From the Institut für Zellbiologie, Abteilung
Molekularbiologie, Universität Tübingen, Auf der
Morgenstelle 15, 72076 Tübingen, Germany, the
§ Institut für Medizinische Strahlenkunde und
Zellforschung (MSZ), Versbacher Strasse 5, 97078 Würzburg,
Germany, and the ¶ Bender + Co Gesellschaft mbH, Dr.
Boehringer-Gasse 5-11, 1121 Wien, Austria
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ABSTRACT |
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Interleukin-5 (IL-5), expressed primarily by
type-2 T helper (Th2) cells, plays an important role in the development
of allergic diseases, such as allergic asthma. Studying the regulation
of IL-5 gene expression by Ets transcription factors, we found that Ets1 and Ets2, but not Elf-1, were able to activate the human IL-5
promoter in Jurkat T-cells. This required the presence of either
phorbol 12-myristate acetate (PMA) plus ionomycin or PMA plus the viral
protein HTLV-I Tax1. By mutation studies, it could be
shown that Ets1 and Ets2 exerted their effects on the IL-5 promoter
through a GGAA motif within the Cle0 element. In myeloid Kasumi cells,
Ets1 and Ets2 failed to stimulate IL-5 promoter activity, unless the
T-cell specific transcription factor GATA3 was added. These results
show, for the first time, that Ets1 and Ets2 are able to cooperate with
GATA3. Both ionomycin and Tax1 increased the combined
effect of GATA3 with Ets1 and Ets2 in the presence of PMA. The data
further demonstrate that, in addition to Ets1, Ets2 is also able to
functionally cooperate with Tax1. The synergism of GATA3
with either Ets1 or Ets2 may play an important role in calcium- or
Tax1-dependent regulation of IL-5 expression in
Th2 cells or in HTLV-I transformed adult T-cell leukemia cells, respectively.
The cytokine interleukin-5
(IL-5)1 activates
eosinophiles (1, 2) and basophiles (3, 4) and seems to be responsible for the development of eosinophilia associated with a number of diseases, including asthma and HTLV-I transformed adult T-cell leukemia
(5-9). Primarily, IL-5 is synthesized by Th2 cells, a subset of T
helper cells, following stimulation with antigens or mitogens (10, 11).
Regulation of IL-5 expression is likely to involve calcium and protein
kinase C-dependent signaling pathways, as a calcium
ionophore and phorbol ester synergistically increase IL-5 expression
and IL-5 promoter activity (12, 13). Activation of Th2 cells is
accompanied by increased activities of phorbol ester responsive AP1 and
calcium responsive NFAT as well as of GATA3 (14). All three
transcription factors have been found to be able to activate the IL-5
promoter (15-18), suggesting that they may play an important role in
controlling IL-5 expression. In retrovirally transformed adult T-cell
leukemia cells, the viral protein Tax1 may contribute to
the production of IL-5 (17). Tax1 has been shown to
deregulate a variety of cellular promoters (for a review, see Ref. 19)
by interacting with various transcription factors, such as CREB,
NF- Ets proteins are transcription factors that share a unique DNA-binding
domain, the Ets domain, allowing these proteins to interact
specifically with GGA(A/T)-based recognition sites (for a review, see
Ref. 33). Ets transcription factors have been found to play a crucial
role in controlling transcription of a variety of genes involved in
important cellular processes, such as proliferation or differentiation.
They have also been shown to contribute to the development of certain
human diseases (for review, see Ref. 34). Some Ets proteins, such as
Ets1, Elf-1, and Fli-1, are primarily expressed in T-cells where they
fulfill important functions, e.g. Ets1 has been reported to
play a crucial role in T-cell survival (35, 36).
