(Received for publication, August 4, 1995)
From the
Interleukin-5 (IL-5) plays a central role in the growth and
differentiation of eosinophils and contributes to several disease
states including asthma. Accumulating evidence suggests a role for cAMP
as an immunomodulator; agents that increase intracellular cAMP levels
have been shown to inhibit production of cytokines predominantly
produced by T helper (Th) 1 cells such as IL-2 and interferon
(IFN-
). In contrast, the production of IL-5, predominantly
produced by Th2 cells, is actually enhanced by these agents. In this
report, we have performed transient transfection experiments with IL-5
promoter-reporter gene constructs, DNase I footprinting assays, and
electrophoretic mobility shift assays to investigate the key regulatory
regions necessary for activation of the IL-5 promoter by dibutyryl cAMP
and phorbol esters in the mouse thymoma line EL-4. Taken together, our
data demonstrate the critical importance of two sequences within the
IL-5 5`-flanking region for activation by these agents in EL-4 cells:
one, a highly conserved 15-base pair element present in genes expressed
by Th2 cells, called the conserved lymphokine element 0 (CLE0; located
between -53 and -39 in the IL-5 promoter), and the other,
two overlapping binding sites for the transcription factor GATA-3 (but
not GATA-4) between -70 and -59. Taken together, our data
suggest that activation via the unique sequence combination GATA/CLE0
results in selective expression of the IL-5 gene in response to
elevated levels of intracellular cAMP.
Interleukin-5 (IL-5) ()is the key cytokine that
regulates the biological functions of eosinophils and contributes to
several human disease states, including asthma(1, 2) .
In both atopic and non-atopic asthma, elevated IL-5 has been detected
in peripheral blood and the
airways(3, 4, 5) . There is considerable
interest in the identification of the transcriptional mechanisms
controlling the synthesis of this cytokine. IL-5 is predominantly
produced by activated T helper 2 (Th2) lymphocytes, although mast cells
and eosinophils have been also shown to produce this
cytokine(2, 6, 7, 8) . Murine T
helper clones are classified into two distinct subsets (Th1 and Th2) on
the basis of their patterns of lymphokine secretion. Whereas IL-5 gene
expression is restricted to the Th2 subset of CD4
cells, which also express IL-4 and IL-10 (but not IL-2 or
interferon-
, which are produced by Th1 cells), GM-CSF is produced
by both Th1 and Th2 cells(9, 10) .
In contrast to
cytokines such as IL-2, IL-5 is strongly induced by factors that raise
intracellular cAMP levels, such as IL-1, prostaglandin
E
, and
forskolin(11, 12, 13, 14) . In
murine Schistosomiasis mansoni infection, vasoactive
intestinal peptide released from eosinophils induces adenylate cyclase
in T cells via vasoactive intestinal peptide receptors, resulting in
IL-5 production(15) . In transient transfection assays,
dibutyryl cAMP (Bt
cAMP)-induced activation of the IL-5
promoter was mimicked by transfection of an expression plasmid encoding
the catalytic subunit of protein kinase A, suggesting that cAMP
stimulates IL-5 transcription via the protein kinase A signaling
pathway(16) . The observation that cAMP increases the
expression of cytokines such as IL-5, while suppressing that of other
cytokines such as IL-2 and interferon-
(IFN-
), suggests a
possible regulatory role for this second
messenger(9, 10, 13, 17, 18) .
The IL-5 promoter contains potential binding sites for multiple transcription factors including NF-AT, AP-1, Oct, and Elf-1(19, 20) . The AP-1- and Elf-1-binding sites together constitute an element called the consensus lymphokine element 0 (CLE0) found in cytokine genes including IL-3, IL-4, IL-5, and granulocyte/macrophage-colony stimulating factor (GM-CSF)(21, 22, 23) . This element plays a crucial role in the regulation of the GM-CSF gene(22, 24, 25) .
