Essential Role of Nuclear Factor (NF)-{kappa}B–inducing Kinase and Inhibitor of {kappa}B (I{kappa}B) Kinase {alpha} in NF-{kappa}B Activation through Lymphotoxin ß Receptor, but Not through Tumor Necrosis Factor Receptor I

Akemi Matsushimaa, Tsuneyasu Kaishob, Paul D. Rennertc, Hiroyasu Nakanod, Kyoko Kurosawad, Daisuke Uchidaa, Kiyoshi Takedab, Shizuo Akirab, and Mitsuru Matsumotoa
a Division of Informative Cytology, Institute for Enzyme Research, University of Tokushima, Tokushima 770-8503, Japan
b Department of Host Defense, Research Institute for Microbial Diseases, and Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation, Osaka University, Osaka 565-0871, Japan
c Department of Immunology and Inflammation, Biogen, Incorporated, Cambridge, Massachusetts 02142
d Department of Immunology and CREST, Japan Science and Technology Corporation, Juntendo University School of Medicine, Tokyo 113-8421, Japan

Correspondence to: Mitsuru Matsumoto, Division of Informative Cytology, Institute for Enzyme Research, University of Tokushima, 3-18-15 Kuramoto, Tokushima 770-8503, Japan. Tel:81-88-633-7432 Fax:81-88-633-7434 E-mail:mitsuru{at}ier.tokushima-u.ac.jp.


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Both nuclear factor (NF)-{kappa}B–inducing kinase (NIK) and inhibitor of {kappa}B (I{kappa}B) kinase (IKK) have been implicated as essential components for NF-{kappa}B activation in response to many external stimuli. However, the exact roles of NIK and IKK{alpha} in cytokine signaling still remain controversial. With the use of in vivo mouse models, rather than with enforced gene-expression systems, we have investigated the role of NIK and IKK{alpha} in signaling through the type I tumor necrosis factor (TNF) receptor (TNFR-I) and the lymphotoxin ß receptor (LTßR), a receptor essential for lymphoid organogenesis. TNF stimulation induced similar levels of phosphorylation and degradation of I{kappa}B{alpha} in embryonic fibroblasts from either wild-type or NIK-mutant mice. In contrast, LTßR stimulation induced NF-{kappa}B activation in wild-type mice, but the response was impaired in embryonic fibroblasts from NIK-mutant and IKK{alpha}-deficient mice. Consistent with the essential role of IKK{alpha} in LTßR signaling, we found that development of Peyer's patches was defective in IKK{alpha}-deficient mice. These results demonstrate that both NIK and IKK{alpha} are essential for the induction of NF-{kappa}B through LTßR, whereas the NIK–IKK{alpha} pathway is dispensable in TNFR-I signaling.

Key Words: alymphoplasia, cytokine signaling, I{kappa}B, Akt kinase, Peyer's patch


