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Blood, Vol. 92 No. 12 (December 15), 1998:
pp. 4529-4538
RAPID COMMUNICATION
Stat6 Inhibits Human Interleukin-4 Promoter Activity in T Cells
By
Steve N. Georas,
John E. Cumberland,
Thomas F. Burke,
Rongbing Chen,
Ulrike Schindler, and
Vincenzo Casolaro
From the Divisions of Pulmonary and Critical Care Medicine and
Allergy and Clinical Immunology, The Johns Hopkins University Asthma
and Allergy Center, Baltimore, MD; and Tularik Inc, South San
Francisco, CA.
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ABSTRACT |
The differentiation of naive T-helper (Th) cells into
cytokine-secreting effector Th cells requires exposure to multiple
signals, including exogenous cytokines. Interleukin-4 (IL-4) plays a
major role in this process by promoting the differentiation of
IL-4-secreting Th2 cells. In Th2 cells, IL-4 gene expression is
tightly controlled at the level of transcription by the coordinated
binding of multiple transcription factors to regulatory elements in the
proximal promoter region. Nuclear factor of activated T cell (NFAT)
family members play a critical role in regulating IL-4 transcription
and interact with up to five sequences (termed P0 through P4) in the
IL-4 promoter. The molecular mechanisms by which IL-4 induces
expression of the IL-4 gene are not known, although the IL-4-activated
transcription factor signal transducer and activator of transcription 6 (Stat6) is required for this effect. We report here that Stat6
interacts with three binding sites in the human IL-4 promoter by
electrophoretic mobility shift assays. These sites overlap the P1, P2,
and P4 NFAT elements. To investigate the role of Stat6 in regulating IL-4 transcription, we used Stat6-deficient Jurkat T cells with different intact IL-4 promoter constructs in cotransfection assays. We
show that, whereas a multimerized response element from the germline
IgE promoter was highly induced by IL-4 in Stat6-expressing Jurkat
cells, the intact human IL-4 promoter was repressed under similar
conditions. We conclude that the function of Stat6 is highly dependent
on promoter context and that this factor promotes IL-4 gene expression
in an indirect manner.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
INTERLEUKIN-4 (IL-4) is a prototypic
immunoregulatory cytokine.1 By virtue of its ability to
induce IgE isotype switching in B cells, mast cell differentiation, and
adhesion molecule expression, IL-4 plays a central role in many
inflammatory responses.2-4 IL-4 is also the primary
cytokine promoting the differentiation of naive T cells into
cytokine-secreting T-helper 2 (Th2) cells.5
Cytokine gene expression in Th2 cells is controlled primarily at the
level of gene transcription,6 and dysregulation of this
process is thought to contribute to the development of allergic
diseases.7 Several transcription factors have recently been
implicated in regulating Th2-restricted IL-4 gene expression (Fig 1).8-13 Nuclear factors of activated T
cell (NFAT) are involved in this process by interacting with up to five
sites in the IL-4 promoter (termed P0 through P4).14,15 The
precise role of individual NFAT family members in regulating IL-4
transcription is currently unknown.16-18
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| Fig 1.
The proximal IL-4 promoter (not drawn to scale). NFAT
binding sites (the P elements) are indicated by open boxes, and the
known Stat6 site is indicated by the solid box. Transcription factors
implicated in Th2-specific IL-4 gene expression are indicated below
their respective binding sites. A binding site for GATA-3 has not yet
been reported.13 NFAT activity is higher in effector Th2
cells ("Activated" NFAT10) and is shown for
simplicity binding only to the P1 NFAT site.
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Using T-cell lines derived from NFAT-reporter transgenic mice,
Rincón and Flavell10 found that NFAT transcriptional
activity is preferentially induced in Th2 cells but not in Th1 cells.
