Cancer Research AACR Conference on Molecular Diagnostics - 2008 Tumor Immunology: New Perspectives
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[Cancer Research 62, 1103-1109, February 15, 2002]
© 2002 American Association for Cancer Research

Immunology

IL-13R{alpha}2, a Decoy Receptor for IL-13 Acts As an Inhibitor of IL-4-dependent Signal Transduction in Glioblastoma Cells1

Shaik Ohidar Rahaman, Pankaj Sharma, Phyllis C. Harbor, M. Javad Aman, Michael A. Vogelbaum and S. Jaharul Haque2

Departments of Cancer Biology, Lerner Research Institute [S. O. R., P. S., P. C. H., M. A. V., S. J. H.], Pulmonary and Critical Care Medicine [P. S., S. J. H.], and Neurosurgery [M. A. V.], Cleveland Clinic Foundation, Cleveland, Ohio 44195, and Department of Cell Biology and Biochemistry, US Army Research Institute of Infectious Diseases, Frederick, Maryland 21702 [M. J. A.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Interleukin (IL)-4 and IL-13 share the type II IL-4 receptor for cell signaling. We show that despite expressing the necessary signaling components, glioblastoma cells failed to respond to either IL-4 or IL-13. This was in part because of the expression of a high-affinity IL-13-binding transmembrane protein IL-13R{alpha}2 that inhibited IL-13-mediated Stat6 activation by acting as a decoy receptor. In contrast, normal human astrocytes that did not express the IL-13R{alpha}2 gene efficiently induced Stat6 activation in response to both IL-4 and IL-13. Transient expression of the IL-13R{alpha}2 transgene in nonexpressing heterologous cells inhibited not only IL-13- but also IL-4-mediated signal transduction and Stat6-responsive gene expression. The inhibition was likely mediated through the physical interaction between the short intracellular domain of the IL-13R{alpha}2 protein and the cytoplasmic domain of the IL-4R{alpha} chain that harbors the Stat6 docking sites. Thus, IL-13R{alpha}2 acts as an inhibitor of IL-4-dependent signal transduction pathways via a novel mechanism that is independent of ligand binding.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
IL-43 produced by activated T lymphocytes, mast cells, and basophils exerts pleiotropic effects on a variety of cell types (1, 2, 3) . IL-4 initiates transmembrane signaling by activating two types of transmembrane receptors (2, 3, 4) . The type I receptor comprises the IL-4R{alpha} subunit that binds to IL-4 and the {gamma}c chain that is shared by IL-2, IL-7, IL-9, and IL-15 (5) . Jak1 and Jak3 constitutively associate with the IL-4R{alpha} and the {gamma}c, respectively (2 , 3 , 5, 6, 7) . However, Jak3 and {gamma}c are not expressed in many nonhematopoietic cells that use the type II receptor for IL-4 signaling (2 , 3 , 5 , 6 , 8) . The type II receptor comprises the IL-4R{alpha} chain and the IL-13R{alpha}1 subunit, a low-affinity receptor for IL-13 that constitutively associates with Jak2 or Tyk2 (2 , 3 , 5 , 9, 10, 11) . IL-13 uses the type II receptor complex for cell signaling. Thus, IL-4 and IL-13 have overlapping biological functions in many cell types that express the type II receptor components (2 , 3) . Binding of IL-4 or IL-13 to the cognate receptor chain leads to the heterodimerization of two receptor subunits. This in turn brings two Jak molecules in an appropriate proximity that allows the trans-phosphorylation of the specific tyrosine residue located in the Jak activation segment (3 , 12) . Trans-phosphorylation promotes the kinase activity of the Jak molecules that is required for the phosphorylation of the downstream substrates in the signaling pathways (12 , 13) . IL-4 and IL-13 activate two intracellular signaling cascades: the Jak-Stat and the IRS-phosphatidylinositol 3'-kinase pathways (2, 3, 4) . Although the IRS-phosphatidylinositol 3'-kinase pathway leads to cell proliferation and survival, the Jak-Stat pathway induces the transcription of genes that contain the Stat6-responsive enhancer element N6-GAS located in their promoters (3 , 14, 15, 16) . On IL-4 or IL-13 stimulation of cells, Stat6 is phosphorylated and forms a homodimer that migrates to the nucleus and binds to N6-GAS to drive the transcription (3 , 16 , 17) .

