Characterization of IL-4 and IL-13 signals dependent on the human IL-13 receptor {alpha} chain 1: redundancy of requirement of tyrosine residue for STAT3 activation

Ritsuko Umeshita-Suyama1,3, Rie Sugimoto1, Mina Akaiwa1, Kazuhiko Arima1, Bin Yu1, Morimasa Wada2, Michihiko Kuwano2, Koichi Nakajima4, Naotaka Hamasaki1 and Kenji Izuhara1

1 Department of Clinical Chemistry and Laboratory Medicine, and
2 Department of Medical Biochemistry, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
3 R & D Institute, UNITIKA, Ltd, 23 Ujikozakura, Uji, 611-0021 Japan
4 Department of Immunology, Osaka City University Medical School, 1-4-3 Asahi-machi, Abeno-ku, Osaka 545-8585, Osaka, Japan

Correspondence to: K. Izuhara (Present address: Department of Biochemistry, Saga Medical School, 5-1-1 Nabeshima, Saga 849-8501, Japan)


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
IL-4 and IL-13 are pleiotropic cytokines whose biological activities overlap with each other. IL-13 receptor {alpha} chain 1 (IL-13R{alpha}1) is necessary for binding to IL-13, and the heterodimer composed of IL-13R{alpha}1 and IL-4R {alpha} chain transduces IL-13 and IL-4 signals; however, the functional mapping of the intracellular domain of IL-13R{alpha}1 is not fully understood. In this study, we constructed wild and mutated types of human IL-13R{alpha}1, and analyzed IL-4 and IL-13 signals using an IL-13R{alpha}1-transfected human B cell line. Expression of IL-13R{alpha}1 evoked STAT3 activation by IL-4 and IL-13, and in stimulated human B cells, on which IL-13R{alpha}1 was highly expressed, IL-4 and IL-13 induced STAT3 activation. Replacement of the two tyrosine residues completely abolished STAT3 activation, although replacing either tyrosine residue alone retained it. Furthermore, we found that the Box1 region and the C-terminal tail of IL-13R{alpha}1 were critical for binding to Tyk2, and activation of Jak1, Tyk2, the insulin receptor substrate-1 and STAT6 respectively. These results suggest that STAT3 activation is involved with IL-4 and IL-13 signals in human B cells along with the activation of STAT6, and that there is a unique sequence in IL-13R{alpha}1 to activate STAT3.

Keywords: B cell, Jak1, Jak3, STAT6


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
IL-13 is a pleiotropic cytokine produced by Th2-type cells and mast cells (1,2). Its biological activities overlap with those of another cytokine, IL-4, and those include anti-inflammatory actions on monocytes, production of chemokines and induction of arachidonic acid-metabolizing enzymes on bronchial epithelial cells. However, IL-4, but not IL-13, induces expansion of Th2-type cells and proliferation of T cells in both the mouse and human systems (1,3,4). IL-4 acts on both mouse and human B cells in the same way, in that it induces class switching including IgE, and expression of CD23 and MHC class II. In contrast, IL-13 behaves on B cells differently in the mouse and human systems: human IL-13 induces class switching toward IgE and IgG4, as well as expression of CD23 and MHC class II on B cells, whereas mouse IL-13 does not exert such actions (1). It still remains undetermined whether this is because mouse IL-13R is not expressed on mouse B cells or because the machinery for mouse IL-13 is impaired in mouse B cells. It has been recently shown that IL-13 has a pivotal role in the defense mechanism against parasite infection (5,6) and that it is involved in the pathogenesis of bronchial asthma (79) in mice. We and others have recently proved that a polymorphism located in the human IL-13 gene is correlated with asthma (10,11).

To date, two IL-13-binding units have been identified, the IL-13 receptor {alpha} chain 1 (IL-13R{alpha}1) and IL-13R{alpha}2 in both the mouse and human systems (1215). Both mouse and human IL-13R{alpha}1 have cytoplasmic domains 60 amino acids long, in which the Box1 region and two tyrosine residues exist (Y402 and Y405) (12,14). One of the tyrosine residues, Y405, is followed at the +3 position by Gln, which represents a consensus STAT3 recruitment and activation motif (16). The IL-13R is composed of the heterodimer consisting of IL-13R{alpha}1 and IL-4 receptor {alpha} chain (IL-4R{alpha}) (14). This heterodimer can also transduce IL-4 signals (17,18), so it is called type II IL-4R. IL-13R{alpha}2 is assumed to act as a decoy receptor because it has a very short cytoplasmic domain (15); however, the details of the function of this molecule remain unclear.

It is known that engagement of the IL-13R by IL-13 induces activation of a variety of signal-transducing molecules (19, 20). These molecules converge into two pathways: the Jak–STAT pathway and the phosphatidylinositol-3 (PI3) kinase pathway. The former includes activation of Jak1, Jak2 and Tyk2, followed by activation of STAT6 (17,2125). The latter includes tyrosine phosphorylation of the insulin receptor substrate (IRS)-1/2, followed by activation of PI3-kinase (17,21,25,26). Analyses of STAT6 knockout mice have verified the necessity of STAT6 for class switching on B cells and expansion of Th2-type cells induced by IL-4, as well as anti-inflammatory actions on monocytes induced by IL-4 and IL-13 (19,20). It has been recently demonstrated that expression of mouse IL-13R{alpha}1 induces activation of STAT3, in addition to STAT6 (27); however, the necessity for tyrosine residues in STAT3 activation has remained unclear. To date, no study has been reported for human IL-13R{alpha}1.