Growing evidence suggests that Th2-specific NFAT cofactors may
specifically enhance IL-4 transcription in these cells. For example, Ho
et al9 found that the proto-oncogene c-maf was
preferentially expressed in Th2 clones and that c-Maf acts
synergistically with NFATp to induce IL-4 production in IL-4-negative
cells. Additionally, Li-Weber et al12 detected a
multiprotein complex containing C/EBP, NFAT, and AP-1 proteins forming
on the P4 element using nuclear extracts from Th2 but not Th1 cells in
electrophoretic mobility shift assays (EMSA).
Exogenous IL-4 is a critical stimulus for the effective differentiation
of naive T cells into IL-4-secreting Th2 cells (for review, see
O'Garra5). The mechanism by which IL-4 induces expression
of the IL-4 gene is currently not known. IL-4 interacts with a
multichain cell-surface receptor (IL-4R) that is expressed by several
cell types, including B cells, T cells, and endothelial cells.4,19 IL-4 binding to the IL-4R chain induces
different intracellular signals, including Jak-mediated phosphorylation of the transcription factor Stat6.20 Stat6 response
elements share a consensus sequence
5 TTCN3/4GAA3 and are
located in the promoter regions of many IL-4-responsive genes.21,22 The fundamental role of Stat6 in IL-4-driven
responses was demonstrated by the phenotype of Stat6-deficient mice in
which IgE synthesis and Th2 responses were abrogated.23-25
Lederer et al11 discovered a Stat6 binding site in the
mouse IL-4 promoter and found that Stat6 bound this sequence in EMSA
using nuclear extracts from IL-4 induced Th2 cells but not Th1 cells.
We and others identified a corresponding site in the human IL-4
promoter ( 169TTCACAGGAA160).26,27
Because multimers of these elements were inducible by IL-4 when linked
to heterologous promoters and transfected into Stat6-expressing B-cell
lines,11,26 it seemed reasonable to conclude that Stat6
would directly enhance IL-4 transcription in T cells.
However, recent studies have suggested that activation of IL-4R
signaling pathways is not required for IL-4 gene expression in effector
T cells. For example, Huang et al28 found that IL-4 did not
enhance transcription driven by a mouse IL-4 promoter construct in
anti-CD3-activated Th2 cells, although the specific role of Stat6 in
regulating the intact IL-4 promoter was not examined in that study.
Additionally, Moriggl et al29 reported that a neutralizing
anti-IL-4 monoclonal antibody (MoAb) actually enhanced anti-CD3-induced IL-4 production in committed Th2 cells. Using Stat6-deficient Jurkat T cells in cotransfection assays, we report here
that, although cotransfected Stat6 strongly enhanced transcription driven by a multimerized response element, the human IL-4 promoter was
significantly repressed under similar conditions. The repressive effects of Stat6 appeared to involve sequence-specific DNA binding, because a Stat6 DNA binding domain mutant failed to inhibit the IL-4
promoter. We describe two novel Stat6 binding sites within the proximal
IL-4 promoter and show that Stat6 and NFAT bind competitively to
overlapping nucleotides.
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MATERIALS AND METHODS |
Plasmid construction.
IL-4 promoter constructs were amplified from human genomic DNA using
the polymerase chain reaction (PCR). A 25-bp 5 primer annealing
265 bp upstream from the transcription start site (tss) according to
Otsuka et al30 was used with a 25-bp 3 primer ending
at position +65. PCR products were sequenced using the dideoxy method
and then ligated into the HindIII and Xba I sites of
pCAT Basic (Promega, Madison, WI) to yield pCAT 265. The
reporter C/EBP-N4 luc contains 4 copies of the composite C/EBP/Stat6
response element from the germline  promoter fused to a thymidine
kinase (TK) minimal promoter driving the firefly luciferase
gene.21 The wild-type Stat6 expression vector (TPU 388) and
the DNA-binding domain mutant vector (TPU522, in which the 3 amino
acids VVI at positions 411 to 413 were replaced by EAA), both driven by
the cytomegalovirus (CMV) promoter, have been described.21
Cell lines and transfections.