Homeostatic control of cytokine-mediated cell signaling in general requires a delicate balance between the molecular events that activate and amplify the signal and the mechanisms that inhibit the generation and transmission of the signal from cell surface to the nucleus. Our long-term interest is to investigate the cellular and molecular mechanisms that govern the negative regulation of IL-4- and IL-13-mediated signal transduction. To that end, we investigated the negative regulation of IL-4- and IL-13-dependent Jak-Stat signaling pathway by two families of Jak inhibitors, the PTP and the SOCS (12 , 18 , 19) . However, the inhibitory functions of PTPs and the SOCS-family proteins are not specific for IL-4- and IL-13-dependent signal transduction pathways.

A high-affinity IL-13-binding transmembrane protein IL-13R{alpha}2 acts as a specific inhibitor of IL-13 signaling likely by functioning as a decoy receptor (20, 21, 22, 23) . The cDNA for IL-13R{alpha}2 was originally isolated from the Caki-1 renal cell carcinoma cell line (20) . IL-13R{alpha}2 protein is expressed at high levels in anaplastic astrocytoma and GBM cells but not in normal astrocytes or low-grade glioma (astrocytoma) cells (24, 25, 26, 27) . IL-13R{alpha}2 does not bind to IL-4 (28 , 29) . IL-4 induces growth arrest in human astroglial cell lines derived from nonneoplastic adult cerebral cortex and from low-grade astrocytomas (26 , 30, 31, 32, 33) . Although GBM cells were capable of binding to IL-4 (34, 35, 36) , they failed to activate Stat6 on IL-4 stimulation (12) .

Here we demonstrate for the first time that IL-13R{alpha}2 functions as an inhibitor of IL-4-dependent signal transduction and Stat6-responsive gene expression. The inhibition is likely mediated through the physical interaction between the short intracellular domain of IL-13R{alpha}2 protein and cytoplasmic domain of the IL-4R{alpha} chain.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cloning of cDNA and Construction of Expression Plasmids.
cDNA for IL-13R{alpha}2 was cloned by PCR amplification using primers for 3' and 5' ends of the cDNA sequence for the mature protein (20) after reverse transcription of RNA from Caki-1 cells using Superscript (Invitrogen). The PCR fragment was inserted in frame into pSecTagC vector (Invitrogen) to be expressed with COOH-terminal tags for Myc and His. Expression of the IL-13R{alpha}2 construct was verified by transient expression in 293T cells and immunoblotting by anti-Myc antibody. The EPOR-IL-4R{alpha} chimeric receptor was prepared as described (19 , 37 , 38) . The deletion mutants of IL-13R{alpha}2 were constructed by PCR technique using specific primers and the IL-13R{alpha}2 cDNA as the template as described (19) .

Antibodies.
Anti-V5 and anti-Myc antibodies were purchased from Invitrogen, and anti-Stat6 antibody was purchased from ZYMED Laboratories, Inc.

Cell Culture.
293T, NIH 3T3, and glioblastoma cell lines were grown in DMEM containing 2 mM glutamine, 10% bovine serum, and 50 mg/liter penicillin and streptomycin. Jurkat cells were grown in RPMI 1640 supplemented with 2 mM L-glutamine, 10% bovine serum, and 50 mg/liter penicillin and streptomycin. NHAs were obtained and maintained in specific growth medium Bullet-Kit from Clonetic-BioWhittaker, Walkersville, MD.

Transfection of Cells.
293T and NIH 3T3 cells (106 cells/10-cm plate) were transfected with the indicated expression plasmids by using calcium phosphate and Lipofectamine Plus (Invitrogen), respectively, as described (19) . T98G cells were transfected by Lipofectamine Plus with appropriate plasmids, and stable IL-4R{alpha}-expressing clones were selected by puromycin (0.5 µg/ml) for 3–4 weeks.

RPA.
Total RNA was isolated from cells using TRIzol reagent according to the manufacturer’s protocol (Life Technologies, Inc., Rockville, MD). Steady-state levels of different receptor transcripts were determined by RPA using the Riboquant system (PharMingen) with a multiprobe template set. Briefly, the hCR-1b template set was used for the T7 polymerase-directed synthesis of high specific activity 32P-labeled antisense RNA probes that include IL-13R{alpha}1, IL-7R{alpha}, IL-9R{alpha}, IL-13R{alpha}2 (also known as IL-13R{alpha}), IL-15R{alpha}, IL-4R{alpha}, {gamma}c, IL-2Rß, IL-2R{alpha}, L-32, and GAPDH. Probe (4 x 105 cpm) was hybridized overnight with 12 µg of total RNA at 56°C. RNA hybrids were digested with RNase A and T1 and purified and resolved by electrophoresis in 6% denaturing polyacrylamide gel.

