From the Institute for Biochemistry, Free University
Berlin, 14195 Berlin, Germany and § Gladstone Institute of
Virology and Immunology, University of California San Francisco,
San Francisco, California 94141-9100
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ABSTRACT |
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Interleukin-9 (IL-9) is a cytokine with
pleiotropic effects on mast cell and T cell lines. It exerts its
effects through the IL-9R complex consisting of IL-9R and the common
c subunit. Here we report functional evidence for
receptor heteromerization for efficient signal transduction, and we
define minimal requirements in the two receptor subunits for IL-9R
function. Tyrosine 336 of the IL-9R
and the membrane-proximal
segment of
c are both crucial for signaling. The
activated IL-9R complex employs the Janus kinases JAK1 and JAK3 for
subsequent activation of the signal transducer and activator
transcription (STAT) factors STAT-1, STAT-3, and STAT-5. This process
is independent of Tyk2. We demonstrate further that the activated STAT
complexes consist of STAT-1 and STAT-5 homodimers and STAT-1-STAT-3
heterodimers. Finally, we show that IL-9R signaling in a T cell line
does not result in detectable mitogen-activated protein kinase
activation and leads to unsustained proliferation. Nonetheless, these T
cells are efficiently protected from dexamethasone-induced apoptosis.
These results further define the molecular architecture of the IL-9R
and its specific connections to various biologic responses.
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INTRODUCTION |
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IL-91 is a pleiotropic cytokine secreted by activated T cells of the TH2 class (for review see Renauld et al. (1)). It is a major factor for mast cell differentiation, but its activity on T cells is less well characterized. It was shown to induce granzyme A and B expression in T cell lines (2). IL-9 is not mitogenic for freshly isolated T cells, but it is active on preactivated T cells and T cell lines (3). In addition, IL-9 stimulates in vitro growth of T cell lymphomas (4), and IL-9 transgenic mice show increased occurrence of thymic lymphomas (5). In humans, dysregulated IL-9 expression has been found in patients with Hodgkin's disease (6) and an autocrine IL-9 loop for proliferation has been demonstrated in Hodgkin's lymphoma-derived cell lines (7). Furthermore, it has been shown that IL-9 can protect thymic lymphoma cells and T cell lines from dexamethasone-induced apoptosis (8, 9). These data together suggest a possible role for IL-9 in tumorigenesis.
IL-9 exerts its effects through the functional IL-9R complex,
consisting of IL-9R (10) and the common
c subunit.
The
c subunit is also utilized in the receptor complexes
for IL-2, IL-4, IL-7, and IL-15 (11-14). Both IL-9R
and
c are members of the hematopoietin receptor superfamily
(15), which share several common motifs, including four canonically
spaced cysteine residues and the WSXWS motif in the
extracellular domain, and the Box1 and Box2 motifs in the intracellular
domain. The IL-9R
was shown previously to associate with
c in the presence of IL-9 (16), and evidence for
functional interaction of the two receptor chains was derived from
studies in which anti-
c antibody treatment abolished IL-9R signaling (17).
Although neither receptor chain has any intrinsic kinase activity, rapid tyrosine phosphorylation of both receptor chains and cellular substrates occurs after receptor engagement. This step is thought to be mediated by the nonreceptor protein tyrosine kinases of the Janus kinase (JAK) family, which are preassociated with the receptor chains (for reviews, see Refs. 18 and 19). Stimulation of the IL-9R was shown to lead to the phosphorylation of JAK1, JAK3, and Tyk2 (20). It remained unclear, however, which of these kinases are necessary for the generation of the signaling response. Phosphorylated tyrosines of the receptor act as docking sites for STAT proteins (signal transducer and activator of transcription), that bind phosphotyrosines via SH2 domains (21, 22). Once bound, they become tyrosine-phosphorylated, dissociate from the receptor, and form homo- or heterodimers with members of their own family via SH2 domain interactions. These activated transcription factor dimers are capable of translocation into the nucleus to bind to specific DNA elements and initiate transcription of target genes. Several studies have suggested that the STATs involved in IL-9R signaling are STAT-1, STAT-3, and STAT-5 (20, 23, 24). However, the specific composition of the STAT·DNA complexes remained unclear.
The major activity of IL-9 on T cells has been proposed to be protection from apoptosis rather than mitogenesis. This has been demonstrated for thymic lymphoma cell lines (8) and T cell lines (9). However, no evidence has been obtained regarding the mechanisms underlying this antiapoptotic activity of the activated IL-9R.