In these studies, we
sought to define precisely the cis-activating elements that
regulate inducible murine IL-5 transcription in EL-4 cells in
response to BtcAMP and PMA, both of which are required for
optimal stimulation of the IL-5 promoter in these
cells(16, 26) . Our data suggest that activation of
the IL-5 promoter by Bt
cAMP and PMA in EL-4 cells requires
sequences within the CLE0 element and also a region located between
-70 and -59 that binds the transcription factor GATA-3. We
speculate that activation via this unique sequence combination confers
the specificity needed for selective expression of the IL-5 gene in
response to elevated levels of intracellular cAMP.
Site-directed mutants were generated following the method of Kunkel using a kit from Bio-Rad(27) . All constructs were confirmed by DNA sequencing.
A fragment containing sequences between -168 and +24
of the IL-5 promoter was used for footprinting assays using techniques
previously described(30) . Labeled fragment (30 fmol) was
incubated at room temperature for 15 min in 100 µl of reaction
buffer containing 10 mM Tris-HCl, pH 7.9, 0.5 mM
EDTA, 5% glycerol, 0.05% Nonidet P-40, 1 mM MgCl
,
0.2% polyvinyl alcohol, 10 µg/ml poly(dI)-poly(dC), in the presence
or absence of nuclear extract prepared from EL-4 cells. At the end of
the incubation, 100 µl of a salt mixture (10 mM MgCl
, 5 mM CaCl
) was added to
each reaction. DNase I (Worthington) was added to a final concentration
of 2.5 or 0.025 unit/ml to tubes with or without nuclear extracts, and
digestion was carried out for 1 min at room temperature. The reactions
were stopped with 200 µl of stop buffer containing 0.2 M NaCl, 0.04 M EDTA, 1% SDS, 125 µg/ml tRNA, and 100
µg/ml proteinase K. The samples were extracted with a mixture of
phenol:chloroform, and the DNA was precipitated with ethanol and
electrophoresed on 8% polyacrylamide, 8.3 M urea gels. For
accurate reading of the footprints, a T+C sequencing reaction of
the labeled strand was electrophoresed in parallel.
Figure 1:
Transcriptional activation of the
murine IL-5 promoter constructs. A, identification and
localization of candidate cis-activating elements in the
murine IL-5 promoter, located in the region immediately 5` to the
transcription start site. Potential binding sites for NF-AT, CLE1, and
CLE0 (which is a composite of AP-1- and Elf-1-binding sites) sites are
indicated. B, a series of luciferase reporter plasmids
containing deletion constructs and site-directed mutations of the IL-5
promoter are shown, with boxes representing the location of
the potential cis-element identified in A. Deletions
include from -545 to -66 bp of the murine IL-5 promoter.
Single mutations include the potential NF-AT, CLE1, and AP-1 sites and
are described in C. The average -fold luciferase induction for
each of the reporter plasmids is shown in the bar graph, with results
representing the average of multiple experiments and normalized for
-galactosidase activity. The absolute basal luciferase activity
was
250-300 relative light units in these experiments and
was similar for all of the promoter constructs. The deviations were no
more than 10% between experiments. C, the locations of the two
overlapping GATA sites (the site that fits the consensus sequence is boxed) and the three site-directed mutations in the IL-5
promoter are identified. Base pair changes are identified with lowercaseletters. A comparison between the human
GM-CSF and the murine and human CLE0 sequences is presented showing a
single base pair difference between the two promoters in the area underlined.
In order to determine
precisely the region in the IL-5 promoter responsible for
transcriptional regulation, EL-4 cells were transiently transfected
with luciferase reporter plasmids incorporating IL-5 gene sequences
ranging from 545 to 66 bp upstream from the transcription start site (Fig. 1B). An 8-h stimulation with BtcAMP
and PMA caused an 8-10-fold increase in transcriptional activity
in the 545-bp construct. While the 168-, 118-, and 91-bp constructs
also gave a similar response,
40-50% activity was induced in
the construct containing 76 bp of sequence upstream of the
transcription start site. However, the 5`-flanking sequence deleted to
-66 was unresponsive to the stimuli (Fig. 1C).