The IL-5 promoter shares with other cytokine promoters, such as the
granulocyte macrophage-colony stimulating factor promoter, the
so-called conserved lymphokine element (Cle0) (37). It is a composite
AP1/Ets element, which supports activation of the granulocyte
macrophage-colony stimulating factor promoter by Ets1 in an AP1/NF- Cell Lines and Plasmids--
Jurkat T-cells and myeloid Kasumi
cells were maintained in RPMI medium supplemented with 10% fetal calf
serum in the absence of any antibiotic.
Plasmids pKCR3-c-ets1, Transient Transfection and Luciferase Assay--
Jurkat and
Kasumi cells were transfected with 5 µg of an IL-5 luciferase
construct by electroporation using a Bio-Rad gene pulser under
conditions, as described previously (45). For expression of Ets1, Ets2,
or Elf-1, 6 µg of the corresponding expression plasmid was added to
the transfection mixture, while for expression of Tax1 or
GATA3, 4 µg of pCTax or 2 µg of RSV/hG3, respectively, was used.
One hour after transfection either PMA (final concentration: 10 ng/ml)
and/or ionomycin (final concentration: 2 µM), both
dissolved in dimethyl sulfoxide, or dimethyl sulfoxide (control) was
added to the cells. After another 6 h cells were harvested. Cell
lysates were assayed for luciferase activity according to Ref. 46.
Preparation of Nuclear Extracts--
Nuclear extracts from
Jurkat cells were prepared essentially as described (28). Briefly,
cells were harvested and washed in phosphate-buffered saline. After
resuspension in 1 ml of buffer A (10 mM Hepes, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl
fluoride) and incubation on ice for 15 min, cells were lysed by
addition of 120 µl of Nonidet P-40 followed by vortexing for 10 s. The nuclei were pelleted by centrifugation at 13,000 rpm for 30 s at room temperature and extracted by addition of buffer C (20 mM Hepes, pH 7.9, 400 mM NaCl, 1 mM
EDTA, 1 mM EGTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride). The nuclear lysate was
cleared by centrifugation at 13,000 rpm for 5 min at 4 °C and stored
at Western Blot Analysis--
Western blot analysis of Jurkat cell
lysates was carried out as described previously (29). Rabbit anti-Ets1
(C-20), anti-Ets1/2 (C-275), or anti-Elf-1 (C-20) serum, all provided
by Santa Cruz Biotechnology, or mouse anti-Tax1 was diluted
to 1:7500, 1:1000, 1:4000, or 1:300, respectively, prior to use.
Anti-IgG horseradish peroxidase and ECL reagents were obtained from
Amersham Corp.
EMSA--
Ets1 baculovirus extract (47) was mixed with 400 pg of
Klenow Ets1 Transactivates the Human IL-5 Promoter in Jurkat T-cells in
the Presence of PMA Plus Ionomycin or PMA Plus
Tax1--
We first studied the ability of Ets1 to regulate
the activity of the human IL-5 promoter. Jurkat T-cells were
transiently transfected with the luciferase reporter plasmid pIL5P.luc
containing a
Importantly, p54ets1 expression was not altered by any of these
treatments (Fig. 1, D and E). Note, however, that
ionomycin affected the production of three smaller proteins (I-III)
that could be recognized by the Ets1 antibody (Fig. 1E).
These proteins probably resulted from an exon VII
domain-dependent/calcium-triggered degradation of
p54ets1 (50-53), a process that Tax1 apparently
could partially prevent (Fig. 1E, compare lanes 2 and 4 with 3 and 5). When we repeated the experiments with the natural
Tax1 was able to increase IL-5 promoter activity 20-fold,
when cells were treated with either PMA alone or PMA plus ionomycin (Fig. 1B). In the presence of PMA alone, a cooperative
effect of Ets1 with Tax1 was observed. Western blot
analysis showed that PMA had a strong up-regulating effect on
cytomegalovirus-promoter driven Tax1 expression (Fig.
1G) suggesting that PMA was mainly required to obtain
Tax1 protein levels sufficient for trans-activation.
The GGAA Motif within the Cle0 Element Is Required for Activation
of the Human IL-5 Promoter by Ets1 and Tax1--
The
We next mutated the GGAA motif of the Cle0 element to AGAA (Fig.