Jurkat T cells (a kind gift of Dr Jack Strominger, Harvard University,
Cambridge, MA) were maintained in complete medium (RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum [FCS; Life Technologies, Gaithersburg, MD] and 50 µg/mL
gentamycin [Life Technologies]). As previously reported, these cells
constitutively express IL-4 mRNA.31 HepG2 cells were
obtained from the ATCC (Rockville, MD) and maintained in
Dulbecco's modified Eagle's medium (DMEM) supplemented
with 10% FCS and 50 µg/mL gentamycin. In cotransfection experiments,
3 × 106 cells were transfected with 1 µg reporter,
2 µg expression or empty vector as a control, and 7 µL Superfect
(Qiagen, Valencia, CA) in 5 mL complete medium and allowed
to recover for 24 hours. Cells were then stimulated with combinations
of the following agonists as indicated in the text for the last 18 hours: 1 µmol/L calcium ionophore (A23187; Calbiochem, San Diego,
CA), 25 ng/mL phorbol-12-myristate-13-acetate
(PMA; Calbiochem), and IL-4 (10 or 50 ng/mL; Peprotech,
Rocky Hill, NJ). Cells were lysed by three freeze-thaw
cycles and reporter gene expression was determined either by measuring
CAT enzyme levels using a sensitive enzyme-linked immunosorbent assay
(ELISA; Boehringer Mannheim, Indianapolis, IN) or by assaying for luciferase activity using standard
techniques (Analytic Luminescence Laboratories, Sparks,
MD). Cell extracts were normalized for protein content using the
Bradford technique (Bio-Rad, Hercules, CA) before assays
for reporter gene expression.
EMSA.
The following 30-bp oligonucleotides and their complements were
synthesized (mutations in the P2 oligonucleotide are indicated as
lowercase letters, and the Stat6 consensus sequence is underlined): 5-ATTGCTGAAACCGAGGGAAAATGAGTTTACAT- TG-3 (P0  69 to
36); 5-TGAGTTTACATTGGAAATTTTCGTTACACCAGATTG-3 (P1
92 to 60);
5-TCTGATTTCACAGGAACATTTTACCTGTTT-3 (P2 wt
175 to 146);
5-gagac-TTTCACAGGAACATTTTACCTGTTT-3 (P2 m1);
5-TCTGAgggacCAGGAACATTTTACCTGTTT-3 (P2 m2);
5-TCTGATTTCAgctcgACATTTTACCTGTTT-3 (P2 m3);
5-TCTGATTTCACAGGActcggTTACCTGTTT-3 (P2 m4);
5-TCTGATTTCACAGGAACATTTgcgtt-TGTTT-3 (P2 m5);
5-TCTGATTTCACAGGAACATTTTTACCcaccg-3 (P2 m6);
5-AATCAGACCAATAGGAAAA- TGAAACCTTTTTAA-3 (P3 201 to 169); and 5-AGTTTCAGCATAGGAAATTACACCATAATTTGC-3
(P4 248 to 216).
The Bcl-6 oligonucleotide (B6BS:
5-GAAAATTCCTAGAAAGCATA-3; donated by Dr Riccardo
Dalla-Favera, Columbia University, New York, NY) has been
described.32 Nuclear extracts were obtained from 5 × 106 Jurkat cells treated without or with IL-4 (20 ng/mL for
20 minutes; Peprotech) using the method of Schrieber et
al.33 EMSAs were performed using 5 µg nuclear protein,
0.8 µg poly (dG-dC) (Amersham Pharmacia Biotech, Piscataway, NJ), and
[ 32P] end-labeled probe in a final volume of 10 µL
per reaction. Free probes and protein-DNA complexes were resolved by
5% polyacrylamide gel electrophoresis (PAGE) with
0.5× TBE. In antibody experiments, extracts were incubated at
room temperature with 1 µL of the following antisera for 30 minutes
after the addition of labeled probe: anti-Stat6 (Santa Cruz Biotech,
Santa Cruz, CA), N70-6 (anti-Bcl-632;
donated by Dr Riccardo Dalla-Favera), or isotype-matched control
antisera.