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The transcription factor nuclear factor (NF)-{kappa}B plays a pivotal role in the regulation of innate immunity, stress responses, inflammation, and the inhibition of apoptosis (1) (2). The activity of NF-{kappa}B is tightly regulated by cytokines and other external stimuli. In most cell types, NF-{kappa}B is present as a heterodimer comprising 50-kD (p50) and 65-kD (p65) subunits and is sequestered in the cytoplasm by a member of the inhibitor of {kappa}B (I{kappa}B) family of inhibitory proteins. NF-{kappa}B activation requires the degradation of I{kappa}B proteins, and the mechanisms of I{kappa}B degradation and subsequent NF-{kappa}B activation have been the subject of intense investigation (3). Those studies have revealed two important classes of kinase involved in this pathway: mitogen-activated protein kinase kinase kinase (MAP3K) and its downstream target, I{kappa}B kinase (IKK) (4) (5). NF-{kappa}B–inducing kinase (NIK) is structurally related to MAP3K and has been identified as a TNFR-associated factor (TRAF)2-interacting protein (6). On the basis of the finding that kinase-inactive mutants of NIK transfected into 293-EBNA cells abolished NF-{kappa}B activation in response to TNF or by cotransfection with type I TNF receptor (TNFR-I), NIK was considered to be involved in an NF-{kappa}B–inducing signaling cascade induced by TNF (6). Subsequently, NIK was demonstrated to phosphorylate IKK{alpha} and IKKß, which directly associate with I{kappa}B{alpha} and specifically phosphorylate it on serines 32 and 36 (4) (5). These studies suggested that interaction of NIK and IKK constitutes an essential step for NF-{kappa}B activation. However, in vivo studies with mutant mice have thrown some doubt on the essential roles of NIK and IKK{alpha} in NF-{kappa}B activation, at least as induced by TNF. The alymphoplasia (aly) mouse is a natural strain with a mutated NIK (7). Despite the NIK mutation, upregulation of vascular cell adhesion molecule 1 (VCAM-1) after stimulation with TNF was present in aly mouse embryonic fibroblasts (EFs) (8). It was also reported that aly mice exhibited similar TNF-mediated endotoxin shock after generalized LPS administration (7). These observations suggested that NIK was not a critical element in the TNF signaling pathway to NF-{kappa}B activation. The in vivo role of IKK in NF-{kappa}B activation was also examined using gene-targeted mice. Although TNF-induced NF-{kappa}B activation was markedly reduced in IKKß-deficient EFs (9) (10), NF-{kappa}B activation from IKK{alpha}-deficient EFs was normal (11) (12) or diminished but still present (13) after stimulation with TNF. Surprisingly, mice deficient in IKK{alpha} showed perinatal death associated with limb and skin abnormalities, suggesting that IKK{alpha} plays an essential role in the regulation of gene expression required for the development of limb and skin rather than for TNF signaling (11) (12) (13). Thus, the role of NIK–IKK{alpha} in NF-{kappa}B activation through TNFR signaling requires further investigation.

The lymphotoxin ß receptor (LTßR) has emerged as a signaling system required for the development of lymphoid organs (14) (15). Although LTßR has been shown to bind TRAF2, -3, -4, and -5, but not TRAF6 (16) (17), and to activate NF-{kappa}B after receptor ligation (18), the molecular mechanisms by which LTßR exerts its biological activities are still poorly understood. aly mice and LT ligand– or LTßR-deficient mice share a unique phenotype, which includes the lack of LN and Peyer's patches (PPs) and a disturbed splenic architecture (7) (14) (15). Therefore, we speculated that NIK plays a role in LTßR signaling. This hypothesis was supported by the demonstration that upregulation of VCAM-1 after stimulation with agonistic anti-LTßR mAb was absent from aly mouse EFs (8).

Phenotypic analyses of mutant and gene-targeted mice, as described above, have unveiled the essential roles of NIK in lymphoid organogenesis and of IKK{alpha} in limb and skin development. However, the roles of NIK and IKK{alpha} in cytokine signaling still remain controversial. We have approached this question with the use of in vivo mouse models. Here, we show that NIK–IKK{alpha} constitutes an essential pathway for the induction of NF-{kappa}B through LTßR, whereas this pathway is dispensable in TNFR-I signaling.


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Mice.
aly/+, aly/aly, and C57BL/6J mice were purchased from CLEA Japan. The mice were maintained under pathogen-free conditions and were handled in accordance with the Guidelines for Animal Experimentation of Tokushima University School of Medicine. The experiments were initiated at 8–12 wk of age. IKK{alpha}-deficient mice were generated by gene targeting as described previously and maintained at Osaka University (11).

Use of EF to Assess Signaling through TNFR-I and LTßR.
EFs were established as described previously (8) (11). EFs from aly/aly mice, IKK{alpha}-deficient mice, and C57BL/6J wild-type mice were cultured in DMEM (GIBCO BRL) supplemented with 10% heat-inactivated FCS (GIBCO BRL), 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin at a density of 7 x 105 cells per 60-mm culture dish. After incubation with control mAb Ha4/8 (2 µg/ml), agonistic anti-LTßR mAb AC.H6 (2 µg/ml; reference (19)), or recombinant human TNF (Genzyme, Inc.), whole cell lysates were harvested from the dish with a lysis buffer containing 1% NP-40 (Sigma-Aldrich) and subjected to Western blot analysis as described previously (20). The following Abs were used: rabbit antipeptide Ab directed against I{kappa}B{alpha} (cat. no. sc-371; Santa Cruz Biotechnology), phospho-specific I{kappa}B{alpha} (cat. no. 9241; New England Biolabs), and polyclonal rabbit Ab against actin (Biomedical Technologies). For blockade of the phosphatidylinositol-3'-OH kinase (PI[3]K)–Akt pathway, EFs were treated with 1 µM wortmannin (Calbiochem) for 30 min before stimulation with TNF. For blockade of proteasome activity, EFs were treated with 100 µM N-acetyl-Leu-Leu-norleucinal (ALLN; Nacalai Tesque) for 1 h.