In this study, we constructed wild and mutated types of human IL-13R{alpha}1, and analyzed IL-13 and IL-4 signals in these types of IL-13R{alpha}1-transfected human B cell line. It turned out that the C-terminal tail of IL-13R{alpha}1 is important for activation of Jak1, Tyk2, IRS-1 and STAT6. We furthermore demonstrated that STAT3 activation by IL-4 and IL-13 occurred when IL-13R{alpha}1 was expressed, which is also the case in primary human B cells. Replacement of either tyrosine residue can still cause STAT3 activation; however, replacement of both tyrosine residues diminished STAT3 activation. These results indicate that there exists a novel signal transduction pathway of IL-13 and IL-4 in human B cells, and that there is a redundancy for requirement of tyrosine residue for STAT3 activation via IL-13R{alpha}1.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Plasmid construction
Full-length human IL-13R{alpha}1 cDNA was cloned from a cDNA library of HeLa cells, as described before (28). Three different nucleotides existed between ours and that published before (14): T -> C at 473, C -> T at 833 and A -> G at 1157 from the starting point of translation. A survey of nucleotide sequencing from peripheral blood cells of five healthy Japanese donors showed C, C and G respectively in all samples, so that only T at 883 was replaced with C, using the Kunkel method (29). A 5'-untranslated region (–10 to –1 nucleotides), modified to fit the Kozak sequence (GCATGCCGCCATG), as well as the signal sequence (1–84 nucleotides) of the human IL-4R{alpha} gene, followed by the Flag sequence, were attached to the N-terminus of the corrected IL-13R{alpha}1, whose signal sequence (1–63 nucleotides) had been deleted (IL-13R{alpha}1Full; Fig. 1AGo). Then this fragment was incorporated into pIRES1hyg (Clontech, Palo Alto, CA) or pME18S (kindly provided by Dr T. Hara, Tokyo University).




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Fig. 1. Schematic model of the mutated types of IL-13R{alpha}1 and expression of the transfected IL-13R{alpha}1. (A) A schematic model of the structures of wild and mutated types of IL-13R{alpha}1. ssIL-4R, TM, Y and F denote the signal sequence of IL-4R{alpha}, transmembrane domain, tyrosine residue and phenylalanine residue respectively. (B) Expression of wild and mutated types of IL-13R{alpha}1 on the transfectants. Each transfectant was incubated with (solid line) or without (dashed line) anti-Flag M2 antibody, followed by FITC-conjugated anti-mouse Ig antibody.

 
Plasmids containing two kinds of truncated IL-13R{alpha}1 were constructed by the PCR method using primers containing a stop codon (common: 5'-ACGGAAACTCAGCCACCTGTG-3', 5'-GGAACTCGAGGGAATATAATAATCTAGAG-3' for IL-13R{alpha}1{Delta}CP, 5'-ATCATTCTCGAGTCCAAACATTTCTTAA-3' for IL-13R{alpha}1{Delta}CT). By this method, the coding regions were terminated at 370 and 385 amino acids from the starting point of translation of IL-13R{alpha}1. These truncated types of IL-13R{alpha}1 were named IL-13R{alpha}1{Delta}CP and IL-13R{alpha}1{Delta}CT respectively (Fig. 1AGo). To replace two tyrosine residues at 402 and 405 amino acids with phenylalanine, we used the PCR method using the primers 5'-GGAAGAAGTTCGACATCTATGAGAAGCAAACCAAGGAG-3' for IL-13R{alpha}1Y402F, 5'-GG-AAGAAGTACGACATCTTTGAGAAGCAAACCAAGGAG-3', for IL-13R{alpha}1Y405F and 5'-GGAAGAAGTTCGACATCTTTGAGAAGCAAACCAAGGAG-3' for IL-13R{alpha}1FF (Fig. 1AGo).

The plasmid encoding Tyk2 incorporated to pRK2 was kindly supplied by Dr K. Shimoda (Kyushu University, Fukuoka, Japan).

Cells and transfection
A human B cell line, DND-39, was provided by Hayashibara Biochemical Laboratories (Fujisaki Cell Center, Okayama, Japan). DND-39 cells were maintained in RPMI 1640 medium supplemented with 10% FCS, 100 µg/ml streptomycin and 100 U/ml penicillin. pGL3-enhancer vector (Promega, Madison, WI) into which the promoter region from –187 to +6 of human I{varepsilon} was inserted, and a blasticidin-resisitant gene (Funakoshi, Tokyo, Japan), were co-transfected to DND-39 cells by electroporation. The resulting stable transfectant was named DND-39/G{varepsilon}. These cells were maintained in a culture medium containing 6 µg/ml of blasticidin S hydrochloride (Funakoshi). Plasmids containing wild and mutated types of IL-13R{alpha}1 were transfected to DND-39/G{varepsilon} cells by electroporation, and stable transfectants were named DND-39/ G{varepsilon}/IL-13R{alpha}1Full, DND-39/G{varepsilon}/IL-13R{alpha}1{Delta}CP, DND-39/G{varepsilon}/IL-13R{alpha}1{Delta}CT, DND-39/G{varepsilon}/IL-13R{alpha}1Y402F, DND-39/G{varepsilon}/IL-13R{alpha}1Y405F and DND-39/G{varepsilon}/IL-13R{alpha}1FF. These cells were maintained in a culture medium containing 6 µg/ml of blasticidin S hydrochloride and 250 µg/ml of hygromycin B (Wako, Osaka, Japan).

COS7 cells were maintained in DMEM supplemented with 10% FCS, 100 µg/ml streptomycin and 100 U/ml penicillin.