Recombinant proteins.
A recombinant fragment of murine NFATp (including 298 amino acids of
the DNA binding domain [DBD] that is highly conserved among different
NFAT family members34) was expressed as a
hexahistidine-tagged protein and extracted as described.35
The NFATp expression vector was kindly donated by Dr Anjana Rao
(Harvard University). Recombinant full-length, in vitro phosphorylated
Stat6 has been described.21
Statistical analysis.
All transfections were performed in duplicate using cells of similar
passage number. Average results of the indicated numbers of independent
experiments were analyzed using the paired Student's t-test
(Statview II Software; SAS Institute, San Francisco, CA), and a P value less than .05 was considered to be statistically significant.
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RESULTS |
The IL-4R signaling pathway is intact in Jurkat T cells.
To investigate the role of Stat6 in regulating transcription of the
intact IL-4 promoter, we studied Jurkat T cells transiently transfected
with different IL-4 promoter reporter constructs. In preliminary
experiments, we did not detect immunoreactive Stat6 in nuclear extracts
from IL-4-activated Jurkat cells in EMSA, suggesting that the cells
used in these experiments express negligible levels of this factor (not
shown). Consistent with this result, IL-4 stimulation alone did not
induce a full-length IL-4 promoter construct (not shown) or the
reporter construct C/EBP-N4 luc (which contains 4 copies of the IgE
C/EBP/Stat6 element driving the firefly luciferase gene;
Fig 2A). However, as previously
reported,21 C/EBP-N4 luc was strongly induced by IL-4 in
Stat6-expressing HepG2 cells (Fig 2B).
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| Fig 2.
The IL-4 receptor signaling pathway is intact in Jurkat T
cells. (A) The luciferase reporter construct C/EBP-N4 luc (see text)
was transiently transfected into Jurkat cells together with a control
(Empty) or Stat6 expression vector. Cells were then stimulated without
( ) or with ( ) IL-4 (50 ng/mL) for 18 hours before assays for
reporter gene expression. In the absence of either cotransfected Stat6
or IL-4 stimulation, C/EBP-N4 luc is not active in Jurkat cells, but it
is highly inducible by IL-4 in cells expressing Stat6. (B) Consistent
with the known expression of Stat6 by Hep G2 cells,21
C/EBP-N4 luc was induced by IL-4 in these cells, but its activity was
further increased by overexpressing Stat6 (note the different scales).
Results are the mean ± SEM of two (B) or three (A) independent
experiments.
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To verify that the IL-4R signaling pathway was otherwise functional in
Jurkat cells, we first analyzed the inducibility of C/EBP-N4 luc in
cells cotransfected with a full-length Stat6 expression vector.
Activity of C/EBP-N4 luc is strongly dependent on the coordinated
binding of C/EBP and Stat6 to adjacent sites.21 Figure 2A
shows that C/EBP-N4 luc was highly induced by IL-4 in Stat6-expressing
Jurkat cells, but not in control cells cotransfected with the
corresponding empty vector. This result confirms the known expression
of C/EBP proteins by Jurkat cells.36 Consistent with
previous findings,21 C/EBP-N4 luc was induced by IL-4 in HepG2 cells in the absence of exogenous Stat6 (Fig 2B). Thus, although
Jurkat cells do not constitutively express functional Stat6 protein,
cotransfected Stat6 is highly induced by IL-4 in these cells.
Stat6 represses transcription driven by the intact IL-4 promoter.