IP and Western Blot Analyses.
For IP, cell extracts were prepared by lysing the cells in ice-cold buffer containing 50 mM Tris (pH 7.9), 150 mM NaCl, 1 mM EDTA, 1 mM DTT, 1% NP40, 10% Glycerol, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, 2 µg/ml pepstatin, and 5 µg/ml aprotinin on ice for 30 min. The cleared supernatant containing 0.5 mg of protein was incubated with 2–5 µg of the indicated antibody immobilized on agarose beads for 12 h at 4°C. The captured beads were boiled in denaturing buffer, and released proteins were analyzed by Western blotting as described (19) .

EMSA and Luciferase Assay.
For EMSA cells were treated with the indicated cytokine, and WCE was prepared as described (39) . EMSA was performed using WCE containing 10–15 µg of protein and 0.2 ng of radiolabeled N5- or N6-GAS oligonucleotide probe as described (12 , 39) . Luciferase activity was determined and normalized as described (18 , 19) .


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Glioblastoma Cells Are Refractory to IL-4 and IL-13 Signaling.
Previously, we found that a number of human glioblastoma cell lines, including T98G, GRE, and M007, were unresponsive to IL-4-dependent signal transduction (12) . Here we show that in contrast to NHAs, different glioblastoma cell lines, including U251, U87-MG (Fig. 1)Citation , A172, and D54 (data not shown), also failed to activate Stat6 in response to both IL-4 and IL-13. These results suggest that unlike normal astrocytes, glioblastoma cells are in general defective in IL-4- and IL-13-dependent signal transduction. Like normal astrocytes, glioblastoma cells expressed IL-4R{alpha} and IL-13R{alpha}1 mRNA but not {gamma}c chain mRNA (Fig. 2A)Citation . The IL-4R{alpha} and IL-13R{alpha}1 physically associate with Jak1 and Jak2 (or Tyk2), respectively (2, 3, 4 , 9, 10, 11) . Glioblastoma cells used in this study showed normal response to both IFN-{alpha} and IFN-{gamma} that use Jak1-Tyk2 and Jak1-Jak2, respectively, indicating that Jak proteins required for IL-4 and IL-13 signaling through the type II receptor were present and functional in the glioblastoma cells (Fig. 3ACitation ; Refs. 17 and 40, 41, 42 ). Stat6 protein was also expressed in the glioblastoma cells (Fig. 3B)Citation . 293T cells transfected with the human Stat6 expression plasmid were used as a positive control for Stat6 (19) . Collectively, these results suggest that glioblastoma cells express either an inhibitor of IL-4-mediated signal transduction or a mutant IL-4R{alpha} chain, which is incapable of supporting the Jak-Stat activation.



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  		Fig. 1. IL-4 and IL-13 fail to activate Stat6 in glioblastoma cells but not in normal astrocytes. U251 and U87-MG human GBM cells, Jurkat human T cells, and NHAs were treated with IL-4 (20 ng/ml) and IL-13 (10 ng/ml) for 30 min or left untreated. Cells were harvested, and WCE was prepared. Stat6 activation was measured by EMSA using 15 µg of protein from NHA and 10 µg of protein from GBM and Jurkat cells, and 0.2 ng of radiolabeled N6-GAS probe derived from the human IL-1Rra promoter that specifically binds to Stat6 homodimer (12 , 18 , 19) . Jurkat cells that are responsive to IL-4, but not to IL-13, served as control.

 


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  		Fig. 2. A, steady-state level of mRNA for IL-4- and IL-13-receptor chains in NHAs and GBM cells. Total RNA was isolated from cells using the TRIzol reagent. RNA sample (12 µg each) was used in RPA for a multiprobe template set obtained from the PharMingen that includes IL-13R{alpha}1, IL-13R{alpha}2, IL-4R{alpha}, {gamma}c, L-32, and GAPDH. The unlabeled bands representing IL-7R{alpha}, IL-9R{alpha}, IL-15R{alpha}, IL-2R{alpha}, and IL-2Rß were also included in the template set. RNase-protected transcripts are indicated by arrowheads on the left and the corresponding undigested probes on the right. L-32 and GAPDH were used as internal controls. In B, overexpression of IL-13R{alpha}2 does not alter the expression of endogenous IL-13R{alpha}1 gene in 293T cells. Cells were transfected with the indicated amount of IL-13R{alpha}2 expression plasmid, and 48-h post-transfection, total RNA was isolated. RNA sample (12 µg) was subjected to RPA analysis using multiprobe template set described above. RNase-protected transcripts are indicated by arrowheads on the left and the corresponding undigested probes on the right. L-32 and GAPDH served as internal controls.