2B), thereby, creating the Ets-binding mutant
To demonstrate that Ets1 can specifically interact with the GGAA motif
of the IL-5 Cle0 element, EMSAs were performed using a
32P-labeled oligonucleotide that corresponded to the IL-5
promoter sequence between Ets2, but Not Elf-1, Is Able to Substitute for Ets1 in
Transactivating the IL-5 Promoter in the Presence of PMA/Ionomycin or
PMA/Tax1--
The G to A mutation of the Cle0 Ets-binding
site not only inhibited the activation of the IL-5 promoter by Ets1,
but also interfered with the ability of Tax1 to stimulate
IL-5 promoter activity (Fig. 2D). This suggests that the
stimulatory effect of Tax1 on the IL-5 promoter required an
Ets protein already present in Jurkat T-cells. Full-length Ets1 and
Elf-1, although able to activate the IL-5 promoter in cells treated
with PMA, failed to stimulate IL-5 promoter activity in the presence of
PMA plus ionomycin or PMA plus Tax1 (Fig. 4A). Interestingly, the PMA/Tax1-mediated activation was even
inhibited by Elf-1 (Fig. 4B). These data show that, in
addition to Ets1, Ets2 is able to activate the IL-5 promoter, while
Elf-1 lacks this ability and rather seems to have the potential to
repress Tax1-induced IL-5 promoter activity.
GATA3 Is Important for Ets1/Ets2-mediated Activation of the IL-5
Promoter--
In the myeloid Kasumi cell line, Ets1 and
Tax1 were found to be unable to activate the IL-5 promoter
under the conditions that allowed these proteins to stimulate promoter
activity in Jurkat cells (Fig. 5). This
may suggest that a T-cell specific factor may have played a role in
Tax1- and Ets1-mediated activation of the IL-5 promoter in
Jurkat cells. A potential candidate is the T-cell specific factor
GATA3, which is highly expressed in Jurkat T-cells (17, 54) and is able
to activate the IL-5 promoter through a proximal GATA site (Fig.
2A), shown to be critical for IL-5 promoter activity (15).
By Western blot analyses of unfractionated and fast protein liquid
chromatography fractionated nuclear extracts, we confirmed that GATA3
is endogenously expressed in Jurkat cells (data not shown). To test the
ability of GATA3 to rescue Ets1/2 and Tax1 stimulatory
activity we analyzed the effects of Ets1/2, Elf-1, and Tax1
on the
Similar to Ets1, Ets2 was unable to activate the IL-5 promoter in
Kasumi cells, unless GATA3 was added (Fig. 5). However, the cooperative
effect of Ets2 with GATA3 was stronger relative to that of Ets1 leading
to an 11-fold induction of IL-5 promoter activity in the presence of
PMA, as compared with a 3-fold stimulation by Ets1 under the same
conditions. Addition of Tax1 or ionomycin further increased
promoter activity 4- or 2-fold, respectively. As found with Jurkat
cells, under optimum conditions, Ets2 was twice as effective as Ets1 in
activating the IL-5 promoter. Interestingly, compared with Ets1, Ets2
also seemed to be the better partner for Tax1, as Ets2
could synergize with Tax1 even in the absence of GATA3.
These data suggest that the interactions of Ets2 with GATA3 and
Tax1 may be stronger than those of Ets1 with these proteins.
In the presence of PMA alone or PMA plus ionomycin, Elf-1 induced IL-5
promoter activity by approximately 2-fold, while it activated the
promoter 3-fold in Kasumi cells treated with PMA plus Tax1.
However, under any of these conditions, co-expression with GATA3 did
not further increase promoter activity, suggesting that Elf-1 is unable
to act in synergy with GATA3.