Procedures to purify human B cells were described before (30). Human B cells were isolated from buffy coats provided by Kyushu University Hospital. Mononuclear cells were separated over Ficoll-Hypaque. B cells were isolated from mononuclear cells with magnetic beads conjugated with anti-CD19 antibody (Dynal, Oslo, Norway). Isolated B cells were cultured in RPMI 1640 medium with 10% FCS, 100 U/ml of penicillin and 100 µg/ml of streptomycin either in the absence or presence of 10 µg/ml of anti-IgM antibody (Cappel, Aurora, OH) and 0.5µg/ml of anti-CD40 antibody (Immunotech, Marseille, France) for the indicated period.

Harvested cells were stimulated with the indicated concentration of IL-4 (provided by Bayer Yakuhin, Kyoto, Japan) or IL-13 (PeproTech, Rocky Hill, NJ) for the indicated period.

Flow cytometry
The washed cells were incubated with anti-Flag M2 antibody (Sigma, St Louis, MO), followed by FITC-conjugated anti-mouse Ig antibody (Zymed, San Francisco, CA). Quantitation was performed using FACScan flow cytometry (Becton Dickinson, San Jose, CA).

Luciferase activity assay
Procedures of luciferase activity assay were performed as described before (31). The cells were incubated with the indicated concentrations of either IL-4 or IL-13 for 24 h. After the cells were washed once by PBS and lysed with reporter lysis buffer (Toyoink, Tokyo, Japan), cell lysates were mixed with luciferase assay reagent (Toyoink).

Immunoprecipitation and Western blotting
Procedures of immunoprecipitation and Western blotting were conducted as previously described (24). For immunoprecipitation, the cells were lysed in lysis buffer containing 1 or 0.5% Triton X-100, followed by incubation with the indicated antibodies. Proteins eluted by boiling with SDS–PAGE sample buffer or cell lysates were applied to SDS–PAGE and transferred electrophoretically to a PVDF membrane (Amersham, Arlington Heights, IL). Proteins were probed with the indicated antibodies and visualized by enhanced chemiluminescence (Amersham).

The antibodies used were anti-STAT3, anti-STAT6, anti-Jak1, anti-Jak3, anti-Tyk2 (Santa Cruz Biotechnology, Santa Cruz, CA), anti-phosphotyrosine (PY) (4G10), anti-IRS-1 (Upstate Biotechnology, Lake Placid, NY), anti-phospho STAT3 (New England Biolabs, Beverly, MA) and anti-Flag M2. Anti-STAT5 was kindly provided from Dr H. Wakao (Helix Research Institute, Kisarazu, Japan).

RT-PCR
Total RNA was extracted from purified human B cells stimulated with 10 µg/ml of anti-IgM antibody and 0.5 µg/ml of anti-CD40 antibody for the indicated period by Isogen (Nippongene, Tokyo, Japan), and then treated with DNase I (Stratagene, La Jolla, CA) and RNase inhibitor (Roche Diagnostics, Tokyo, Japan). Reverse transcrition was performed using a TaKaRa RNA PCR kit (Takara Shuzo, Otsu, Japan).

The competitor for ß-actin was constructed with cDNA as a template using the primers 5'-CCTCGCCTTTGCCGATCCGTGATGGTGGGCATGGGT-3' and 5'-ATGAGGTAGTCAGTCAGGTCCCG-3', corresponding to the portion between –49 and –32 attached to that between +126 and +143 (underlined) and that between +568 and +546 from the starting point of translation respectively. Consequently, the product of the competitor for ß-actin was composed of the portion between –49 and –32 attached to that between +126 and +568. The PCR reaction was performed with cDNA as a template using the primers corresponding to the portion between –49 and –32 and that between +568 and +546. This was done in the presence of various amounts of competitors after an initial 5 min denaturation at 94°C, followed by 35 cycles of 94°C for 45 s, 57°C for 45 s and 72°C for 45 s. After the amount of total RNA was normalized based on that of ß-actin, the competitive PCR for IL-13R{alpha}1 was performed. The competitor for IL-13R{alpha}1 was constructed with cDNA as a template using the primers 5'-CAGTGTAGCACCAATGAGAGTGAG-3' and 5'-TCAGGTTTCACACGGGAAGTTAAAG-3', corresponding to the portion between +301 and +324 and that between +686 and +662 from the starting point of translation respectively. Then the portion between +353 and +404 was cleaved by NsiI. Consequently, the product of the competitor for IL-13R{alpha}1 was composed of the portion between +301 and +352 attached to that between +405 and +686 (334 bp). The PCR reaction was performed with cDNA as a template using the above primers in the presence of various amounts of competitors after an initial 5 min denaturation at 94°C, followed by 45 cycles of 94°C for 45 s, 57°C for 45 s and 72°C for 45 s (386 bp).


