bound Indo-1 and unbound or FL-5/FL-4 vs. time. Full-scale deflection of the Ca²⁺ flux was measured by the addition of 3.0 µg/mL ionomycin.

	Acknowledgements

 
We thank Dr T. Muta (University of Tohoku) for hTLR2 cDNAs, Ms T. Yoshioka and M. Ohtsu for technical assistance, and Ms Y. Nishi for manuscript preparation. This study was supported by special Grants-in-Aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (NIBIO), the Naito Memorial Foundation, the Takeda Science Foundation, the Mochida Memorial Foundation, the Kato Memorial Foundation, the Kanae Foundation for the promotion of Medical Science, Clinical Research Foundation, the Osaka Cancer research Foundation, Mitsubishi Pharma Research Foundation and the Ichiro Kanehara Memorial Foundation.

	Footnotes

 
Communicated by: Tetsuya Taga

^* Correspondence: E-mail: yakihiko{at}bioreg.kyushu-u.ac.jp

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Accepted: 15 November 2007

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