In the present study chimeric receptors between the erythropoietin
(EPO) extracellular domain and the IL-9R and
c
intracellular domains were created and tested, along with several
mutants of these chimeras, in COS-7 cells. The role of JAKs in IL-9R
signaling was investigated in the human fibrosarcoma cell lines U4A and U1A that are deficient in JAK1 and JAK3, and JAK3 and Tyk2,
respectively. Using these systems, the minimal requirements for IL-9R
signaling were defined. Finally, a murine T helper cell line was stably transfected with chimeric receptors, and the effect of receptor stimulation was investigated by assays for STAT-DNA binding,
antiapoptotic activity, and proliferation. These data suggest that
signaling through the IL-9R blocks dexamethasone-induced activation of
components of the cell death machinery, possibly due to activation of
the antiapoptotic STAT-3 and STAT-5.
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MATERIALS AND METHODS |
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Cell Lines and Reagents--
The IL-2-dependent
murine T helper cell line HT-2 (ATCC) was cultured in RPMI 1640, supplemented with 10% fetal bovine serum, 55 µM
2-mercaptoethanol, 2 mM L-glutamine, and 200 units/ml recombinant human IL-2. Transfections were performed by
electroporation (950 µF, 300 V, on a Bio-Rad Genepulser II) using
1 × 107 cells and 20 mg of DNA; stable transfectants
were obtained by selection in G418 (1 mg/ml, Life Technologies, Inc.).
Clones isolated by limiting dilution were screened by Northern blot
analysis to identify cell lines stably expressing the transfected
receptor subunit. Stable transfectants expressing two receptor subunits were derived from cells already expressing either EPO or EPO
YF; clones were obtained by selection in G418 and hygromycin B (Boehringer Mannheim) and screened by Northern blot analysis.
Plasmids and Constructs--
All receptor cDNAs were
subcloned into the expression vectors pCMV4, pCMV4neo, or pCMV4hygro.
Chimeric EPO and receptor mutants were generated as described
elsewhere (25). The EPO9 chimera was constructed in a similar way using
a NheI site at the fusion junction between EPOR
extracellular domain and the transmembrane region of the human
IL-9R
. The EPO9Y3F mutant was generated using polymerase chain
reaction overlap methodology as described previously (25). Native human
IL-9R
cDNA was a kind gift of Dr. J.-C. Renauld.
Proliferation Assays-- Conventional 72-h [3H]thymidine incorporation assays were performed using triplicate cultures of 5 × 104 cells per sample. Cells were incubated for the indicated time period with the indicated amount of factor. 1 µCi of [3H]thymidine was added for the last 4 h of incubation. Data are expressed as a percentage of [3H]thymidine incorporation of cells treated with 10 nM IL-2.
Apoptosis Assays-- 5-10 × 106 cells were incubated for 24 h in medium without IL-2, or stimulated with 10 nM IL-2, 50 units/ml EPO, or 1 µM dexamethasone (Sigma), respectively. Cells were harvested, washed with phosphate-buffered saline, and immediately used for staining. Staining with fluorescein isothiocyanate-conjugated annexin 5 and propidium iodide was performed using the ApoAlert kit (CLONTECH). Samples were analyzed on a Becton Dickinson FACScan. The percentage of dead (annexin 5- and propidium iodine-positive cells) and actively dying cells (annexin 5-positive and propidium iodide-negative) was determined by gating on the intact cell population, excluding cellular debris.
Electrophoretic Mobility Shift Assays (EMSA)-- 20-40 × 106 cells were rested in serum-free medium containing 1% bovine serum albumin for 4 h and stimulated as described for 10 min (COS-7, U4A, U1A) or 15 min (HT-2). Cells were lysed, and nuclear extracts were prepared as described previously (26).
DNA binding studies were performed with 1 × 105 cpm of probe, 3 µg of poly(dI-dC) (Boehringer Mannheim) and the indicated amounts of nuclear extracts on a nondenaturing 5% polyacrylamide gel. The IgG Fc receptor STAT response element (FcMAPK Assay-- 10-20 × 106 cells were rested for 6 h and stimulated as described. In vitro MAPK assays were performed using the MAPK assay kit (New England Biolabs). In short, cells were lysed and activated Erk1/Erk2 MAPK was immunoprecipitated using an antiphospho-MAPK antibody. Kinase assays were performed using an Elk1-glutathione S-transferase fusion protein as a substrate with subsequent immunoblotting for phosphorylated Elk1.