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Generation of transfectants and expression of the transfected IL-13R{alpha}1
To generate IL-13R{alpha}1-expressing transfectants, we employed a human B cell line, DND-39. Flow cytometry confirmed that this cell line expressed IL-4R{alpha} and {gamma}c, but RT-PCR showed that it did not express IL-13R{alpha}1 (data not shown). Plasmids coding six kinds of IL-13R{alpha}1 were transfected by electroporation, and several positive clones for each IL-13R{alpha}1 were obtained. The expression levels of the transfected IL-13R{alpha}1 were almost the same in each clone of DND-39/G{varepsilon}/IL-13R{alpha}1Full8C, DND-39/G{varepsilon}/IL-13R{alpha}1{Delta}CP, DND-39/G{varepsilon}/IL-13R{alpha}1{Delta}CT and DND-39/G{varepsilon}/IL-13R{alpha}1Y405F, although clones of DND-39/G{varepsilon}/IL-13R{alpha}1FF and DND-39/G{varepsilon}/IL-13R{alpha}1402F expressed comparatively lower levels of IL-13R{alpha}1 (Fig. 1BGo). DND-39/G{varepsilon}/IL-13R{alpha}1Full8C was used for the following experiments as DND-39/G{varepsilon}/IL-13R{alpha}1Full. Another clone of DND-39/G{varepsilon}/IL-13R{alpha}1Full, DND-39/G{varepsilon}/IL-13R{alpha}1 FullE5, expressed almost the same level of IL-13R{alpha}1 as DND-39/G{varepsilon}/IL-13R{alpha}1FF (Fig. 1BGo).

STAT6 activation of the transfectants
It has been confirmed that STAT6 has a pivotal role for IL-4 and IL-13 signals (19,20). To clarify the role of the C-terminal tail of IL-13R{alpha}1 in STAT6 activation, we first analyzed it using four kinds of transfectants: mock-transfected DND-39, DND-39/G{varepsilon}/IL-13R{alpha}1Full, DND-39/G{varepsilon}/IL-13R{alpha}1{Delta}CP and DND-39/G{varepsilon}/IL-13R{alpha}1{Delta}CT. The mock-transfected DND-39 cells induced tyrosine phosphorylation of STAT6 by IL-4, but not by IL-13, whereas DND-39/G{varepsilon}/IL-13R{alpha}1Full did so by both IL-4 and IL-13, demonstrating that the transfected IL-13R{alpha}1 transduced the IL-13 signal (Fig. 2AGo). In contrast, tyrosine phosphorylation of STAT6 by IL-4 declined and that by IL-13 was not detected in either DND-39/G{varepsilon}/IL-13R{alpha}1{Delta}CP or DND-39/G{varepsilon}/IL-13R{alpha}1{Delta}CT (Fig. 2AGo). The amounts of immunoprecipitated STAT6 were almost the same among the transfectants (Fig. 2AGo). These results clearly indicated that the C-terminal tail of IL-13R{alpha}1 was essential for STAT6 activation by IL-13 and affected that by IL-4.




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Fig. 2. Activation of STAT6 induced by IL-4 and IL-13 in the transfectants. (A) Immunoprecipitates of STAT6 stimulated by 10 ng/ml of IL-4 or 50 ng/ml of IL-13 for 10 min of each transfectant were probed with anti-PY antibody (upper panel) or anti-STAT6 antibody (lower panel). (B) Luciferase activities induced by IL-4 and IL-13 in the transfectants. Each transfectant was incubated with the indicated concentration of either IL-4 (dashed bars) or IL-13 (closed bars) for 24 h. Each experiment was done with three samples and the mean values are shown.

 
As the I{varepsilon} promoter region contains the binding site for STAT6, we next analyzed the transcription activity of IL-4 and IL-13 on the I{varepsilon} promoter in the transfectants. The mock-transfected DND-39 cells displayed the transcription activity of IL-4 on the I{varepsilon} promoter in a dose-dependent manner, but not that of IL-13, whereas DND-39/G{varepsilon}/IL-13R{alpha}1Full displayed that of both IL-4 and IL-13 in a dose-dependent manner (Fig. 2BGo). In contrast, the transcription activity of IL-4 on the I{varepsilon} promoter decreased and that of IL-13 disappeared in both DND-39/G{varepsilon}/IL-13R{alpha}1{Delta}CP and DND-39/G{varepsilon}/IL-13R{alpha}1{Delta}CT, in line with the results of STAT6 activation (Fig. 2BGo). Several clones of each transfectant showed the same tendency (data not shown). These results suggest that the transcription activity of IL-4 and IL-13 on the I{varepsilon} promoter parallels the STAT6 activation.

Activation of Jak molecules and tyrosine phosphorylation of IRS-1 of the transfectants
It is widely known that Jak molecules locate upstream of STAT molecules and that activation of Jak molecules is followed by activation of STAT molecules (32,33). To elucidate the effect of the C-terminal tail of IL-13R{alpha}1 for STAT6 activation, we next analyzed activation of Jak molecules induced by IL-4 and IL-13 in the transfectants. Jak1 has been shown to be activated by both IL-4 and IL-13, and associate with IL-4R{alpha} upon stimulation of IL-4 (3437). The mock-transfected DND-39 cells induced tyrosine phosphorylation of Jak1 by IL-4, but not by IL-13, whereas DND-39/G{varepsilon}/IL-13R{alpha}1Full did so by both IL-4 and IL-13 (Fig. 3AGo). In contrast, tyrosine phosphorylation of Jak1 by IL-13 was not detected and that by IL-4 was markedly inhibited in both DND-39/G{varepsilon}/IL-13R{alpha}1{Delta}CP and DND-39/G{varepsilon}/IL-13R{alpha}1{Delta}CT (Fig. 3AGo). It has been shown that Jak3 constitutively associates with {gamma}c, and is tyrosine phosphorylated upon stimulation of IL-4 (36,38). Activation of Jak3 occurred by IL-4 stimulation in the mock-transfected DND-39 cells and activation of Jak3 by IL-4 was slightly decreased in DND-39/G{varepsilon}/IL-13R{alpha}1Full or DND-39/G{varepsilon}/IL-13R{alpha}1{Delta}CP or DND-39/G{varepsilon}/IL-13R{alpha}1{Delta}CT, whereas IL-13 did not cause tyrosine phosphorylation of Jak3 in any of the four transfectants (Fig. 3BGo). It has been demonstrated that Tyk2 is tyrosine phosphorylated by both human and mouse IL-13 (17,21,23,25). Activation of Tyk2 was induced by IL-4 and IL-13 only in DND-39/G{varepsilon}/IL-13R{alpha}1Full; however, it was not induced by IL-4 or IL-13 in the mock-transfected DND-39, DND-39/G{varepsilon}/IL-13R{alpha}1{Delta}CP or DND-39/G{varepsilon}/IL-13R{alpha}1{Delta}CT (Fig. 3CGo). The amounts of immunoprecipitated Jak1, Jak3 and Tyk2 were almost the same among the transfectants. (Fig. 3AGo–C). Jak2 was also activated as the same manner as Jak1 in the transfectants (data not shown).