 


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  		Fig. 3. In A, Jak1, Jak2, and Tyk2 proteins are functional in glioblastoma cells. T98G, U251, U87-MG, and A172 cells were treated with IFN-{alpha}2 (1000 units/ml) and IFN-{gamma} (500 units/ml) for 30 min or left untreated. WCE containing 15 µg of protein and 0.2 ng of radiolabeled IRF-1 GAS (N5-GAS) probe was used in EMSA as described above. IFN-{alpha} induced three DNA-protein complexes, of which the top one contains Stat3 homodimer, the middle complex contains Stat1-Stat3 heterodimer, and the bottom one contains Stat1 homodimer (12) . IFN-{gamma} induced one DNA-protein complex that contains Stat1 homodimer. In B, Stat6 protein is expressed in glioblastoma cells. Extracts derived from human GBM cells T98G, U251, D54, and U87-MG; NHAs; and human fetal kidney cells 293T expressing the human Stat6 transgene were subjected to Western blot analysis. Protein (75 µg) was resolved in each lane of a 10% SDS-polyacrylamide gel. Proteins transferred on Immobilon-P membrane were probed with a monoclonal anti-Stat6 antibody. Immunodetection was performed using enhanced chemiluminescence reagents.

 
IL-13R{alpha}2 Protein Inhibits both IL-13- and IL-4-mediated Signal Transduction and Gene Expression.
Human glioblastoma cells were reported to express high levels of IL-13R{alpha}2 mRNA and protein as determined by RT-PCR and radiolabeled IL-13-binding assay, respectively (25, 26, 27) . Although IL-13R{alpha}2 mRNA is expressed in glioblastoma cells, it is not expressed in normal primary astrocytes (Fig. 2A)Citation . As the IL-13R{alpha}2 protein has been shown to function as a high-affinity (100–300-fold higher affinity than IL-13R{alpha}1) nonsignaling receptor for IL-13 (20, 21, 22, 23) , it was expected that IL-13-mediated Jak-Stat signaling would be inhibited in glioblastoma cells (Fig. 1)Citation .

To examine the possibility that IL-4-signaling can be blocked by the expression of the IL-13R{alpha}2 protein, an IL-13R{alpha}2 transgene was coexpressed in 293T cells with Stat6 by transient transfection. 293T cells express IL-4R{alpha} and IL-13R{alpha}1 but not the functional endogenous Stat6 protein (Fig. 2BCitation ; Refs. 16 and 19 ). Post-transfection (48 h), cells were treated with IL-4 (20 ng/ml) or IL-13 (10 ng/ml) for 30 min, and Stat6 activation was measured by EMSA. The results show that IL-4-dependent activation of Stat6 was markedly inhibited by IL-13R{alpha}2 expression (Fig. 4A)Citation . In a control experiment, an expression plasmid encoding an unrelated cytokine receptor human IFNGR-2 when coexpressed with Stat6 did not inhibit IL-4-mediated activation of Stat6 in 293T cells (data not shown). To examine whether the expression of IL-13R{alpha}2 has any effect on the expression of endogenous IL-13R{alpha}1 and exogenous Stat6 (tagged with V5-epitope), RPA for IL-13R{alpha}1 and immunoblot analysis for Stat6 were performed (Fig. 2BCitation and 4BCitation ). The results show that IL-13R{alpha}2-mediated inhibition of IL-4-dependent activation of Stat6 was not because of any alterations in the expression of either IL-13R{alpha}1 or Stat6. Similar results were obtained when IL-13R{alpha}2 was expressed in NIH 3T3 cells (data not shown).