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RESULTS |
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Reconstitution of IL-9R Function in COS-7 Cells--
To evaluate
the minimal requirements for efficient signaling in the IL-9R system a
chimeric receptor approach was employed in the background of COS-7
cells. Expression plasmids encoding EPO9 (chimeric IL-9R subunit
containing extracellular domain of EPOR) and EPO
(chimeric
c
chain containing extracellular domain of EPOR) were cotransfected into
these cells together with various components of the signaling machinery
and STAT activation was investigated. After stimulation with EPO,
nuclear extracts were prepared, followed by EMSA with a STAT-specific
binding element (Fc
R1). DNA binding activity was observed only when
both chimeric receptor chains EPO9 and EPO
were present (Fig.
1A), and was not detected when
either receptor chain alone was present. Thus, signaling through the
IL-9R apparently depends on heteromultimerization of the two receptor
subunits; based on the structure of EPOR these presumably represent
dimers, but higher order multimers may exist. Endogenous JAKs and
cotransfected JAK3 were necessary and sufficient for the generation of
a signal by this receptor complex. In the absence of cotransfected
STAT, a single band corresponding to endogenous STAT-1 (as identified
by antibody supershift analysis, data not shown) was present;
cotransfection of STAT-5 resulted in a second more slowly migrating
band, which was identified as STAT-5 by antibody supershift analysis
(data not shown). These data show that EPO9 and EPO
apparently must
form heteromers for efficient signal transduction resulting in STAT
binding to DNA. This signaling complex requires JAK3 and is able to
activate both STAT-1 and STAT-5.
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Reconstitution of Native IL-9R Function--
To confirm these
findings and to clarify further the role of the JAKs in IL-9R function,
U4A and U1A cells were employed in a similar reconstitution approach.
U4A is a somatic mutant of the human 2FTGH cell line that fails to
express JAK3 and JAK1 but contains JAK2 and Tyk2 (27). In U4A cells,
IL-9-mediated induction of STAT-5 was reconstituted by the simultaneous
transfection of IL-9R,
c, JAK1, JAK3, and STAT-5
(Fig. 2). No signal was observed when any
of these components were omitted from the transfection, indicating that
endogenous Tyk2 or JAK2 cannot substitute for JAK1 or JAK3 in this
system (Fig. 2). Furthermore, replacement of either wild-type JAK1 or
JAK3 with a kinase-inactive mutant that lacks the catalytic lysine
(JAK1 K907A or JAK3 K851A, respectively) (28) abolished downstream
induction of STAT-5. These findings strongly suggest that the primary
kinases required for IL-9R function are JAK1 and JAK3, and that Tyk2 is
dispensable for this activity.
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Signaling by a Chimeric IL-9R Induces STAT-1, STAT-3, and STAT-5 in
a T Cell Line--
The EPO9 and EPO/EPO
YF receptor chains were
stably transfected into the IL-2-dependent murine T helper
cell line HT-2 to investigate IL-9R signaling in a T cell context.
After stimulation of stable transfectants with EPO, nuclear extracts
were prepared and analyzed by EMSA with the Fc
R1 probe. DNA binding
activity was detected only in cells expressing both chimeric receptor
chains, EPO9 and EPO
(Fig. 3). The
resulting triple band vanished when excess unlabeled oligonucleotide
was used as a competitor. STAT activation was sustained over a 60-min
stimulation period, and the signal became weaker with prolonged
stimulation vanishing after 90 min (data not shown). The tripartite
band was also observed in cells transfected with vectors encoding EPO9
and EPO
YF (Fig. 3). To determine further the nature of the observed
bands, supershift analysis was performed with antibodies against
phosphotyrosine and against STAT-1, STAT-3, and STAT-5 (Fig. 3).