To further define the key cis-regulatory elements in the
IL-5 promoter, transient transfection experiments using site-directed
mutants were performed (Fig. 1C). Mutations in NF-AT
and CLE1 decreased transcriptional activity modestly (Fig. 1B). It has been well established that activation
of NF-AT occurs via a Ca-dependent
mechanism(33) . Previous work indicated that elevations in
[Ca
]
play a minimum role in
modulating the transcriptional regulation of the IL-5 gene, suggesting
that NF-AT probably does not play a major role in the transcriptional
activity of IL-5(34, 35, 36) . It is
important to note, however, that activation of the NF-AT element in
EL-4 cells by PMA alone, in the absence of a
Ca
-activated pathway, is probably due to a
constitutive increase in [Ca
]
in EL-4 cells(37) . In these studies, the preservation of
near full activity in the deletion construct p91IL5luc, compared to the
545-bp construct, further supports the absence of a critical
contribution by NF-AT-activated pathways in the activation of IL-5 gene
transcription(34, 35, 36) .
Fig. 1B shows that a mutation in the AP-1 sequence
within the CLE0 element eliminated activity of the IL-5 promoter. This
observation indicated that transcription factors binding to the AP-1 or
overlapping sequences within the CLE0 element are critical (but not
sufficient) for transcriptional activity of IL-5 in response to
BtcAMP and PMA.
Figure 2:
Binding of EL-4 cell nuclear proteins to
the IL-5 promoter between -168 and +1. DNase I protection
experiments were performed with an IL-5 promoter fragment containing
sequences between -168 and +24, labeled with P
on the sense strand. Labeled DNA fragments were incubated without (laneO) or with nuclear extracts from EL-4 cells
uninduced (laneU) or induced with Bt
cAMP
and PMA for 8 h (laneI). The sequence from -88
to -41 is shown in detail with footprints identified by lines to the right of the figure.
Figure 3:
Binding of inducible EL-4 cell nuclear
proteins to potential cis-activating elements of the murine
IL-5 promoter. A, a radiolabeled oligonucleotide containing
the IL-5 CLE0 sequence (see Fig. 1C) was used in EMSAs
with nuclear extracts prepared from EL-4 cells left unstimulated or
stimulated for 8 h with BtcAMP (1 mM) and PMA (25
ng/ml). Bands representing CLE0 binding activity are identified with arrows. Competitive binding assays were performed with
unlabeled CLE0, classical AP-1, mutant CLE0 (mElf-1), and
mutant CLE0 (mAP-1) probes. The antisera that were used were
against the Jun family, cJun, JunB, JunD, the Fos family, and the p50
subunit of NF-
B. B, a radiolabeled oligonucleotide
incorporating the region from -93 to -54 bp of the IL-5
promoter, exclusive of the potential NF-AT, CLE1, and CLE0 binding
sites was used in EMSAs with nuclear extracts as described in A. An arrow identifies a band representing specific
binding activity within this sequence. Competition with unlabeled
``self,'' IL-5 CLE0, classical AP-1, NF-
B, and muIL-5
NF-AT/CLE1 oligonucleotides is shown. Antisera against the Jun and Fos
families were utilized.
Figure 4: The 20-bp oligonucleotide spanning nucleotides -73 to -54 (73/54) competes for binding of nuclear proteins to the longer 40-bp oligonucleotide. The probe was the same as in Fig. 3B. A 100-fold molar excess of the following unlabeled oligonucleotides were used as competitors: self (lane3), two shorter oligonucleotides derived from this 40-bp oligonucleotide containing the region between -91 and -74 and between -73 and -54 (lanes4 and 5, respectively), an overlapping oligonucleotide containing the sequence between -82 and -67 (lane9), and three mutant oligonucleotides m1, m2, and m3 (lanes 6-8; the specific mutations are indicated by lowercase letters). All other conditions were as described in the legend to Fig. 3.