Similar data were obtained, when the experiments were repeated with the
We show here for the first time that in the presence of PMA, Ets1
and Ets2, but not Elf-1, are able to cooperate with GATA3 to
synergistically activate the IL-5 promoter. Our results further suggest
that Tax1 or ionomycin can enhance this Ets/GATA3 synergy. Based on these data, we propose that IL-5 expression is regulated by
the concerted action of at least three transcription factors, Ets1/2,
AP1, and GATA3, whose combined activities can be modulated by
Tax1 or by a putative ionomycin-regulated cellular factor
(Fig. 6). In this model, we suggest PMA
to be needed for recruitment of AP1 to the Cle0 element. Both Ets1 and
Ets2 are able to cooperate with AP1 to activate a variety of promoters
and to mediate PMA-dependent activation (15, 39, 55-59).
In Jurkat cells, PMA rapidly increases c-jun and
c-fos steady-state mRNA levels, which is followed by an
accumulation of c-Fos and c-Jun proteins reaching maximum levels after
6 h (60, 61). In addition, co-expression of c-Fos and c-Jun was
shown to eliminate the requirement for PMA for
Cle0-dependent Ets1 trans-activation of the granulocyte
macrophage-colony stimulating factor promoter in Jurkat cells (39)
suggesting again that PMA stimulates AP1 activity in Jurkat cells.
Furthermore, in the presence of both Tax1 and PMA, c-Jun
was demonstrated to be a strong activator of the human IL-5 promoter
(17). Like PMA, Tax1 may have also activated AP1, as it can
up-regulate c-fos and c-jun expression (61-64),
activate the c-Jun N-terminal kinase-1 (65), and increase AP1 binding
activity (66-68). In addition, Tax1 may have directly affected Ets1 activity by binding to Ets1 (29) and also by inhibiting exon VII-dependent degradation of Ets1 (Fig.
1E). Furthermore, Tax1 may have promoted Ets-AP1
and/or Ets-GATA3 interactions by forming ternary complexes with these
proteins, as previously shown for Ets1 and Sp1 (29). Finally,
Tax1 may have facilitated the interaction of Ets with CBP
and p300 (69) as was shown for the binding of CREB to these co-factors
(25, 70, 71).
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
B, SRF, NF-Y, and Ets1 (20-29), and with basal factors, such as
TBP, TFIIA, and TAFII 28 (30-32).
B
dependent manner (37-39). This raises the question of whether Ets
proteins may also be able to regulate the IL-5 promoter. In order to
address this possibility, we performed transient transfection studies
with Jurkat T-cells and the myeloid Kasumi cell line. We found that
Ets1 and Ets2, but not Elf-1, were able to activate the IL-5 promoter
in Jurkat cells in the presence of either PMA plus ionomycin or PMA
plus Tax1. Using Kasumi cells, we could show that Ets1 and
Ets2 needed to cooperate with GATA3 for their stimulatory activities on
the IL-5 promoter. These synergisms were enhanced in the presence of
ionomycin or HTLV-I Tax1. The data suggest an important
role for Ets proteins in the regulation of the IL-5 expression in Th2
lymphocytes and HTLV-I transformed leukemic cells.