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  		Fig. 4. In A, IL-13R{alpha}2 inhibits both IL-4- and IL-13-mediated activation of Stat6. 293T cells were cotransfected with 2 µg of human Stat6 expression plasmid and varying amounts of human IL-13R{alpha}2 expression plasmid as indicated. Post-transfection (48 h), cells were treated with IL-4 (20 ng/ml) or IL-13 (10 ng/ml) for 30 min or left untreated. WCE containing 15 µg of protein was subjected to EMSA using 0.2 ng of N6-GAS probe as described above. In B, overexpression of IL-13R{alpha}2 does not alter the expression of Stat6 transgene in 293T cells. Cells were cotransfected with 2 µg of human Stat6 expression plasmid (V5-tagged) with the indicated amount of human IL-13R{alpha}2 expression plasmid. Post-transfection (48 h), cell lysates were prepared, and 75 µg of protein were resolved in a 10% SDS-polyacrylamide gel for Western blot analysis using anti-V5 antibody.

 
To determine whether IL-13R{alpha}2 expression regulates the transcription of IL-4-responsive genes, an IL-4-responsive luciferase reporter construct (16 , 18 , 19) and the IL-13R{alpha}2 expression plasmid (at varying amounts) were cotransfected into 293T cells, along with the Stat6 expression plasmid. The results clearly indicate that IL-4-responsive luciferase activity was markedly inhibited by the expression of the IL-13R{alpha}2 transgene in a dose-dependent manner (Fig. 5)Citation . Human IFNGR-2 did not produce any dose-dependent and specific inhibition of IL-4-mediated induction of luciferase activity when coexpressed with Stat6 in 293T cells (data not shown). Taken together, these data indicate that IL-13R{alpha}2 functions as an inhibitor of IL-4-dependent signal transduction and gene expression.



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  		Fig. 5. IL-13R{alpha}2-mediated inhibition of IL-4-dependent promoter activity. 293T cells were cotransfected with 2 µg of human Stat6 expression plasmid, 4 µg of Stat6-responsive luciferase reporter or control plasmid, and varying amounts of human IL-13R{alpha}2 expression plasmid as indicated. Post-transfection (24 h), cells were trypsinized and split into two halves. After 12 h, IL-4 (20 ng/ml) was added to one plate, whereas the other plate was left untreated as control. Post-IL-4 treatment (15 h), cells were harvested for the measurement of luciferase activity. Luciferase activity was normalized based on protein concentration. The data are presented as mean +/-SD of three independent experiments.

 
Intracellular Domain of IL-13R{alpha}2 Protein Confers the Inhibition of IL-4 Signaling.
IL-13R{alpha}2 does not bind to IL-4 (28 , 29) ; however, it forms a complex with IL-13R{alpha}1 in cells that are exposed to IL-13 but not to IL-4 (24) . This rules out the possibility that IL-13R{alpha}2 inhibits IL-4 signaling by sequestering either IL-4 or IL-13R{alpha}1. To understand the mechanisms underlying the IL-13R{alpha}2-mediated inhibition of IL-4 signaling, we prepared a chimeric transmembrane protein composed of the extracellular plus the transmembrane domains of the murine EPOR and the cytoplasmic domain of the human IL-4R{alpha} chain (37 , 38) . This chimeric receptor (EPOR-IL-4R{alpha}) when coexpressed with Stat6 protein in 293T cells supported EPO-dependent activation of Stat6 (19) . Here we show that IL-13R{alpha}2 inhibited EPO-dependent activation of Stat6, which was mediated through the EPOR-IL-4R{alpha} chimeric receptor expressed in 293T cells (Fig. 6A)Citation . Immunoblot analysis using Stat6 antibody revealed that IL-13R{alpha}2-mediated inhibition of EPO-dependent Stat6 activation was not because of the reduced expression of the Stat6 transgene (Fig. 6B)Citation . Induction of Stat6-responsive luciferase gene expression by EPO signaling through the EPOR-IL-4R{alpha} chimeric receptor was also markedly inhibited in 293T cells (data not shown). The human IL-13R{alpha}2 is a 380 amino acid transmembrane protein with 341 amino acid ectodomain and a very short cytoplasmic tail that contains only 17 amino acids (20) . These results suggest that the 17 amino acid cytoplasmic domain of the IL-13R{alpha}2 protein functions as an inhibitor of IL-4- or IL-13-dependent activation of the Jak-Stat pathway. To test this, we prepared mutant IL-13R{alpha}2 constructs that lack the COOH-terminal 3 and 6 amino acids. The mutant IL-13R{alpha}2 protein lacking COOH-terminal 6 but not 3 amino acids when coexpressed with Stat6 in 293T cells failed to produce marked inhibition of IL-4-dependent activation of Stat6 compared with the wild-type IL-13R{alpha}2 (Fig. 7A)Citation . Western blot analysis revealed that the expression of either wild-type or mutant IL-13R{alpha}2 protein did not affect the level of expression of the transfected Stat6 transgene in 293T cells (Fig. 7B)Citation .