A close inspection of the sequence between -73 and -54 revealed the sequence AGATAA, between nucleotides -70 and -65, which fit the consensus sequence WGATAR (W = A/T and R = A/G) for the GATA family of transcription factors. We noticed an overlapping TGATTG sequence on the complementary strand, which also was in close agreement with the GATA consensus sequence except for T at the +1 position. To investigate whether this region contributed to the formation of the DNA-protein complex, mutations were introduced in three different regions (m1, m2, and m3; see Fig. 4). m1 contained mutations in the distal GATA sequence, m2 in the proximal sequence, while m3 was mutated in both the sequences. Used at a 100-fold molar excess, m2 appeared to compete better than the other two. When we repeated the experiment with 100- and 200-fold excess of the same oligonucleotides, the rank order of the ability to compete was m2 > m1 > m3 (Fig. 5). We also used consensus binding sequences for three additional transcription factor family proteins, C/EBP, Oct, and CREB/ATF, none of which, even at 200-fold excess, competed for binding to the probe (Fig. 5).
Figure 5: Binding of nuclear proteins to the 20-bp 73/54 oligonucleotide. The competitor oligonucleotides m1, m2, and m3 were used at two concentrations, while the oligonucleotides containing the consensus sequences for the binding of C/EBP, Oct, and CREB/ATF family proteins were used at a 200-fold molar excess. All other conditions were as described in the legend to Fig. 3.
We next used oligonucleotides containing wild-type or mutant GATA
sequence to investigate whether complexes I or II contained GATA
proteins. As illustrated in Fig. 6, while an oligonucleotide
containing wild-type GATA sequence displayed impressive competition,
mutations within the GATA sequence abolished this competition. Since,
among the GATA factors, GATA-3 has been reported to be predominantly
expressed by T lymphocytes (with some expression also in the developing
central nervous system), we used a GATA-3-specific antibody (which does
not cross-react with either GATA-1 or 2), to further characterize the
protein that bound to the AGATAA sequence in the IL-5 promoter. The
anti-GATA-3 antibody but not the control NF-B-specific antibody
supershifted both the complexes (Fig. 6, lane6). We also performed the reciprocal experiment using the
oligonucleotide containing the wild-type GATA sequence as the probe. As
is evident in lane12, the IL-5 sequences between
-73 and -54 efficiently competed for binding of GATA-3 to
the consensus GATA sequence. The specific localization of the
GATA-binding sequence to nucleotides between -70 and -59 in
the IL-5 promoter also explained the complete loss of inducibility of
the promoter when deleted to -66 (Fig. 1B).
Figure 6: Binding of GATA-3 to the 20-bp 73/54 oligonucleotide. The competitor oligonucleotides were used at a 100-fold molar excess. All other conditions were as described in the legend to Fig. 3.
While these studies were in progress, Yamagata et al.(43)
reported the involvement of GATA-4 in the activation of the human IL-5
gene in the ATL-16T cell line. We, therefore, performed EMSA using a
GATA-4-specific antibody that does not cross-react with any of the
presently characterized GATA proteins(32) . As illustrated in Fig. 7, while the anti-GATA-3 antibody again supershifted the
specific DNA-protein complexes (lane3), the
anti-GATA-4 antiserum did not supershift or inhibit formation of either
complex (lane5). In the reciprocal situation as
well, although anti-GATA 4 antiserum specifically inhibited complex
formation by affinity-purified GST-GATA-4 (lane8),
anti-GATA-3 antibody had no effect on complex formation (lane9). Taken together, these data suggest that GATA-3 (but
not GATA-4) and Fos/Jun (JunB and JunD but not c-Jun) proteins, that
bind to the GATA and CLE0 elements, respectively, play crucial roles in
the induction of the IL-5 promoter in response to BtcAMP
and PMA in EL-4 cells.
Figure 7: GATA-3 and not GATA-4 is the IL-5 DNA-binding protein in EL-4 nuclear extracts. 1 µl each of preimmune or immune serum was used in lanes4 or 7 and lanes5 or 8, respectively, while 0.2 µg of affinity-purified GST-GATA-4 protein was used in lanes6-9. All other conditions were as described in the legend to Fig. 3.
The results presented in this study demonstrate that sequences within the CLE0 element are critical but not sufficient for activation of the IL-5 promoter in response to cAMP agonists and phorbol esters. We have identified a second region between -70 and -59, which contains two overlapping GATA sites, disruption of which (as in the -66 deletion construct) abrogates activation of the promoter.