MATERIALS AND METHODS
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ABSTRACT
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MATERIALS AND METHODS
RESULTS
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EBets1
243-330, pCTax, pCM5-Tax, and
pCM7-Tax for expression of p54ets1, p46ets1,
WT-Tax1, M5-Tax1, or M7-Tax1,
respectively, are described elsewhere (40-43). The plasmids
EBets2
and
EBelf-1 for expression of Ets2 or Elf-1, respectively, were
generous gifts from Jacques Ghysdael. The plasmid RSV/hG3 for
expression of human GATA3 was kindly provided by James D. Engel. For
construction of the
494/+44 IL-5 promoter/luciferase plasmid
(pIL5P.luc), the IL-5 promoter fragment was first amplified by
polymerase chain reaction using the primers as described previously (13), followed by cloning this fragment into a luciferase-containing vector (44). For generation of the deletion mutant
106/+44 IL-5
promoter, the internal HindIII/BsmI fragment,
containing the IL-5 promoter sequence from
494 to
82, was replaced
by an oligonucleotide bearing the IL-5 specific sequence from
106 to
82. For creating the G to A mutation at position
44 of the
antisense strand, we polymerase chain reaction amplified an
approximately 500-base pair long fragment containing the IL-5 promoter
fragment from
81 to +44 and part of the luciferase gene by
using the following primers:
5'-GGCATTCTCTATCTGATTGTTAGAAATTATTCATTTCTTCAAAGACAG-3' (the
mutated nucleotide is underlined) and
5'-AATTGAAGAGAGTTTTCACTGCATACGACGATTCTGTGATTTGTATTC-3'. The
fragment was cloned into the polymerase chain reaction 2.1 vector
(Invitrogen) and, after cutting with BsmI and
XbaI, inserted into the
106/+44 IL-5 luciferase construct
creating the
106/+44 EM IL-5 promoter luciferase plasmid.
80 °C.
-32P-labeled IL-5 WT (sense strand:
5'-GTTAGAAATTATTCATTTCCTCAAAGA-3'), corresponding to the IL-5 promoter
sequence from nucleotides at position
63 to
37, in the presence of
2.5% CHAPS (48), 1 mM Tris, pH 7.5, 3 mM
Hepes, pH 7.9, 70 mM NaCl, 0.25 mM EDTA, 0.15 mM EGTA, and 0.01% bovine serum albumin, and incubated for
5 min at room temperature. For competition experiments, an IL-5
specific oligonucleotide carrying a C to A mutation at
44 (IL-5 EM)
or unlabeled IL-5 WT was used in a 100-fold molar excess over the probe. Electrophoresis was carried out as described (49).
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
494/+44 fragment of the IL-5 promoter either in the
presence or absence of an expression plasmid coding for p54ets1
(pKCR3-c-ets1). As shown in Fig.
1A, Ets1 was able to increase IL-5 promoter activity approximately 3-fold, when cells were
simultaneously treated with PMA and ionomycin. Withdrawal of either one
or both of these agents prevented activation of the IL-5 promoter by
Ets1. In addition to ionomycin, HTLV-I Tax1 was able to act
in concert with PMA to stimulate Ets1 activity (Fig. 1B).
For this stimulatory effect on Ets1, Tax1 had to be active,
as two inactive Tax1 mutants, M5 and M7 Tax1
(43), failed to support trans-activation of the IL-5 promoter by Ets1
(Fig. 1C). At the same time, wild-type and mutant
Tax1 were expressed at the same level (Fig.
1F).
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Fig. 1.
Ets1 is able to trans-activate the human IL-5
promoter in PMA/ionomycin or Tax1-treated Jurkat
cells. A-C, Jurkat cells were transfected with 5 µg
of the 494/+44 IL-5 promoter/luciferase plasmid in the presence or
absence of 6 µg of pKCR3-c-ets1 and/or 4 µg of pCTax and treated
with PMA and/or ionomycin (iono) or left untreated
(control), as indicated. Seven hours after transfection,
cell extracts were assayed for luciferase activity (RLU,
relative light units) as described under "Materials and Methods."
Bars represent the average luciferase activity calculated
from four independent experiments. In panel C, WT, M5, and
M7 denote wild-type, D22A/C23S, and C29A/P30S mutant Tax1
proteins, respectively. D-G, Western blot analyses of
extracts from Jurkat cells treated as indicated. Blots were probed with
an antibody either directed to Ets1 (D and E) or
Tax1 (E-G). In panel E, I, II, and
III denote bands, which likely correspond to degradation products of
Ets1. The asterisk indicates a nonspecific band.
VII Ets1 mutant protein
p46ets1, we found that exon VII indeed was required for the
production of proteins I-III, but was dispensable for Ets1 activation
of the IL-5 promoter (data not shown). This demonstrates that proteins I-III were not important for the Ets1 effect on the IL-5 promoter. Collectively, these data show that certain conditions were required to
allow Ets1 activation of the IL-5 promoter in Jurkat cells.