Isotype-matched antibodies resulted in no change of pattern. Treatment
with antiphosphotyrosine antibodies resulted in a complete loss of all
three bands, due to prevention of formation of STAT dimers via SH2
domain-phosphotyrosine interactions. When extracts were treated with
anti-STAT-1 antibody, the two lower bands were lost, whereas
anti-STAT-3 treatment led to selective loss of the middle band and some
diminution of the lower band. Anti-STAT-5 antibody resulted in slower
migration of the upper band. The anti-STAT-1 and anti-STAT-3 antibodies presumably are blocking antibodies, preventing either STAT-dimer formation or DNA binding, resulting in loss of the band. In contrast, the anti-STAT-5-antibody apparently binds to the STAT-5·DNA complex, which leads to a slower migration of the band on the gel. These data
suggest that the STAT complexes formed upon stimulation of the IL-9R
consist of STAT-5 and STAT-1 homodimers (upper and
lower bands, respectively), STAT-1-STAT-3 heterodimers
(middle band), and possibly STAT-3 homodimers (bottom
band).
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Signaling through the Chimeric IL-9R Complex Inhibits
Dexamethasone-induced Apoptosis--
To test whether IL-9R stimulation
protected HT-2 cells from glucocorticoid-induced apoptosis, stable
cell lines were treated with dexamethasone and evaluated for apoptotic
responses by flow cytometry analysis of cells stained with fluorescein
isothiocyanate-labeled annexin 5. HT-2 cells were starved of IL-2 for
24 h and treated with either dexamethasone alone or with
dexamethasone and EPO. IL-2 withdrawal, as well as additional
dexamethasone treatment, led to the onset of apoptosis after ~4 h
(data not shown), resulting in a massive number of apoptotic cells
after 24 h (Fig. 4). Treatment with
dexamethasone increased the percentage of dead and dying cells
considerably above the level induced by IL-2 withdrawal. Stimulation of
the chimeric receptor complex with EPO led to a complete blockade of
apoptosis caused by dexamethasone, but had no influence on the degree
of apoptosis caused by IL-2 starvation. EPO-mediated inhibition of
dexamethasone-dependent apoptosis was not observed in cells
expressing a single receptor subunit (EPO or EPO9, Fig.
5, A and B,
respectively), but only in cells expressing both EPO9 and EPO
or EPO9 and EPO
YF (Fig. 4, C and D,
respectively).
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The Signal Delivered by the Chimeric IL-9R Does Not Sustain
Proliferation in HT-2 Cells--
Since reduction of the percentage of
annexin 5-positive cells by treatment with EPO could have been due to
an increased overall cell number, the proliferative signal generated by
the IL-9R was investigated. HT-2 cells transfected with either EPO9 or
EPO alone displayed no proliferative response to EPO in the absence of IL-2 as measured by [3H]thymidine incorporation (Fig.
5). Both, EPO
/9 and EPO
YF/9, however, showed vigorous DNA
synthesis after 24 h in a dose-dependent fashion.
Nevertheless, this response was not sustained and diminished to
background levels after 72 h (Fig. 5). Prolonged cultivation in
EPO without IL-2 did not lead to generation of
EPO-dependent clones.
Activation of Erk1/Erk2 MAPK Does Not Take Place in Response to
EPO--
To test whether the proliferative response after chimeric
receptor stimulation with EPO was associated with activation of MAPKs,
in vitro kinase assays of immunoprecipitated Erk1/Erk2 MAPK
were performed. Unstimulated HT-2 EPO/9 cells showed no detectable
MAPK activity as shown by Western blotting for in vitro phosphorylated Elk1 substrate (Fig. 6).
However, upon stimulation with IL-2, strong activation of MAPK was
observed. Stimulation of the chimeric EPO
/9 receptor complex with
EPO did not result in substantial induction of MAPK activity. These
data suggest that the signaling pathways leading to proliferation are
different in the IL-2R and IL-9R systems, and apparently are incomplete in the IL-9R system in this cellular context.
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DISCUSSION |
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These studies demonstrate that the functional IL-9R complex
consists of heteromers of c and IL-9R
, as confirmed
in a variety of cellular backgrounds and by various measurements of
signal transduction. In a T cell line, STAT activation, induction of DNA synthesis and protection from apoptosis were observed exclusively upon engagement of both receptor subunits. The data presented here with
various mutants of the
c or IL-9R
cytoplasmic tail are also
consistent with the "trigger driver" model proposed for signaling
by various chimeric cytokine receptors (30). In this model, the
c chain acts in the IL-2, IL-4, IL-7, IL-9, and IL-15 receptors to initiate the signaling response upon engagement of ligand
by conveying a kinase (JAK3) into the receptor complex. Within the
IL-9R, the specificity of the resulting signaling response appears to
be coupled to tyrosine residues within the IL-9R
chain.