In agreement with a previous report by Lee et
al., BtcAMP and PMA synergistically activated the IL-5
promoter (16) . In a recent report, Lee et al. have
implicated the NF-AT element in the IL-5 5`-flanking region in
induction of the gene by Bt
cAMP and PMA. In their studies,
mutations in the NF-AT site led to an
80% reduction in activation
of the IL-5 promoter by these stimuli. Activation of NF-AT is coupled
to increased levels of
[Ca
]
(33, 38, 39) .
This requirement, however, can be bypassed in EL-4 cells due to
constitutive increases in
[Ca
]
(37) . Our studies
indicate that, although mutations in the NF-AT site in the context of
the 545-bp promoter lead to
50% reduction in activity of the
promoter (which is approximately equivalent to an 80% reduction when
compared to the 1.2-kilobase pair construct), the deletion construct
with the end point just 3` to this site (at -91) retains full
transcriptional activity compared to the 545-bp construct, leading to a
conclusion that the NF-AT site is not critical for induction of the
promoter (Fig. 1B). In support of this, our DNase I
footprinting studies did not reveal any binding of proteins across the
NF-AT site in the IL-5 promoter, although distinct footprints were
generated on the AP-1 and the GATA sites (Fig. 2). CsA blocks
the nuclear translocation of NF-AT and has been shown to inhibit the
expression of genes such as IL-2 and IL-4, which require NF-AT for
induction of gene expression (33, 38, 39) .
Although Lee et al.(40) used CsA to demonstrate
inhibition of binding of proteins to the NF-AT site in the IL-5
promoter, they did not investigate whether CsA indeed inhibits IL-5
gene expression in functional (transfection) experiments. Recently,
Lacour et al.(41) reported inhibition of NF-AT
induction by cAMP. Data previously reported by the Arai group also
demonstrated that the NF-AT site in the IL-2 promoter is a target for
inhibition of IL-2 promoter activation by cAMP, presumably via
inhibition of calcineurin activity(42) . However, this is only
possible if distinct NF-AT species simultaneously control inhibition of the IL-2 gene and activation of the
IL-5 gene by cAMP in the same cells (EL-4), as the authors also
suggest(40) .
Our studies establish a critical role for the
GATA sequence in activation of the IL-5 promoter in EL-4 cells and also
directly demonstrate binding of GATA-3 to the IL-5 promoter. In the
studies reported by Lee et al.(40) , deletion of the
promoter to -62 and mutation of the GATA element led to complete
abrogation of activation of the promoter by BtcAMP and PMA.
We have established that the activation of the murine IL-5 gene in EL-4
cells involves binding of GATA-3, but not GATA-4, in contrast to the
involvement of GATA-4 in the activation of the IL-5 gene in the ATL-16T
cell line(43) .
GATA-3 and GATA-4 are members of the GATA
family of transcription factors that bind to the consensus sequence WGATAR
via a highly conserved
C
zinc finger
domain(44, 45, 46) . Four GATA members, 1
through 4, have been described in vertebrates. The transcription factor
GATA-3 is expressed most abundantly in T-lymphocytes and the developing
central nervous
system(46, 47, 48, 49, 50) .
In contrast, GATA-4 is predominantly expressed in the heart, gut
epithelium, and reproductive organs (51, 52, 53, 54) . GATA-3 has been
shown to play a crucial role in the transcriptional regulation of T
cell receptor-related
genes(46, 47, 48, 49) . DNA-binding
studies with bacterially expressed GATA proteins and oligonucleotides
containing randomized GATA sequences indicate that GATA sequences
containing G at the +2 position, such as the one found in the IL-5
promoter between -70 and -65, may have a relatively lower binding affinity for the GATA-binding protein than
sequences containing A at the +2 position(45) . However,
the data of Ko and Engel (45) indicate that the lower binding
affinity of a site can be compensated for if this site overlaps another
GATA site. Indeed, as shown in Fig. 1, the IL-5 gene does have
overlapping GATA sites, one between -70 and -65 (which fits
the consensus sequence) and another located between -65 and
-59. Although the latter site has an intact GAT core but has a T
instead of A in the +1 position, it appears that this substitution
can still be selected by GATA-3, particularly within overlapping GATA
sites(45) . Overlapping GATA sites have been also previously
identified in many erythroid expressed genes such as the chicken
-globin promoter(55) . Overlapping and or multiple GATA
sites appear to confer increased GATA binding activity and may play a
key role in promoting full transcriptional
activity(55, 56, 57) .