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Fig. 3. Activation of Jak molecules and IRS-1 induced by IL-4 and IL-13 in the transfectants. Immunoprecipitates of Jak1 (A), Jak3 (B), Tyk2 (C) and IRS-1 (D) stimulated by 10 ng/ml of IL-4 or 50 ng/ml of IL-13 for 10 min of each transfectant were probed with anti-PY antibody (upper panel) or anti-Jak1, Jak3 and Tyk2 antibodies (lower panel). In (D), cell lysates for immunoprecipitates of IRS-1 were stained with Coomassie brilliant blue.

 
It has been already shown that stimuli of both IL-4 and IL-13 lead to tyrosine phosphorylation of IRS-1/2, important for recruitment of PI3-kinase to IL-4R{alpha} (20). To examine the effect of the C-terminal tail of IL-13R{alpha}1 for tyrosine phosphorylation of IRS-1/2, we analyzed tyrosine phosphorylation of IRS-1 and IRS-2 of the transfectants. Neither expression nor tyrosine phosphorylation of IRS-2 by IL-4 or IL-13 was detected in these transfectants (data not shown). The mock-transfected DND-39 cells induced tyrosine phosphorylation of IRS-1 by IL-4, but not by IL-13, whereas DND-39/G{varepsilon}/IL-13R{alpha}1Full did so by both IL-4 and IL-13 (Fig. 3DGo). In contrast, tyrosine phosphorylation of IRS-1 significantly decreased with either IL-4 or IL-13 in both DND-39/G{varepsilon}/IL-13R{alpha}1{Delta}CP and DND-39/G{varepsilon}/IL-13R{alpha}1{Delta}CT as well as Jak1 activation (Fig. 3DGo), which is compatible with previous reports that IRS-1/2 is a substrate of Jak1 (37,39,40). The amounts of the protein of the applied samples were normalized. Protein staining of the membrane showed that invariable amounts of proteins were applied for each sample (Fig. 3DGo). These results suggested that both IL-4 and IL-13 were capable of inducing tyrosine phosphorylation of IRS-1 in DND-39 cells and that the C-terminal tail of IL-13R{alpha}1 was critical.

Association of Tyk2 with IL-13R{alpha}1
It has been shown that Box1 regions located at the proximal regions of the cytoplasmic domains of the cytokine receptors are necessary for binding to Jak molecules (41). The result that expression of IL-13R{alpha}1 was essential for activation of Tyk2 prompted us to investigate whether the Box1 region in the cytoplasmic domain of IL-13R{alpha}1 is required for association of Tyk2 with IL-13R{alpha}1. We co-transfected Tyk2 and either IL-13R{alpha}1Full or IL-13R{alpha}1{Delta}CP or IL-13R{alpha}1{Delta}CT into COS7 cells. The constitutive association of Tyk2 with IL-13R{alpha}1Full and IL-13R{alpha}1{Delta}CT, but not IL-13R{alpha}1{Delta}CP, was detected and stimulation of IL-13 enhanced the association of Tyk2 with IL-13R{alpha}1Full, but not IL-13R{alpha}1{Delta}CT (Fig. 4Go). The expression levels of Tyk2 and the receptors were invariant (Fig. 4Go). These results demonstrated that the Box1 region in IL-13R{alpha}1 was critical for the association with Tyk2 and that engagement of IL-13R{alpha}1 by IL-13 enhanced this association.



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Fig. 4. Association of Tyk2 with IL-13R{alpha}1. COS7 cells co-transfected with Tyk2 and the indicated type of IL-13R{alpha}1 were stimulated by 50 ng/ml of IL-13 for 10 min. Immunoprecipitates with anti-Flag M2 antibody (upper and middle panels) or cell lysates (lower panel) were probed with anti-Tyk2 antibody (upper and lower panels) or anti-Flag M2 antibody (middle panel).

 
STAT3 activation in the transfectants
To explore the possibility that IL-4 and IL-13 induce activation of STAT molecules other than STAT6, we next studied tyrosine phosphorylation of STAT molecules induced by either IL-4 or IL-13 in the transfectants. Neither expression nor tyrosine phosphorylation of STAT1, 2 and 4 induced by either IL-4 or IL-13 was observed in these transfectants (data not shown). Expression of STAT5 was detected; however, neither IL-4 nor IL-13 caused tyrosine phosphorylation of STAT5 in these transfectants (Fig. 5AGo). In contrast, tyrosine phosphorylation of STAT3 was induced by IL-4 and IL-13 only in DND-39/G{varepsilon}/IL-13R{alpha}1Full, although it was not evoked by IL-4 or IL-13 in the mock-transfected DND-39, DND-39/G{varepsilon}/IL-13R{alpha}1{Delta}CP and DND-39/G{varepsilon}/IL-13R{alpha}1{Delta}CT (Fig. 5BGo). The amounts of immunoprecipitated STAT3 were almost the same among the samples (Fig. 5BGo). These results clearly suggest that expression of IL-13R{alpha}1 was required for both IL-4 and IL-13 to activate STAT3 and that the C-terminal tail of IL-13R{alpha}1 was essential.