Use of NF-{kappa}B Reporter Assay to Assess Signaling through TNFR-I and LTßR.
EFs cultured in a 35-mm culture dish (2 x 105 cells) were transfected with 2 µg of a reporter plasmid comprising three repeats of the NF-{kappa}B site upstream of a minimal thymidine kinase promoter and a luciferase gene in the pGL-2 vector (Promega), together with 2 µg of ß-actin promoter–driven ß-galactosidase expression plasmid. Transfected cells were incubated in the presence of recombinant human TNF (100 U/ml) or agonistic anti-LTßR mAb AC.H6 for 8 h. After 24 h, the cells were harvested in PBS and lysed in a luciferase lysis buffer, LC-ß (Piccagene). Luciferase assays were performed with a luminometer (Lumat LB 9507; Berthold). Activity was normalized to ß-galactosidase activity, and data were expressed as the fold activation compared with stimulation by control anti-KLH hamster mAb Ha4/8.

Assessment of LTßR Expression on EFs with Flow-cytometric Analysis.
EFs were incubated with anti-LTßR mAb AF.H6 (19) or control mAb Ha4/8. After washing twice with PBS, cells were incubated with FITC-conjugated anti–hamster IgG mAb (clone G94-56; PharMingen). Cells were analyzed with a FACSCaliberTM flow cytometer (Becton Dickinson) with CELLQuestTM software. Mouse mAb–producing hybridoma cells were used as negative control.

Assessment of PP Formation with Whole-Mount Immunohistochemistry.
Whole-mount immunohistochemistry for the detection of PP was performed as described (21). In brief, 2% paraformaldehyde (pH 7.4)-fixed intestines from 18.5 days postcoitus (d.p.c.) embryos were incubated with mAb against VCAM-1 (PharMingen) and then with horseradish peroxidase (HRP)-conjugated anti–rat Ig (Tago Immunologicals). Color development for bound HRP was done with diaminobenzidine.

Assessment of Association between NIK and IKK{alpha}.
Proteins were expressed by transfecting expression constructs with the indicated cDNAs into COS-7 cells. Extracts were prepared 30 h after transfection. Immunoprecipitation and Western blot analysis was performed as described previously (20) (22). Full-sized, Flag-tagged wild-type NIK (6), Flag-tagged aly-type NIK, and Myc-tagged IKK{alpha} (22) were expressed in pCR3 vectors (Invitrogen); aly-type NIK cDNA was generated by the introduction of an amino acid substitution (G860R) into the COOH-terminal region of human NIK (7) by site-directed mutagenesis. Anti-Flag mAb (clone M2) and anti-Myc mAb (clone 9E10) were from Sigma-Aldrich and Santa Cruz Biotechnology, respectively.


  Results and Discussion
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Materials and Methods
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Retained TNFR-I Signaling in NIK-mutant EFs.
NIK was originally identified as a kinase that participates in an NF-{kappa}B–inducing signaling cascade induced by TNF, CD95, and IL-1 (6). To assess the impact of NIK mutation in TNF responsiveness, we treated EFs from both wild-type and aly mice with human TNF, which signals only through mouse TNFR-I (23), and assessed NF-{kappa}B activation by Western blot analysis. Rapid I{kappa}B{alpha} degradation concomitant with the appearance of phosphorylated I{kappa}B{alpha} was observed with similar kinetics in EFs from both wild-type and aly mice (Fig 1 A). 30 min after stimulation with TNF, I{kappa}B{alpha} started to recover similarly in both wild-type and aly mice (Fig 1 B). I{kappa}Bß degradation in response to TNF was also indistinguishable between wild-type and aly mice (data not shown). We also tested TNF responsiveness by titrating the TNF concentration between 0.1 and 100 U/ml. In this range, TNF sensitivity assessed by I{kappa}B{alpha} degradation, and I{kappa}B{alpha} phosphorylation was indistinguishable between wild-type and aly mice (Fig 1 C). Using an NF-{kappa}B–binding oligonucleotide probe in an electrophoretic mobility shift assay (EMSA), we also observed a very similar level of NF-{kappa}B activation between wild-type and aly mice in TNF-stimulated EFs or TNF-stimulated thymocytes (data not shown). Furthermore, IL-1 and IL-6 production from TNF-stimulated EFs was indistinguishable between wild-type and aly mice (data not shown). These results demonstrate that NF-{kappa}B activation through TNFR-I is not affected by the NIK mutation.