We next analyzed the ability of Stat6 to transactivate the intact IL-4
promoter. Figure 3 shows that a full-length
human IL-4 promoter construct (pCAT 265) was consistently inhibited by
IL-4 in Stat6-cotransfected Jurkat cells. To determine whether
unstimulated Jurkat cells lacked an activation-induced Stat6 cofactor
or whether Stat6 needed a further activation signal, which is necessary
for IL-4 transcription, we next analyzed the effects of IL-4 in
activated cells cotransfected with Stat6 and pCAT 265. As previously
reported,37,38 a calcium-dependent signal alone was
sufficient to maximally induce the IL-4 promoter (Fig 3). PMA
downregulated IL-4 promoter activity, which we previously found was due
to the displacement of NFATp from the human P1 sequence by induced
nuclear NF- B heterodimers.37 Interestingly, IL-4
consistently inhibited calcium-induced promoter activity in
Stat6-cotransfected cells and almost completely repressed the promoter
in combination with PMA (Fig 4). Thus, even
in conjunction with activation of intracellular calcium and
PKC-signaling pathways, Stat6 inhibited transcription driven by the
full-length IL-4 promoter.
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| Fig 3.
Stat6 inhibits transcription driven by the intact IL-4
promoter. pCAT 265, which contains all of the known NFAT P elements
including the P2 Stat6 site (solid box), was transfected into
unstimulated Jurkat cells together with a control (Empty) or a Stat6
expression vector, and the cells were incubated without () or with
() IL-4 (50 ng/mL) for 18 hours before assays for reporter gene
expression by ELISA. Results are expressed relative to the constitutive
activity of pCAT 265 without IL-4 and are the mean ± SEM of four
independent experiments. IL-4 significantly downregulated promoter
activity only in Stat6-cotransfected cells.
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| Fig 4.
Stat6 does not synergize with other signals to activate
the IL-4 promoter. Methods were similar to those described in Fig 3,
except that cells were also stimulated with calcium ionophore ( ),
PMA (), both ( ), or no agonists () with or without IL-4 as
indicated. In the presence of IL-4, reporter activity was consistently
decreased in Stat6-cotransfected cells for each condition examined.
Note that the combination of PMA and IL-4 almost completely repressed
the promoter. Results are from one experiment performed in duplicate
and are representative of three.
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To map the IL-4 promoter element(s) necessary for Stat6-mediated
transcriptional repression, we used a smaller promoter construct in
additional cotransfection experiments. This construct (pCAT 145)
contains 145 bp of the human promoter, including the P1 and P0 NFAT
elements, but lacks the previously described Stat6 binding site
(169TTCACAGGAA160). Interestingly,
pCAT145 activity was significantly inhibited by IL-4 in both resting
and stimulated Stat6 cotransfected cells (Fig 5). Importantly, an expression vector
encoding a Stat6 DNA binding domain mutant (TPU522, see Materials and
Methods) did not inhibit pCAT145 activity in either resting or
activated cells (Fig 5, right side). These results suggested that
Stat6-induced repression involved binding sites located downstream of
the known Stat6 sequence and that the DNA binding ability of Stat6 was
required for this effect.
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| Fig 5.
Stat6 inhibits a minimal IL-4 promoter construct. Jurkat
cells were cotransfected with a minimal promoter construct lacking the
known P2 Stat6 element (pCAT 145) with or without a wild-type (wt) or
DNA-binding domain mutant (DBD mut) Stat6 expression vector as
indicated. Cells were stimulated for 18 hours with calcium ionophore
() with or without IL-4 as indicated, followed by cell lysis and
analysis of reporter gene expression by ELISA. Results are expressed
relative to CAT production in unstimulated cells and are the mean ± SEM of four (DBD mut) or seven (wt) independent experiments. *P < .05.
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The human IL-4 promoter contains multiple overlapping Stat6 and NFAT
binding sites.
The previously described Stat6 binding site is contained within the P2
NFAT element (Fig 6). We and others have
shown that this element can support the cooperative binding of NFAT and
AP-1 proteins.39,40 As shown in Fig 6, the 3-half of
the Stat6 site (5GAA3) overlaps
the 5-end of the NFAT site
(5GGAA3). In view of our
functional data showing repression of a construct lacking the P2
element (Fig 5), we speculated that Stat6 could interact with
additional P elements from the IL-4 promoter. To test this hypothesis,
we analyzed the ability of recombinant Stat6 to bind similar length
oligonucleotides including the five known IL-4 P elements by EMSA.