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Fig. 5. Tyrosine phosphorylation of STAT3 induced by IL-4 and IL-13 in the transfectants. Immunoprecipitates of STAT5 (A) or STAT3 (B) stimulated by 10 ng/ml of IL-4 or 50 ng/ml of IL-13 for 10 min of each transfectant were probed with anti-PY antibody or anti-STAT5 antibody or anti-STAT3 antibody.

 
Activation of STAT3 by IL-4 and IL-13 in stimulated human B cells
We next explored the possibility that STAT3 is activated by IL-4 and IL-13, dependent on expression of IL-13R{alpha}1, in not only the human B cell line, but also in primary human B cells. It has been revealed that co-stimulation of Ig and CD40 receptors markedly induces expression of IL-13R{alpha}1 in human B cells (42). We first confirmed that stimulation of purified B cells with anti-IgM antibody and anti-CD40 antibody augmented the amount of IL-13R{alpha}1 mRNA. After the amount of cDNA was normalized based on that of ß-actin, competitive PCR for IL-13R{alpha}1 was performed. The amount of cDNA for IL-13R{alpha}1 started to increase 6 h after stimulation of anti-IgM antibody and anti-CD40 antibody by ~10-fold, and then the amount reached levels up 100-fold 24 h after the stimulation (Fig. 6AGo). Using purified B cells unstimulated and stimulated with anti-IgM antibody and anti-CD40 antibody, we investigated whether IL-4 and IL-13 activate STAT3. In the stimulated, but not unstimulated B cells, IL-4 and IL-13 induced tyrosine phosphorylation of STAT3 by Western blotting (Fig. 6BGo). The amounts of STAT3 themselves were increased in the stimulated B cells, compared to unstimulated B cells (Fig. 6BGo). These results clearly showed that both IL-4 and IL-13 activated STAT3 in human B cells stimulated with anti-IgM antibody and anti-CD40 antibody.




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Fig. 6. Activation of STAT3 by IL-4 and IL-13 in stimulated human B cells. (A) RT-PCR for IL-13R{alpha}1. Competitive PCR was performed in the presence of the indicated amounts of competitors with cDNA from purified human B cells unstimulated and stimulated with 10 µg/ml of anti-IgM antibody and 0.5 µg/ml of anti-CD40 antibody for the indicated period. (B) Tyrosine phosphorylation of STAT3 induced by IL-4 and IL-13 in unstimulated and stimulated human B cells. Cell lysates of unstimulated and stimulated human B cells activated by either 10 ng/ml of IL-4 or 100 ng/ml of IL-13 for 15 min were blotted by anti-phospho STAT3 antibody (upper panel) or anti-STAT3 antibody (lower panel).

 
Effects of the tyrosine residues of IL-13R{alpha}1 on IL-4 and IL-13 signals
It is well known that most cytokine receptors are phosphorylated on tyrosine residues by stimuli and it is important for many signal-transducing molecules to be recruited to the receptors (32). IL-13R{alpha}1 possesses two tyrosine residues in the cytoplasmic portion, at 402 and 405 amino acids. As both of these tyrosine residues were absent in IL-13R{alpha}1{Delta}CT, it was assumed that impairment of the IL-13 signal in DND-39/G{varepsilon}/IL-13R{alpha}1{Delta}CT is due to the lack of these tyrosine residues, although we could not detect tyrosine phosphorylation of IL-13R{alpha}1 (data not shown). Particularly, the sequence around Y405 was compatible with the YXXQ motif, the consensus sequence for binding and activation of STAT3 (16). To test these possibilities, we investigated the IL-4 and IL-13 signals in DND-39/G{varepsilon}/IL-13R{alpha}1Y402F, DND-39/G{varepsilon}/IL-13R{alpha}1Y405F and DND-39/G{varepsilon}/IL-13R{alpha}1FF. The transcription activity on the I{varepsilon} promoter and activation of STAT6 by IL-4 and IL-13 were almost the same between DND-39/G{varepsilon}/IL-13R{alpha}1Full and these three transfectants (Fig. 7A and BGo). Tyrosine phosphorylation of Tyk2 by IL-4 and IL-13 in DND-39/G{varepsilon}/IL-13R{alpha}1FF was invariant compared to DND-39/G{varepsilon}/IL-13R{alpha}1Full (Fig. 7CGo). In contrast, activation of Jak1 by IL-4 was slightly diminished and that by IL-13 was significantly attenuated in DND-39/G{varepsilon}/IL-13R{alpha}1FF, whereas it was invariant in DND-39/G{varepsilon}/IL-13R{alpha}1Y402F and DND-39/G{varepsilon}/IL-13R{alpha}1Y405F (Fig. 7DGo). Activation of IRS-1 by IL-13 was also blocked in DND-39/G{varepsilon}/IL-13R{alpha}1FF as well as Jak1 (data not shown). Furthermore, it is noteworthy that tyrosine phosphorylation of STAT3 induced by either IL-4 or IL-13 completely disappeared in DND-39/G{varepsilon}/IL-13R{alpha}1FF, although IL-4 and IL-13 could activate STAT3 in both DND-39/G{varepsilon}/IL-13R{alpha}1Y402F and DND-39/G{varepsilon}/IL-13R{alpha}1Y405F (Fig. 7EGo). The comparisons with another clone of DND-39/G{varepsilon}/IL-13R{alpha}1Full, DND-39/G{varepsilon}/IL-13R{alpha}1FullE5 expressing lower IL-13R{alpha}1 and longer exposure of the membranes showed the same results, indicate that these results were not due to relatively lower expression of the receptor in DND-39/G{varepsilon}/IL-13R{alpha}1FF (data not shown). These results suggest that these tyrosine residues were essential for activation of STAT3 and Jak1, but not Tyk2 and STAT6, and that the existence of either tyrosine residue alone was enough to activate these molecules.