494/+44 IL-5 promoter fragment contains eight GGA(A/T) core motifs
(E), all of which could potentially interact with Ets1 (Fig.
2A). To analyze the importance
of the Cle0 GGAA motif for Ets1-mediated activation, we first removed
the sequence between
494 and
106 (Fig. 2A). This
deletion altered neither Ets1- nor Tax1-dependent activation (Fig. 2C).
This suggests that the seven GGA(A/T) motifs upstream of nucleotide of
position
106 were dispensable for stimulation of promoter activity by
these proteins. However, compared with the
494/+44 IL-5 promoter, the
106/+44 promoter fragment had a reduced ability to respond to PMA
plus ionomycin. This is likely due to the fact that the
ionomycin-responsive NFAT site at
116 is missing in the smaller
promoter construct (Fig. 2A). On the other hand, removal of
the NFAT site did not impair the ability of the IL-5 promoter to
respond to Ets1 in a PMA/ionomycin-dependent manner. This
suggests that ionomycin had two different effects on the IL-5 promoter.
One was required for activation of the promoter by Ets1, while the
other was likely to be mediated by activation of NFAT.
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Fig. 2.
The GGAA motif within the Cle0 element is
essential for trans-activation of the IL-5 promoter by Ets1.
A, a schematic illustrating the three IL-5 promoter
fragments used here. Potential Ets responsive elements (E),
the Cle0 element as well as the binding sites for NFAT, GATA (GATA3 and
GATA4), and AP1 are indicated. B, sequences (sense strands)
of the wild type (a and b) or the Ets mutant Cle0
element (c) in promoter fragments 494/+44 (a)
and
106/+44 (b) or
106/+44 (c), respectively.
The sequence GGAA was mutated to AGAA in the
106/+44 EM
IL-5 promoter fragment (c). C and D,
the activities of the
494/+44 IL-5 promoter (a)
versus the
106/+44 promoter (b) (C)
and those of the
106/+44 promoter (b) versus
the
106/+44 EM promoter (c) (D) were compared
under various conditions. Bars represent the average values
of three independent experiments. E, mutation within the
GGAA motif of the Cle0 element abrogates Ets1 binding. EMSAs were
performed with a p54ets1 baculovirus extract and a
32P-labeled oligonucleotide (IL-5 WT) corresponding to the
IL-5 promoter sequence between
63 and
37. For competition studies,
either unlabeled IL-5 WT (lane 3) or a GGAA mutant version
of this oligonucleotide (IL-5 EM, lane 4) was added to the
EMSA reaction mixture in a 100-fold molar excess over the probe.
106/+44 EM IL-5 promoter (Fig. 2A). As shown in Fig. 2D,
this mutation completely abrogated the ability of Ets1 to
trans-activate the IL-5 promoter.
63 and
37. By using a
p54ets1-enriched baculovirus extract, we could show that Ets1
was able to bind to this probe (Fig. 2E, lane 2). The
formation of the Ets1 complex could be prevented by the addition of
unlabeled wild type
63/
37 oligonucleotide (IL-5 WT) (lane
3), but not by adding an Ets mutant version of this
oligonucleotide (IL-5 EM) that contained a mutation at the first G of
the GGAA motif (lane 4). These results show that Ets1
interacts with the IL-5 Cle0 element by contacting its GGAA motif.
These data suggest that responsiveness of the IL-5 promoter to Ets1
relied on the Ets-binding site within the Cle0 element.
VII Ets1 are endogenously expressed in Jurkat cells (Fig.
3A). Treatment with PMA or PMA plus ionomycin, which allowed Tax1 to trans-activate the
IL-5 promoter, did not significantly change the expression levels of these Ets1 proteins relative to control conditions (Fig.