The IL-9R and its associated JAK1 and JAK3 apparently activate a
specific signaling program involving STAT-1 homodimers, STAT-1-STAT-3 heterodimers and STAT-5 homodimers as shown by antibody supershift analysis of nuclear extracts of stable transfectants. Furthermore, activation of these STAT complexes is linked to tyrosine 336 of IL-9R and does not depend upon tyrosines of the
c
subunit. Therefore, as in the IL-2, IL-4, and IL-7 receptors,
specificity in the signaling program appears to be driven largely
through the longer, non-
c subunit of the receptor.
Consistent with earlier studies demonstrating JAK1 and JAK3 activation (20, 23) by IL-9, these kinases were observed to be crucial for generating STAT·DNA complexes in the IL-9R system. In the U4A system, simultaneous transfection of both JAK1 and JAK3 (along with the receptor chains and STAT-5) effectively reconstituted IL-9-mediated STAT-5 induction. Since these cells express Tyk2 endogenously, it is evident that Tyk2 could not replace either JAK1 or JAK3 to support IL-9R function. Additionally, the catalytic integrity of both of these kinases was essential for IL-9R function. These findings are consistent with those observed in the reconstitution experiments, in which endogenous Tyk2 and JAK1 were insufficient to support IL-9R function. Moreover, experiments with the U4A cell line lacking Tyk2 revealed definitively the dispensability of Tyk2 for IL-9R function. Therefore, although Tyk2 may be activated detectably by IL-9 in some cellular contexts, it does not appear to play a major role in IL-9R signaling function.
IL-9R signals through chimeric receptors were found to stimulate short-term DNA synthesis of HT-2 cells. Strong proliferative effects of IL-9 have been observed only in mast cells and activated T-cells. Non-sustained proliferation, as observed here, could be due to a variety of signaling constraints that may be cell context-specific. The failure of the IL-9R to activate Erk1/Erk2 MAPK as measured in the short-term assay in this system is one such example, and other cell-specific pathways also need to be explored.
Perhaps the most important function mediated by IL-9 is prevention of
apoptosis, which was shown here to depend upon dimerization of the
IL-9R and
c subunits of the IL-9R. The molecular
mechanism underlying the antiapoptotic effect remains to be elucidated. STAT-3 and/or STAT-5 may play a role, since they have been implicated in antiapoptotic effects in other signaling systems. For example, it
was demonstrated that overexpression of a dominant-negative form of
STAT-3 inhibited induction of the antiapoptotic gene bcl-2 upon
stimulation of the IL-6R complex (31). Moreover, STAT-5 has been shown
to bind to the activated glucocorticoid receptor and to inhibit
glucocorticoid receptor-mediated gene transcription in COS-7 cells
(32). Further studies are needed to clarify these and other possible
mechanisms with regard to the IL-9R. Since apoptosis in general plays
an important role in tissue homeostasis and regulation of the immune
response (33), clarifying mechanisms by which immune regulatory
cytokines exert antiapoptotic activity and proliferative actions
remains an important goal. The system presented here should prove
useful in addressing these questions.
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ACKNOWLEDGEMENTS |
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We thank Drs. Ian Kerr and George Stark for
the U4A and U1A cell lines, Dr. Jean-Christophe Renauld for the human
IL-9R cDNA, and Amy Corder, John Carroll, Brian Clark, and
Jessica Diamond for their excellent help in preparing this manuscript.
We also thank K. Mark Ansel for critical comments regarding this
manuscript.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant R01 GM54351 (to M. A. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Supported by a gift from Ligand Pharmaceuticals Inc. (to M. A. G.).
Supported by the National Institutes of Health Medical
Scientist Training Program and the Biological Sciences Program of the University of California, San Francisco.
** Supported by the National Institutes of Health Medical Scientist Training Program and the Biomedical Sciences Program of the University of California, San Francisco.
To whom correspondence should be addressed: Gladstone Institute
of Virology and Immunology, P. O. Box 419100, San Francisco, CA
94141-9100. Tel.: 415-695-3775; Fax: 415-826-1514; E-mail: Mark_Goldsmith{at}quickmail.ucsf.edu.
1 The abbreviations used are: IL, interleukin; JAK, Janus kinase; STAT, signal transducer and activator of transcription; EPO, erythropoietin; EPOR, EPO receptor; MAPK, mitogen-activated protein kinase; Erk, extracellular signal-regulated kinase; EMSA, electrophoretic mobility shift assay.
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REFERENCES |
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