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Fig. 7. Effects of the tyrosine residues of IL-13R{alpha}1 on IL-4 and IL-13 signals. (A) Luciferase activities induced by IL-4 and IL-13 in the transfectants. Each transfectant was incubated with the indicated concentration of either IL-4 (dashed bars) or IL-13 (closed bars) for 24 h. Each experiment was done with three samples and the mean values are shown. (B–E) Tyrosine phosphorylation of Jak molecules and STAT3 and 6 induced by IL-4 and IL-13 in the transfectants. Immunoprecipitates of STAT6 (B), Tyk2 (C), Jak1 (D) and STAT3 (E) stimulated by 10 ng/ml of IL-4 or 50 ng/ml of IL-13 for 10 min were probed with anti-PY antibody (upper panel) or anti-STAT6, Tyk2, Jak1 and STAT3 antibodies (lower panel).

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In this study, we transfected various kinds of human IL-13R{alpha}1 to a human B cell line, and then analyzed activation of Jak molecules, IRS-1 and STAT molecules in the transfectants to clarify the functional role of the cytoplasmic domain, particularly the tyrosine residues of human IL-13R{alpha}1 on IL-4 and IL-13 signals in human B cells.

It had been controversial whether IL-4 and IL-13 evoke STAT3 activation (23,4346). Very recently, it was reported that expression of mouse IL-13R{alpha}1 in myeloid cell lines led to STAT3 activation by both IL-4 and IL-13 (27). In this study, we also demonstrated that expression of human IL-13R{alpha}1 enabled both IL-4 and IL-13 to activate STAT3 using a human B cell line (Fig. 5Go), and in stimulated human B cells, on which IL-13R{alpha}1 was highly expressed, but not unstimulated B cells, both IL-4 and IL-13 induced STAT3 activation (Fig. 6Go). Not only the amounts of IL-13R{alpha}1 mRNA, but also expression of STAT3 itself, were augmented in stimulated human B cells, compared to unstimulated cells (Fig. 6A and BGo), indicating that the amounts of both IL-13R{alpha}1 and STAT3 regulated STAT3 activation by IL-4 and IL-13 in human primary B cells.

Y405 on IL-13R{alpha}1 corresponds to the tyrosine residue of the YXXQ motif, which is the consensus sequence for binding and activation of STAT3 (16). Our present results—that STAT3 activation by IL-4 and IL-13 was completely blocked in DND-39/G{varepsilon}/IL-13R{alpha}1FF, whereas it was induced in DND-39/G{varepsilon}/IL-13R{alpha}1Y402F and DND-39/G{varepsilon}/IL-13R{alpha}1Y405F (Fig. 7Go)—suggest that there is a redundancy for the requirement of tyrosine residues for STAT3 activation. Our results were in line with the previous report that the single replacement of the tyrosine residue at 402 amino acid in mouse IL-13R{alpha}1, corresponding to Y405 in human IL-13R{alpha}1, caused only a partial decrease of STAT3 activation (27). The mechanism by which Y402 in IL-13R{alpha}1 is involved in STAT3 activation remains unclear. It has been demonstrated that cysteine can be substituted for glutamine to generate a STAT3 activation motif (47); however, to date, there is no report that the YXXY sequence could be another STAT3 activation motif. It would be possible that as Y402 and Y405 are located close together, the SH2 domain of STAT3 can sterically recognize Y402 the same as Y405. Analyses of the crystal structure of intracellular domain of IL-13R{alpha}1 would clarify this point.

STAT3 has been shown to be activated by various kinds of cytokines and growth factors (32), and several target genes of STAT3 have been already identified, such as junB, IRF1, STAT3, p19INK4D and c-myc (4851). The physiological roles of STAT3 in B cells are not fully understood, but it has been shown that STAT3 is involved in Ig production, an anti-apoptotic effect, and a proliferation effect in B cell lines and myeloma cells (50,5254), and it is assumed that STAT3 activation is correlated with induction of CD23, ICAM-1 and lymphotoxin-{alpha} by engagement of CD40 in human B cells (55). To date, the functional role of STAT3 on IL-4 and IL-13 signals in stimulated human B cells remains to be resolved, whereas that of STAT6 is well investigated (19,20). It would be possible that STAT3 and STAT6 independently correlate with IL-4 and IL-13 signals or alternatively that STAT3 and STAT6 co-operatively act to transduce IL-4 and IL-13 signals. Further studies aimed at clarifying this point are awaited.