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  		Fig. 6. In A, intracellular domain of IL-4R{alpha} is involved in the IL-13R{alpha}2-mediated inhibition of Stat6 activation in response to EPO-stimulation. 293T cells were cotransfected with 2 µg of Stat6, 5 µg of EPOR-IL-4R{alpha} chimeric receptor and indicated amount of human IL-13R{alpha}2 expression plasmids. Post-transfection (48 h), cells were treated with EPO (5 units/ml) for 30 min or left untreated. WCE containing 15 µg of protein was subjected to EMSA using 0.2 ng of N6-GAS probe as described above. In B, overexpression of IL-13R{alpha}2 does not alter the expression of Stat6 transgene in 293T cells. Cells were cotransfected with 2 µg of human Stat6 expression plasmid with the indicated amount of human IL-13R{alpha}2 expression plasmid, and cell lysates were prepared after 48 h. Protein (75 µg) was resolved in a 10% SDS-polyacrylamide gel for Western blot analysis using a monoclonal anti-Stat6 antibody.

 


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  		Fig. 7. In A, cytoplasmic domain of the IL-13R{alpha}2 protein confers the inhibition of IL-4-dependent activation of Stat6. 293T cells were cotransfected with 2 µg of Stat6 and 4 µg of wild-type or mutant (in which three or six amino acids were deleted from the COOH terminus) IL-13R{alpha}2 expression plasmids. Post-transfection (48 h), cells were treated with IL-4 (20 ng/ml) for 30 min or left untreated. WCE containing 15 µg of protein was subjected to EMSA using 0.2 ng of N6-GAS probe as described above. In B, overexpression of neither wild-type nor mutant IL-13R{alpha}2 protein alters the expression of Stat6 transgene in 293T cells. Cells were cotransfected with 2 µg of human Stat6 expression plasmid and 4 µg of wild-type or mutant IL-13R{alpha}2 expression plasmid, and cell lysates were prepared after 48 h. Protein (75 µg) was resolved in a 10% SDS-polyacrylamide gel for Western blot analysis using a monoclonal anti-Stat6 antibody.

 
To address how the short cytoplasmic domain of IL-13R{alpha}2 confers the inhibition of IL-4 signaling, we performed a protein-protein interaction assay. 293T cells were cotransfected with the EPOR-IL-4R{alpha} (tagged with V5 epitope) and the IL-13R{alpha}2 (tagged with c-Myc epitope) expression constructs. Post-transfection (48 h), cell lysates were prepared and subjected to IP with either anti-V5 or anti-c-Myc antibodies followed by immunoblot analyses with either anti-c-myc or anti-V5 antibodies, respectively. The results show that the chimeric EPOR-IL-4R{alpha} receptor protein coimmunoprecipitated the IL-13R{alpha}2 protein when coexpressed in 293T cells (Fig. 8)Citation . Taken together, these data suggest that the IL-13R{alpha}2-mediated inhibition of IL-4 signaling was conferred by the short cytoplasmic domain of this protein.



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  		Fig. 8. Physical association between IL-4R{alpha} and the IL-13R{alpha}2. 293T cells were cotransfected with expression plasmids for IL-13R{alpha}2 (COOH-terminally tagged with Myc-epitope) and EPOR-IL-4R{alpha} chimeric receptor (COOH-terminally tagged with V5 epitope) for Lanes 1 and 4. Lanes 2 and 6, cells were cotransfected with plasmids for EPOR-IL-4R{alpha} chimeric receptor and the empty vector for IL-13R{alpha}2. Cells were cotransfected with expression plasmids for IL-13R{alpha}2 and empty vector for EPOR-IL-4R{alpha} chimeric receptor for Lanes 3 and 5. Each plasmid (5 µg) was used for transfection of cells. Post-transfection (48 h), cell lysates were prepared and subjected to IP and immunoblot (IB) analyses using antibodies as indicated.