Figure 7 shows that recombinant Stat6 bound
30-bp oligonucleotides containing the P1, P2, and P4 NFAT sites, but
not the P0 or P3 sites.
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| Fig 6.
The P2 element contains overlapping binding sites for
Stat6 and NFATp. (A) Alignment of the human IL-4 P2 NFAT element with
canonical binding sites for NFAT (overline) and Stat6 (underline). Note
that the 3-end of the Stat6 sequence overlaps the 5-end
of the NFAT site. (B) The ability of recombinant Stat6 to interact with
wild-type (wt) and mutated probes (see Materials and Methods) was
determined by EMSA. Stat6 no longer bound the m2-m4 oligonucleotides,
confirming the overlapping nature of the Stat6 and NFAT binding
sites.
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| Fig 7.
The IL-4 promoter contains multiple Stat6 binding sites.
(A) The ability of recombinant Stat6 to bind oligonucleotides
containing the human P elements was determined by EMSA. Stat6 bound the
P1, P2, and P4 probes (solid arrow). An additional slowly migrating
complex (open arrow) formed on the P1 oligonucleotide (and occasionally
on the P2 probe; see Fig 8). The relative migration of the free probes,
which were of similar length (see Materials and Methods) and
radiolabeled with similar specific activity, is not shown in this
figure. (B) Alignment of the P elements that supported Stat6 binding
with the composite C/EBP-Stat6 site from the germline IgE promoter. The
P2 oligonucleotide is shown in opposite orientation than in Fig 2.
Binding sites for NFAT, Stat6, and C/EBP are indicated by boxes. C/EBP
proteins may interact with the P0, P1, and P4 NFAT elements, although
the precise nucleotide binding sites have been reported only for the P4
sequence.36
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Based on sequence homology and mutational analysis (Fig 6), we
predicted that Stat6 and NFAT would bind competitively to overlapping sites in the IL-4 promoter. To test this hypothesis and to exclude any
combinatorial interactions of these factors on the IL-4 promoter, we
analyzed the effect of Stat6 on the ability of the NFATp DBD (see
Materials and Methods) to interact with oligonucleotides containing the
P1 and P2 elements in EMSA. As expected, NFATp readily bound both
probes (Fig 8). Interestingly, increasing
amounts of Stat6 displaced NFATp from its cognate sites on both
oligonucleotides. Displacement of NFAT from the P1 element by Stat6 may
provide an explanation for the repressive effects of Stat6 on pCAT 145.
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| Fig 8.
Stat6 binds competitively with NFAT to the P1 and P2
elements. (A) The ability of a recombinant fragment of NFATp (see
Materials and Methods) to bind oligonucleotide probes containing the P1
and P2 elements in the presence of increasing concentrations of
recombinant Stat6 was determined by EMSA. The relative mobilities of
each factor are indicated. Serial twofold dilutions of Stat6 were
examined against a constant amount of NFATp. n.s., nonspecific. (B) The
relative intensities of observed bands were analyzed by densitometry
and expressed relative to intensity of the NFAT complex for each
oligonucleotide in the absence of Stat6 (which was defined as 1).
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DISCUSSION |
The molecular basis by which IL-4 induces the production of Th2
cytokines is currently not known. Experiments with Stat6-deficient mice
have conclusively demonstrated a requirement for this IL-4-inducible transcription factor during the differentiation of naive T cells into
Th2 cells.23-25 However, recent studies have found that
activation of the IL-4R-signaling pathway is not required for the
induction of IL-4 gene expression in committed Th2
cells.28,29,41 Additionally, Stat6 can repress IL-4 gene
expression in Th1 cells by binding a cell-type specific silencer in the
3 untranslated region (UTR).42 Thus, the precise
role of Stat6 in regulating IL-4 expression is currently not clear.