Jak1 has been shown to be activated by both IL-4 and IL-13, and associate with IL-4R{alpha} upon stimulation of IL-4 (3437). The present results—that tyrosine phosphorylation of Jak1 induced by IL-13 was completely abolished in DND-39/G{varepsilon}/IL-13R{alpha}1{Delta}CT and DND-39/G{varepsilon}/IL-13R{alpha}1{Delta}CP, and severely impaired in DND-39/G{varepsilon}/IL-13R{alpha}1FF (Figs 3A and 7DGoGo)—suggested that IL-13R{alpha}1 was involved in the activation mechanism of Jak1 and that its C-terminal tail, particularly the tyrosine residues, was important for the activation. The existence of either tyrosine residue alone was enough for Jak1 activation as well as STAT3 activation (Fig. 7DGo). Thus far, a mechanism requiring the tyrosine residues of IL-13R{alpha}1 for activation of Jak1 remains unknown. It may be important for the binding of Jak1 to IL-13R{alpha}1 or, alternatively, some molecule, which is correlated with Jak1 activation, may be recruited to this site. STAT3 was not co-immunoprecipitated with Jak1 (data not shown). The present result—that tyrosine phosphorylation of Jak1 by IL-4 was impaired in DND-39/G{varepsilon}/IL-13R{alpha}1{Delta}CT and DND-39/G{varepsilon}/IL-13R{alpha}1{Delta}CP compared to DND-39/G{varepsilon} mock cells—suggested that competition between IL-13R{alpha}1 and {gamma}c for IL-4R{alpha} occurred (Fig. 3AGo). This notion was supported by the results that STAT6 or Jak3 activation was higher in DND-39/G{varepsilon} mock cells than DND-39/G{varepsilon}/IL-13R{alpha}1{Delta}CT and DND-39/G{varepsilon}/IL-13R{alpha}1{Delta}CP or the other three transfectants respectively (Figs 2A and B, and 3BGoGo).

It had been already demonstrated that Tyk2 is tyrosine phosphorylated by both human and mouse IL-13 (17,21,23,25); however, it was controversial whether IL-4 also induces tyrosine phosphorylation of Tyk2 (17,21,23,25,35,37). The present result showed that expression of IL-13R{alpha}1 enabled both IL-4 and IL-13 to activate Tyk2 by engaging IL-4 and IL-13 receptors containing IL-13R{alpha}1 as a component (Fig. 3CGo), in line with the previous results that in IL-13R-expressing cells, IL-4 can activate Tyk2 (17,21,23,25). The present results—that IL-13R{alpha}1Full and IL-13R{alpha}1{Delta}CT, but not IL-13R{alpha}1{Delta}CP, constitutively associated with Tyk2 (Fig. 4Go)—clearly showed that the Box1 region of IL-13R{alpha}1 was the binding site for Tyk2, so that Tyk2 was not tyrosine phosphorylated in DND-39/G{varepsilon}/IL-13R{alpha}1{Delta}CP (Fig. 3CGo). The result that the association of Tyk2 with IL-13R{alpha}1 was enhanced by IL-13 and that this enhancement decreased in IL-13R{alpha}1{Delta}CT (Fig. 4Go) may indicate that the C-terminal tail of IL-13R{alpha}1 is required for its optimal conformational change by the ligands, important for association and activation of Tyk2, so that Tyk2 activation may be diminished in DND-39/G{varepsilon}/IL-13R{alpha}1{Delta}CT (Fig. 3CGo).

It has been shown that Jak3 constitutively associates with {gamma}c and is tyrosine phosphorylated upon stimulation of IL-4 (36,38). The present result—that tyrosine phosphorylation of Jak3 was not induced by IL-13 (Fig. 3BGo)—was in line with the previous results that {gamma}c is not involved in IL-13R (21,24,56), and that a heterotrimer composed of IL-13R{alpha}1, IL-4R{alpha} and {gamma}c does not exist (18,27). Analyses of cross-linking using IL-4 labeled with 125I in DND-39/G{varepsilon}/IL-13R{alpha}1Full also showed the existence of the heterodimers, but not the heterotrimer (data not shown).

It is well known that upon stimulation, STAT6 is recruited to phosphorylated tyrosine residues of IL-4R{alpha} and then is tyrosine phosphorylated, followed by localization to the nucleus (20,57). It has been already reported that Jak1 and Jak3 are correlated with STAT6 activation when stimulated by IL-4 (19,40,58,59); however, it remained to be resolved whether Tyk2 is also involved in STAT6 activation. The results that activation of STAT6 by IL-13 was retained as well as that of Tyk2 in DND-39/G{varepsilon}/IL-13R{alpha}1FF, whereas activation of Jak1 by IL-13 was significantly blocked in this transfectant (Fig. 7BGo–D), strongly supported the notion that Tyk2 is also involved in STAT6 activation. In DND-39/G{varepsilon}/IL-13R{alpha}1{Delta}CT and DND-39/G{varepsilon}/IL-13R{alpha}1{Delta}CP, in which activation of both Tyk2 and Jak1 by IL-13 was completely blocked, STAT6 activation was not detected (Fig. 2Go), suggesting that the C-terminal tail of IL-13R{alpha}1 was critical for tyrosine phosphorylation of not only Jak1, Tyk2 and IRS-1, but also of STAT6 induced by IL-13.


    Acknowledgments
 
We thank Dr Toshihiko Akimoto for critical support of this study and Dr Kazuya Shimoda for giving us the plasmid. We also thank Dr Dovie R. Wylie for critical review of this manuscript.


    Abbreviations
 
IL-13R{alpha} IL-13 receptor {alpha} chain
IL-4R{alpha} IL-4 receptor {alpha} chain
IRS insulin receptor substrate
PI3 phosphatidylinositol 3
PY phosphotyrosine

    Notes
 
Transmitting editor: K. Arai

Received 10 May 2000, accepted 11 July 2000.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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