 
As shown above, IL-13R{alpha}2 was expressed at varying levels in different glioblastoma cells. To examine if overexpression of the IL-4R{alpha} chain relieved the IL-13R{alpha}2-mediated inhibition of IL-4 and IL-13 signaling in glioblastoma cells, we prepared stable T98G cell lines expressing the human IL-4R{alpha} cDNA. Two representative cell lines stably expressing the IL-4R{alpha} transgene and a vector control cell line were treated with IL-4 (20 ng/ml) or IL-13 (10 ng/ml) for 30 min or left untreated, and Stat6 activation was measured by EMSA. The results revealed that overexpression of the IL-4R{alpha} chain in T98G cells restored the IL-4- but not IL-13-mediated signal transduction (Fig. 9)Citation . These data suggest that IL-4 signaling could be restored by titration of intracellular domains of the endogenous IL-13R{alpha}2 protein leaving its ectodomain fully functional in sequestering the added IL-13 molecules. Thus, IL-13R{alpha}2 appears to negatively control the IL-4 and IL-13 signaling through both overlapping and distinct mechanisms.



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  		Fig. 9. IL-4 but not IL-13 activates Stat6 in GBM cells overexpressing the IL-4R{alpha} transgene. T98G cells were cotransfected with human IL-4R{alpha} expression plasmid or the empty vector and an expression plasmid for puromycin resistance gene. Stable clones were isolated by drug selection (0.5 µg/ml puromycin). Two representative clones, IL-4R{alpha}/C4 and IL-4R{alpha}/C6, were treated with IL-4 (20 ng/ml) or IL-13 (10 ng/ml) for 30 min. WCE containing 15 µg of protein was subjected to EMSA using 0.2 ng of N6-GAS probe as described above.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In principle, cytokine-mediated cell signaling may be negatively controlled at multiple levels of the signaling cascades by a number of molecules acting through different mechanisms (19 , 43, 44, 45) . Transmembrane receptors for IL-4 and IL-13, as well as many other cytokines, use the Jak-family tyrosine kinase for cell signaling (3, 4, 5 , 17 , 40, 41, 42) . Activated Jak-catalyzed tyrosine autophosphorylation and tyrosine phosphorylation of receptors and downstream substrates are essential for the generation, amplification, and transmission of cytokine signals from the cell surface to the nucleus (13 , 17 , 40, 41, 42) . PTPs and the SOCS-family proteins inhibit the kinase activity of Jaks through their recruitment to the Jak-receptor complexes (18 , 19) . The recruitment is mediated by their specific and direct binding to the Jaks and/or receptor chains (19 , 43, 44, 45) . Given that four Jak-family proteins are redundantly used by a number of cytokine receptors, the PTP and SOCS molecules that inhibit Jak activity by directly binding to Jak proteins cannot act as specific inhibitors of a particular cytokine signaling pathway.

Nonsignaling cytokine receptors expressed in a soluble- or membrane-bound form that sequester the cognate cytokine molecules may function as decoy receptors to attenuate signal transduction by a particular cytokine (21 , 46) . Specific binding of such decoy receptors to their cognate cytokines is capable of maintaining the specificity of inhibition of the signaling pathways. IL-4 and IL-13 are encoded by adjacent loci in human chromosome 5q31–33 and are predominantly expressed in antigen-activated type 2 T-helper cells (47) . These two cytokines have overlapping biological functions in many cell types by virtue of sharing a common receptor complex (2, 3, 4) . Negative regulation of both IL-4 and IL-13 signaling pathways by Shp-1, SOCS-1, or SOCS-3 is not specific for IL-4 and IL-13 signaling pathway (18 , 19) . In contrast, IL-13R{alpha}2 functions as a decoy receptor for IL-13 because of its high affinity binding to this cytokine (20, 21, 22, 23, 24) . IL-13R{alpha}2 does not bind to IL-4 (28 , 29) . For the first time, we demonstrate herein that the short cytoplasmic domain of the IL-13R{alpha}2 binds to the cytoplasmic domain of the IL-4 receptor {alpha} chain and thereby confers the inhibition of IL-4- and IL-13-mediated signal transduction. Our data show that IL-13R{alpha}2 is a more potent inhibitor for IL-13 than IL-4, because IL-13 signaling is inhibited by both the ectodomain and the endodomain of the IL-13R{alpha}2 protein, whereas IL-4 signaling is inhibited only by the endodomain (Figs. 6Citation 7Citation 8)Citation .