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Figure 1. NF-{kappa}B activation in response to TNF is retained in EFs from NIK-mutant mice. EFs from wild-type mice (WT) and aly mice (aly) were stimulated with TNF (100 U/ml), and cells were harvested at the indicated time points (A and B). I{kappa}B{alpha} degradation (detected by anti-I{kappa}B{alpha} Ab) and I{kappa}B{alpha} phosphorylation (detected by phospho-specific anti-I{kappa}B{alpha} Ab) was assessed by Western blot analysis. Arrow indicates phosphorylated I{kappa}B{alpha} (A). EFs were stimulated with different concentrations (Conc.) of TNF ranging from 0.1 to 100 U/ml as indicated, and cells were harvested 7 min after stimulation (C). EFs were stimulated with TNF (100 U/ml) with or without prior treatment with 1 µM wortmannin (D). The same blot was probed with anti-actin Ab (bottom).

Recently, it was demonstrated that Akt serine–threonine kinase is involved in the activation of NF-{kappa}B by TNF (24). Although the results described above do not suggest a role for NIK in TNFR-I signaling, it is possible that NIK plays an important role in NF-{kappa}B activation in combination with Akt. We therefore tested the combined effect of NIK and Akt in the NF-{kappa}B–inducing pathway downstream of the TNFR-I. I{kappa}B{alpha} degradation occurred 10 min after TNF stimulation in wild-type EFs, even in the presence of 1 µM wortmannin, a sufficient concentration for blockade of the PI(3)K–Akt pathway (references (24) and (25); Fig 1 D). This suggests that Akt, the downstream target of PI(3)K, by itself has no major role in NF-{kappa}B activation by TNF. Additionally, no obvious effect of wortmannin on I{kappa}B{alpha} degradation was observed in aly mouse EFs, indicating that NF-{kappa}B activation by TNF can occur even when the functions of both NIK and Akt are inhibited. We failed to observe phosphorylation of Akt in response to TNF when we probed the same blot with phospho-specific anti-Akt Ab in this experimental setting (data not shown). These results suggest that neither NIK nor Akt is essential for NF-{kappa}B activation by TNF and that other IKK kinase(s) can substitute for NIK and Akt in NF-{kappa}B activation by TNF, at least in EFs. Elucidation of the IKK kinase(s) that activates IKK in response to TNF awaits further study.

Indispensable Role of NIK and IKK{alpha} in NF-{kappa}B Activation through LTßR.
We intended to evaluate the role of NIK in TNFR-I and LTßR signaling with the use of in vivo mouse models, rather than introducing enforced gene-expression systems. To this end, we transfected EFs (which express both TNFR-I and LTßR) only with a reporter plasmid that has three repeats of the NF-{kappa}B site upstream of a minimal thymidine kinase promoter and a luciferase gene, and then stimulated the transfected EFs with TNF or agonistic anti-LTßR mAb (AC.H6). EFs from both wild-type and aly mice showed upregulation of luciferase activity in response to human TNF (Fig 2 A). The normal level of TNF responsiveness in aly mice is consistent with the results shown above (Fig 1). In contrast to TNF stimulation, NF-{kappa}B activation in response to agonistic anti-LTßR mAb was significantly reduced in aly mouse EFs, indicating that NIK is involved in LTßR signaling (Fig 2 A).