Th2-restricted IL-4 gene expression is thought to be controlled at the
level of transcription by the coordinated interactions of transcription
factors binding to a proximal promoter region. Regulatory elements
within the promoter have been shown not only to bind nuclear factors
unique to Th2 cells, but also to be preferentially induced in Th2
cells.8-13 The observation that Stat6 can interact with a
consensus sequence from both the mouse11 and
human26 IL-4 promoters suggested that this factor might
provide a direct link between IL-4R activation and IL-4 gene
expression. However, a conclusion of our studies is that Stat6 might
only facilitate IL-4 gene expression in T cells in an indirect fashion.
This report, showing for the first time the specific effect of Stat6 on
the intact IL-4 promoter, contains several novel observations. First,
we found that, although IL-4 receptor-mediated signals are faithfully
transduced in Jurkat T cells (Fig 2), Stat6 was unable to transactivate
the proximal human IL-4 promoter in these cells (Figs 3 through 5).
Together with previous studies showing that IL-4 can induce multimers
of the P2 Stat6 site linked to heterologous
promoters,11,26,28 our findings suggest that the
transactivation potential of Stat6 is dependent on promoter context.
This conclusion is in keeping with recent analyses of Stat6 and the
germline IgE43 and  -casein genes44 and with the study of Huang et al,28 who found that Stat6
differentially regulated multimers of the P2 Stat6 element in M12 B
cells depending on the minimal promoter construct used.
Second, we found that transcription driven by two deletion constructs
of the human IL-4 promoter was consistently inhibited by IL-4 in
Stat6-expressing Jurkat T cells. Interestingly, recent studies suggest
that IL-4 can inhibit IL-4 gene expression in a negative feedback
fashion. For example, Moriggl et al29 found that committed
Th2 cells secreted more IL-4 when restimulated with anti-CD3 in the
presence of a neutralizing anti-IL-4 MoAb. Additionally, IL-4 seemed
to inhibit anti-CD3-induced transcription driven by the full-length
mouse IL-4 promoter in a Th2 clone.28 Taken together, these
results suggest that activated Stat6 might downregulate IL-4 expression
in effector Th cells and emphasize the need to distinguish between Th2
differentiation and IL-4 gene expression (see below).
Third, we have identified two novel Stat6 binding sites (within the P1
and P4 elements) in the IL-4 promoter. Although these sites do not
contain the consensus Stat6 binding site
(5-TTCN4GAA-3) defined by binding site
selection assays, a significant fraction (10/42) of sequences selected
by Stat6 in these assays contained single nucleotide substitutions
within the dyad half-sites.45 Thus the ability of Stat6 to
bind the P1 oligonucleotide
(5-TTCN4GTA-3) is not surprising.
The reason why Stat6 bound the P4 element but not the P0 element is
less apparent, because they contain similar noncanonical dyad half
sites, although they do differ in the spacer region.
Fourth, the demonstration that Stat6 and NFAT can bind competitively to
the IL-4 promoter provides evidence of a previously unreported
interaction between these two factors. Given the fundamental role of
NFAT family members in regulating IL-4 gene expression, this
observation provides a plausible explanation for the observed inhibitory effects of Stat6 on transcription driven by the IL-4 promoter. This would be similar to the recently described antagonism of
NF-B by Stat6 in the E-selectin promoter.46 Because the P1 element in particular plays a major role in activating IL-4 transcription,8,47 competition by Stat6 for NFAT binding to this element might have particularly repressive effects. Alternatively, we cannot exclude the possibility that Stat6 might actively repress the
basal transcription complex or that it might also bind to and titrate
away from the promoter a factor necessary for maximal IL-4
transcription.
Stat6 might require a coactivator not expressed by Jurkat cells to
maximally transactivate the IL-4 promoter. For example, Stat6
cooperates with both C/EBP21 and NF-B/Rel
proteins48 to promote germline IgE
transcription. The fact that C/EBP-N4 luc is highly induced (Fig 2)
argues that the inability of Stat6 to activate IL-4 transcription in
our experiments is not due to the lack of C/EBP proteins and confirms
the known expression of C/EBP in Jurkat cells.36
Additionally, we have shown that PMA-activated Jurkat cells contain
abundant nuclear NF-B.37 We cannot formally exclude the
requirement for as yet unidentified Stat6 coactivators necessary for
IL-4 transcription, although the studies by Huang et al28
argue against the existence of such factors in a differentiated Th2
clone. Another possibility is that Stat6 might require additional posttranslational modification to achieve full transcriptional competency in Jurkat cells. However, the observations that (1) PMA
enhanced the repressive effects of Stat6 (Fig 4) and (2) C/EBP-N4 luc
was inducible by IL-4 in Stat6-cotransfected Jurkat cells (Fig 2) argue
against this explanation.