IL-4 and IL-13 stimulate or inhibit cell proliferation in a tissue-specific fashion (48 , 49) . IL-4 induces growth arrest in astrocytes and low-grade gliomas (26 , 30, 31, 32, 33) . The antimitogenic effect of IL-4 is mediated through a p21(Waf1/Cip1)-dependent up-regulation of p27(Kip1) level in normal glial cells, and in primary astrocytic tumors, p27 protein levels are reduced and are almost absent in glioblastomas (32 , 50) . We have found previously that glioblastoma cell lines failed to activate Stat6 in response to IL-4, but on expression of the functional IL-4R{alpha} transgene glioblastoma cell line, T98G became partly responsive to IL-4 (12) . Using commercially available antibody, we could not detect IL-4R{alpha} protein in T98G cells. These observations suggested that T98G cells did not express the functional IL-4R{alpha} chain, and the complementation of T98G cells by the functional IL-4R{alpha} gene rendered the responsiveness to IL-4 (12) . By using sensitive methods like RT-PCR (data not shown) and RPA, we were able to show that both subunits of the type II IL-4 receptor were expressed in T98G and in other glioblastoma cell lines (Fig. 2)Citation . Here we demonstrate that both IL-4R{alpha} and IL-13R{alpha}1 are expressed in normal astrocytes as well as in all of the glioblastoma cell lines we have examined (Fig. 2)Citation . Puri et al. (24 , 25 , 34, 35, 36) also demonstrated the expression of IL-4R{alpha} in normal astrocytes and glioblastoma cells by radiolabeled ligand binding assay and RT-PCR technique. We have shown here that IL-4 response at the level of Stat6 activation, as measured by its specific interaction with N6-GAS, is present in normal astrocytes but not in glioblastoma cells (Fig. 1)Citation . Surprisingly, we have found that IL-4 activated Stat3 molecules in all of the glioblastoma cells we have examined but not in normal astrocytes.4 The mechanism underlying the unusual signaling through the IL-4 receptor in IL-13R{alpha}2-expressing glioblastoma cells is presently under an active investigation. Puri et al. (51 , 52) have extensively used the IL-13R{alpha}2 protein expressed on the surface of glioblastoma cells for the targeted delivery of a recombinant IL-13-Pseudomonas endotoxin chimeric protein that induces cytotoxicity in these cells. However, the physiological significance of IL-13R{alpha}2 expression in glioblastoma and in other tumor cells, including prostate carcinoma cells (24 , 53) , remains unclear. Experimental animals coimplanted with glial tumor cells and cells that were engineered to produce IL-4 via viral vector had significantly improved survival compared with animals bearing only the tumor cells (54, 55, 56) . An international, population-based, case-control study has identified a statistically significant inverse association between glioma and atopy (57) . Type 2 T-helper cells largely contribute to pathophysiology of atopy by producing IL-4 and IL-13 (4 , 58 , 59) . Infiltrated T lymphocytes could be a source of IL-4 and IL-13 in the brain. These studies suggest that inhibition of IL-4 and IL-13 signaling by the IL-13R{alpha}2 facilitates the growth of high-grade glioma cells. Further investigation to define the role of IL-13R{alpha}2 in IL-4-mediated signal transduction and cell cycle progression of glioblastomas is warranted.


   ACKNOWLEDGMENTS
 
We thank Dr. Ulrike Schindler for the luciferase constructs and the Schering-Plough Research Institute for human IL-4. We also thank Drs. Bryan Williams, Thomas Hamilton, Serpil Erzurum, and Baisakhi Raychaudhuri for careful reading of and critical comments on the manuscript.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by Grant R01-GM60533 (to S. J. H.) from NIH. Back

2 To whom requests for reprints should be addressed, at Department of Cancer Biology, NB-40, Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195. Phone: (216) 445-6622; Fax: (216) 445-6269; E-mail: haquej{at}ccf.org Back

3 The abbreviations used are: IL, interleukin; EMSA, electrophoretic mobility shift assay; GBM, glioblastoma multiforme; PTP, protein tyrosine phosphatase; SOCS, suppressor of cytokine signaling; {gamma}c, {gamma} common; EPOR, erythropoietin receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IFN, interferon; IP, immunoprecipitation; Jak, Janus kinase; RPA, RNase protection assay; RT-PCR, reverse transcription-PCR; Stat, signal transducer and activator of transcription; WCE, whole cell extract; NHA, normal human astrocyte. Back

4 S. O. Rahaman et al., submitted for publication. Back

Received 4/ 9/01. Accepted 12/ 3/01.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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