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Figure 2. Impaired NF-{kappa}B activation in response to LTßR stimulation in NIK-mutant and IKK{alpha}-deficient EFs. Wild-type EFs and aly mouse EFs were transfected with NF-{kappa}B reporter plasmids and stimulated with TNF or agonistic anti-LTßR mAb. 8 h later, NF-{kappa}B activation was assessed by the measurement of luciferase activities. Data were expressed as fold activation compared with stimulation by control mAb, and the results were plotted as the mean ± SEM for a total of four independent experiments. White and black bars represent wild-type EFs and aly mouse EFs, respectively (A). EFs from wild-type, aly, and IKK{alpha}-deficient mice (IKK{alpha}-/-) were stimulated with agonistic anti-LTßR mAb for 1 h, and I{kappa}B{alpha} phosphorylation was assessed by Western blot analysis (B, top). For the assessment of the basal level of NF-{kappa}B activation, EFs were treated with ALLN alone. Phosphorylated I{kappa}B{alpha} is indicated as P. NS, nonspecific bands. The same blot was probed with anti-actin Ab (B, bottom). TNF stimulation induced I{kappa}B{alpha} phosphorylation in IKK{alpha}-deficient EFs as in wild-type and aly EFs (C). LTßR expression assessed by flow-cytometric analysis with anti-LTßR mAb (D, thick line) was similar among wild-type, aly, and IKK{alpha}-deficient EFs (D). Anti-KLH mAb Ha4/8 (D, thin line) and mouse hybridoma cells (top left) were used as negative control.

With the combination of EFs and agonistic anti-LTßR mAb, signals for NF-{kappa}B activation assessed by EMSA were not strong enough to evaluate the role of NIK in NF-{kappa}B activation through the LTßR (our unpublished observation). Involvement of NIK in LTßR signaling was therefore examined by detection of I{kappa}B{alpha} phosphorylation in response to agonistic anti-LTßR mAb. In wild-type EFs stimulated with agonistic anti-LTßR mAb for 1 h, I{kappa}B{alpha} phosphorylation was easily detected by Western blot analysis (Fig 2 B). In contrast, aly mouse EFs showed minimal, if any, phosphorylated I{kappa}B{alpha} after LTßR stimulation. Taken together, these results demonstrate that NIK is essential for NF-{kappa}B activation in LTßR signaling, which accounts for the abnormal lymphoid organogenesis in aly mice.

We have previously demonstrated that EFs isolated from IKK{alpha}-deficient mice can activate NF-{kappa}B in response to TNF and IL-1, suggesting that IKK{alpha} is not essential for either the TNF or IL-1 signaling pathways (11). The dispensable role of IKK{alpha} in NF-{kappa}B activation through TNFR-I was also confirmed by the detection of phosphorylated I{kappa}B{alpha} in IKK{alpha}-deficient EFs after TNF stimulation (Fig 2 C). Because NIK is essential for LTßR signaling, as demonstrated above, and NIK has been shown to phosphorylate IKK{alpha} (26), it is important to determine whether IKK{alpha} is also essential for LTßR signaling. We therefore treated EFs from IKK{alpha}-deficient mice with agonistic anti-LTßR mAb and assessed NF-{kappa}B activation by the detection of phosphorylated I{kappa}B{alpha}. We found that IKK{alpha}-deficient EFs showed no I{kappa}B{alpha} phosphorylation after LTßR stimulation, suggesting that NIK–IKK{alpha} constitutes an important pathway in LTßR signaling (Fig 2 B). LTßR expression assessed by flow-cytometric analysis with anti-LTßR mAb (AF.H6) was similar among wild-type, aly, and IKK{alpha}-deficient EFs (Fig 2 D). The basal level of NF-{kappa}B activation, assessed by the treatment of EFs with ALLN alone, which blocks the degradation of phosphorylated I{kappa}B{alpha} by proteasomes (3), was reduced in aly mice and reduced more profoundly in IKK{alpha}-deficient mice (Fig 2 B).