3A). Therefore, it is possible that Tax1
cooperated with Jurkat cell-derived Ets1, when no Ets1 had been added
exogenously. Alternatively, a different Ets transcription factor, such
as Ets2 or Elf-1, which are also expressed by Jurkat cells (Fig.
3B), could instead have mediated the effect of
Tax1 on the IL-5 promoter. To study this possibility we
tested the effects of ectopically expressed Ets2 and Elf-1 on the IL-5
promoter. We found that Ets2 was twice as potent as Ets1 in its ability
to activate the IL-5 promoter in Jurkat cells, causing a 6-10-fold
increase in promoter activity in the presence of PMA plus ionomycin
(Fig. 4A) or PMA plus
Tax1 (Fig. 4B), respectively. Interestingly, the
cooperative effect of Ets2 with Tax1 was stronger than that
of Ets1 with Tax1 (Fig. 4B). Ets2 also had a
significant stimulatory effect on the promoter in the presence of PMA
alone (Fig. 4A).
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Fig. 3.
Jurkat cells express Ets1, Ets2, and Elf-1
endogenously. Nuclear extracts from Jurkat cells were analyzed for
expression of Ets1, Ets2, or Elf-1 by the Western blot technique.
A, comparison of the Ets1 levels in the nuclei of Jurkat
cells treated either with PMA (P), ionomycin (I),
PMA/ionomycin (P/I), or with dimethyl sulfoxide
(Co) for 5 h. B, in addition to Ets1,
significant levels of Ets2 and Elf-1 proteins were also detectable in
Jurkat cell nuclear extracts. The Western blot was subsequently probed
with anti-Ets1, anti-Ets1/2, and Elf-1 antibody. Note that the antibody
( -Ets1/2) used for detection of Ets2 also recognized Ets1.
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Fig. 4.
In addition to Ets1, Ets2, but not Elf-1, is
able to activate the IL-5 promoter. Transient transfection
experiments were performed with Jurkat cells as described under
"Materials and Methods." Each bar represents the average
value of two to three independent experiments.
494/+44 IL-5 promoter in myeloid Kasumi cells, when GATA3 was
overexpressed. As shown in Fig. 5, overexpression of GATA3 had no
effect on the IL-5 promoter (Fig. 5). However, when combined with Ets1
in the presence of PMA a 3-fold increase in promoter activity was
observed. This suggests that Ets1 was able to cooperate with GATA3 to
trans-activate the IL-5 promoter. Further addition of Tax1
or ionomycin strongly increased this effect resulting in a 14- or
11-fold induction of promoter activity, respectively. Neither Ets1 nor
GATA3 alone was able to efficiently activate the IL-5 promoter under
these conditions. This shows that Tax1 and ionomycin could
only exert their effects on Ets1 and GATA3, when both of these
transcription factors were present. It suggests that Tax1
and ionomycin strengthened the functional interaction between Ets1 and
GATA3.
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Fig. 5.
GATA3 is required for Ets1- or
Ets2-dependent activation of the IL-5 promoter in myeloid
Kasumi cells. For transient transfection, Kasumi cells were
treated the same way as Jurkat cells. Each bar represents
the average value of two to three independent experiments.
106/+44 IL-5 promoter fragment (data not shown). However, when the
Ets mutant 106/+44 EM promoter fragment was used instead, Ets1 and Ets2
failed to synergize with GATA3 and/or Tax1 (data not
shown). These results show that the Cle0 Ets-binding site is essential
for cooperative effects of Ets1 or Ets2 with GATA3 and/or
Tax1.
DISCUSSION
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ABSTRACT
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DISCUSSION
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Fig. 6.
A model depicting a suggested mechanism by
which Tax1 or ionomycin could increase the cooperative
effect of GATA3 and Ets1 or that of GATA3 and Ets2 on the IL-5
promoter. Ets1 and Ets2 are known to physically interact with
Tax1 or AP1, respectively (29, 88), as indicated by
small circles. Proposed interactions between proteins are
shown as double-headed arrows. X refers to a
postulated cellular factor, which, in Th2 cells, may mediate the
ionomycin-stimulated effect on the activity of Ets1 or Ets2.