Finally, it is possible that other factors inhibit the transactivation
potential of Stat6 on the IL-4 promoter. For example, it has recently
been suggested that Bcl-6, a transcriptional repressor deregulated in
many lymphomas,32,49 can specifically inhibit the ability
of Stat6 to transactivate gene expression. In fact, Bcl-6-deficient
mice were found to have enhanced IL-4 production and Th2
responses.50 To exclude the possibility that Bcl-6 was inhibiting transactivation of the IL-4 promoter by Stat6 in Jurkat cells, we assayed for Bcl-6 binding to both the P2 element and a
consensus Bcl-6 site using Jurkat nuclear extracts in EMSA. Using
conditions known to support Bcl-6 binding,32 we did not detect Bcl-6 using two specific antisera (not shown). Additionally, IL-4 gene expression was not inhibited in Jurkat cells expressing inducible Bcl-6 protein (Dr Riccardo Dalla-Favera, personal
communication, Spring 1998). Thus, we conclude that the
inability of Stat6 to activate IL-4 transcription in Jurkat T cells is
not due to the presence of the specific Stat6 antagonist Bcl-6.
The IL-4R transduces other signals in addition to the Jak-mediated
tyrosine phosphorylation of Stat6. For example, the I4R motif of the
IL-4R subunit leads to the IRS-1-dependent phosphorylation of the
nonhistone chromosomal protein HMGI/Y.51 Phosphorylation of
HMGI/Y in B cells inhibits its ability to bind DNA and results in the
derepression of germline IgE transcription.43,52 HMGI/Y has
recently been shown to downregulate the IL-4 promoter by competing with
NFAT for binding to the P1 element.53 Thus, activation of
this IL-4R signaling pathway would be expected to de-repress the IL-4
promoter. However, our observation that Stat6 also displaces NFAT from
the P1 element (Fig 8) might explain why this did not occur in our
experiments.
It is worth emphasizing that IL-4-dependent differentiation of naive
Th cells into effector Th2 cells involves prolonged exposure to
multiple concomitant signals emanating from the T-cell receptor, costimulatory molecules, and possibly other APC-derived
cytokines.5,54-56 The precise role of Stat6 in this process
requires further study. We have shown that Stat6 does not directly
transactivate the IL-4 promoter, which it actually represses in cells
transcribing the IL-4 gene. Together with recent analyses of committed
Th cells,28,29 our results suggest that Stat6 may rather
facilitate the acquisition of an IL-4-producing phenotype in
differentiating Th cells in an indirect fashion. In this regard,
investigating the regulation of other lineage-specific Th2
transcription factors by Stat6 may be helpful.
| |
ACKNOWLEDGMENT |
The authors thank Dr Riccardo Dalla-Favera for assistance with the
Bcl-6 experiments, Dr Anjana Rao for the NFATp expression vector, and
Dr Marcia Wills-Karp (Johns Hopkins University, Baltimore, MD) for
helpful suggestions.
| |
FOOTNOTES |
Submitted July 2, 1998;
accepted October 5, 1998.
Supported by Grant No. AI01152 from the National Institutes of Health
and Research Grant No. 056-N from the American Lung Association.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Steve N. Georas, MD, Room 4B.41, The Johns
Hopkins Asthma & Allergy Center, 5501 Hopkins Bayview Circle,
Baltimore, MD 21224; e-mail: sgeoras{at}welchlink.welch.jhu.edu.
| |
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Abstract
Introduction