The essential role of IKK{alpha} in LTßR signaling was examined by investigation of lymphoid organogenesis in IKK{alpha}-deficient mice. Because IKK{alpha}-deficient mice show perinatal death associated with abnormal limb and skin development, lymphoid organogenesis in IKK{alpha}-deficient mice was assessed by the development of PP from 18.5 d.p.c. embryos. PP formation in control embryos (n = 8) was easily detected by whole-mount immunohistochemistry with mAb against VCAM-1 (Fig 3 A); VCAM-1+ cells accumulate at the site of PP development starting from 15.5 d.p.c. and can be used as a stromal marker for PP formation (21). In contrast, no PP formation was detected in intestines isolated from IKK{alpha}-deficient embryos (n = 7; Fig 3 B). A similar lack of VCAM-1+ cell accumulation in embryonic intestines has been demonstrated in aly mice (21). This result shows that LTßR signaling is fundamentally impaired in IKK{alpha}-deficient mice. Together, these findings are important in providing clear evidence that IKK{alpha} is involved in cytokine receptor signaling in vivo.



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Figure 3. Lack of PP development in IKK{alpha}-deficient mice. Embryonic intestines isolated from control littermates (A) and IKK{alpha}-deficient mice (B) were stained with anti–VCAM-1 mAb. Arrows indicate the sites of PPs. Original magnification, x10.

It is important to note that abnormal lymphoid organogenesis in IKK{alpha}-deficient mice is not due to defective receptor activator of NF-{kappa}B (RANK) signaling, because mice deficient in RANK have PPs despite their lack of peripheral LNs (27); RANK activates NF-{kappa}B by recruiting TRAF6, which has not been observed to associate with LTßR (16) (17). It remains possible, however, that there exist other undefined NIK–IKK{alpha}-activating receptor pathways involved in lymphoid organogenesis beyond LTßR.

The above data strongly suggest that NIK and IKK{alpha} together control LTßR signaling with a close mechanistic relationship in their pathway. We have therefore reasoned that impaired LTßR signaling in aly mice may be due to defective interaction between mutated NIK and IKK{alpha}. To investigate this, NIK and IKK{alpha} were coexpressed in COS-7 cells, and protein interactions were assessed by immunoprecipitation. Association of wild-type NIK with IKK{alpha} was easily detected (Fig 4). In contrast, association of aly type NIK, which corresponds to a G855R substitution in mice, with IKK{alpha} was disrupted by the mutation, providing further support for the role of NIK–IKK{alpha} as an essential pathway for NF-{kappa}B activation in LTßR signaling. Despite this finding, the possibility remains that aly mice might have a different phenotype from NIK-null mutation mice.



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Figure 4. Disruption of interaction with IKK{alpha} by the aly-type NIK mutation. Protein extracts from COS-7 cells transfected with the indicated cDNAs were immunoprecipitated with anti-Myc mAb and detected with either anti-Flag mAb (top) or anti-Myc mAb (center). Expression of NIK was verified by Western blot analysis of total cell lysates with anti-Flag mAb (bottom). aly-type NIK (aly) has an amino acid substitution (G860R) in the COOH-terminal region. Minus (-) indicates transfection with empty vectors.

It is unclear whether TRAFs mediate all of the signaling activities of LTßR. In fact, mice deficient in TRAF2, -3, or -5 show LN development (28) (29). In support of the dispensable role of TRAFs in LTßR signaling, recent mutational analyses of the cytoplasmic region of LTßR have demonstrated a TRAF-independent mechanism of NF-{kappa}B activation through LTßR (30). Consistent with this idea, TRAF–NIK interaction in COS-7 cells as assessed by immunoprecipitation was not affected by the aly mutation (our unpublished observation), although the aly mutation resides in a putative TRAF-binding domain of NIK (31). Elucidation of the molecular mechanisms by which NIK becomes activated after LTßR stimulation will be critical for understanding the biological nature of LTßR signaling.


  Acknowledgements
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We thank Drs. J.L. Browning, D. Wallach, T. Takemori, and K. Miyazono for providing us with reagents. We thank Drs. K. Honda, M. Shono, S. Noji, H. Ohuchi, T. Matsuzaki, K. Tsuchida, Z.-g. Liu, and C.F. Ware for valuable suggestions. We also thank Ms. M. Kimura for technical assistance.

This work was supported in part by Special Coordination Funds and Grant-in-Aid for Scientific Research of the Ministry of Education, Culture, Sports, Science, and Technology, the Japanese Government, and by the Japan Research Foundation for Clinical Pharmacology.

Submitted: 2 November 2000
Revised: 18 January 2001
Accepted: 24 January 2001


  References
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Acknowledgements
References

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