Ionomycin may have mimicked Tax1 action by recruiting a factor (proposed factor X in Fig. 6) with a similar function as Tax1. Alternatively, ionomycin may have stimulated Ets-dependent activation of the IL-5 promoter by activating Ras (72, 73). Ras is known to super-activate Ets1 and Ets2 and to induce transcription through AP1/Ets composite elements (74-76). Activation of calcium has also been reported to activate CBP (77) and may, as proposed for Tax1, have promoted CBP-Ets interaction.
It is unlikely, however, that Tax1 and ionomycin exerted
their stimulatory effects on Ets1 or Ets2 by activating NFAT (78-85), which can bind upstream of GATA3 (Fig. 2B). This is
suggested by the fact that removal of the NFAT site at 116 (Fig.
2B) did not influence the Ets1 or Ets2 response of the IL-5
promoter. Nor did PMA, ionomycin or Tax1 seem to have an
effect on the binding of Ets1 and Ets2 to the Cle0 element as suggested
from EMSA data obtained with nuclear extracts from Jurkat cells (data
not shown).
Although able to form a complex with AP1 on the Cle0 element (37),
Elf-1 failed to cooperate with GATA3 in Kasumi cells and to activate
the IL-5 promoter in Jurkat cells. Interestingly, Elf-1 (but not Ets1)
was reported not to be able to activate the Cle0-regulated granulocyte
macrophage-colony stimulating factor promoter, whose activity is
dependent upon NF-B instead of a GATA factor, either (38, 39). It
might suggest that Elf-1 may act as a repressor on
Cle0-dependent transcription by competing with stimulatory
Ets factors, such as Ets1 or Ets2, for binding to the Cle0 element.
This notion is supported by the finding that Elf-1 inhibited activation
of the IL-5 promoter by Tax1, which depended upon the Cle0
Ets-binding site (Fig. 4).
Interestingly, as previously reported for Ets1 (29, 45), Ets2 was found here also to be able to cooperate with Tax1. The functional interaction of Ets2 with Tax1 seemed to be even stronger than that of Ets1 with Tax1. Although adult T-cell leukemia cells have been reported to express Ets1 (86), the Ets1 expression level has been found to vary among HTLV-I transformed cell lines.2 The potential to cooperate with both Ets1 and Ets2 may render Tax1 less dependent upon the expression of a particular Ets protein regarding its ability to trans-activate Ets-regulated promoters.
Collectively, these data suggest an important role for Ets proteins,
such as Ets1 and Ets2, in the regulation of IL-5 expression. Ets2 may
be of particular importance in activated Th2 cells, as its expression
is up-regulated upon antigenic stimulation of T-cells, while, at the
same time, the Ets1 level declines (87). For
Tax1-dependent IL-5 expression in HTLV-I
transformed leukemic cells, Ets1 and Ets2 may be equally important.
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ACKNOWLEDGEMENT |
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We thank Jacques Ghysdael for providing
plasmids EBets2 and
EBelf-1 and Douglas Engel for RSV/hG3.
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FOOTNOTES |
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* This work was supported by Boehringer Ingelheim International, Dr. Mildred Scheel-Stiftung Grant 10-1058-NO2, and the Fonds der Chemischen Industrie.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.
To whom correspondence should be addressed. Tel.:
49-7071-297-8893; Fax: 49-7071-295359; E-mail:
juergen.dittmer{at}uni-tuebingen.de.
2 J. Dittmer, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: IL-5, interleukin-5; Th2 cells, type-2 T-helper cells; Cle0, conserved lymphokine element 0; PMA, phorbol 12-myristate 13-acetate; EMSA, electromobility shift assay; HTLV-I, human lymphotropic leukemia virus I; CHAPS, 3-[(3-cholamidopropyl) dimethyl ammonio]-propanesulfonate.
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REFERENCES |
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