It remains
undetermined from our data if both or one of the two potential GATA-3
sites are critical for protein binding. The finding that the m3
mutation, which disrupted both GATA sites, showed the least competition
in our gel shifts suggests but does not prove that both sites may be
critical for optimal binding. Both the IL-5 GATA sequence-containing
probe and the oligonucleotide containing the GATA consensus sequence
formed two complexes I and II with EL-4-nuclear extracts. It is
possible that complexes I and II represent monomeric and dimeric forms
(the latter induced by stimulation of cells) of the same protein. The
requirement of the CLE0 element for activation of the promoter suggests
that the GATA region alone is not sufficient for IL-5 promoter
activation. In a similar fashion, in the T cell receptor- gene,
GATA-3 needs to interact with proteins bound to nearby sites for
enhancer function(58) .
Among the cytokine genes, potential
GATA binding sequences are present in the 5`-flanking regions of the
IL-3, IL-4, GM-CSF, and IFN- genes (but not in the IL-2 gene).
There is no previous evidence for functional importance of this site in
any of the other genes. In the case of the IFN-
gene, deletion
into the GATA sequence had little effect on activity of the
promoter(59) . To the best of our knowledge, this is the first
report of a functional role for GATA-3 in the transcriptional
regulation of a T cell cytokine gene. It will be interesting to
determine in future studies with T cell clones and primary T
lymphocytes whether human T cells utilize GATA-3 for inducible
expression of the IL-5 promoter.
Naora et al.(60) have suggested that the TCATTT element, which overlaps with the AP-1-like element within the CLE0 element in the IL-5 promoter, is important for induction of IL-5 gene expression in response to mitogens and PMA. The TCA sequence in the TCATTT element overlaps with the AP-1 site, while the TTT sequence overlaps with the Elf-1-binding site within the CLE0 element. In EMSAs reported by Naora et al.(60) , mutation of the TCATTT element to cgAaTT (which also mutated the AP-1 element) abolished protein binding. However, the authors did not use antibodies against the AP-1 proteins to determine whether this complex contained AP-1 family proteins(60) . It is possible that the TTT sequence, immediately adjacent to the AP-1 sequence, has a permissive role only(61, 62) . Wang et al.(25) observed that the inducible transcription factors that bound to the GM-CSF CLE0 element contain JunB and c-Fos. Our gel shift assays show that specific antisera to the AP-1 family members JunB and JunD and an antiserum that recognizes members of the Fos family significantly inhibit formation of complexes I and II with the IL-5 CLE0 element. However, since there was a small amount of residual DNA binding activity in both of these complexes that was not abolished by any of these antisera, we cannot rule out the possibility that there are less abundant related proteins present in both the complexes. Complex III may comprise proteins similar to the constitutive proteins that Wang et al. had detected with the GM-CSF CLE0 probe (25) .
Although the 5`-flanking region of the IL-5 gene bears some homology to the promoter of other cytokines such as GM-CSF and IL-4, the divergent regulation of these cytokines suggests that different mechanisms control their synthesis, particularly in response to elevations in intracellular cAMP levels(12, 13, 18, 63) . Identification of a distinct combination of regulatory elements, i.e. GATA/CLE0 for cAMP activation of the IL-5 promoter, is therefore consistent with the differential effect of cAMP on expression of the IL-5 gene. Ultimately, these findings may begin to explain how IL-5 gene expression is uniquely regulated by cAMP in T lymphocytes and how it becomes dysregulated in several common disease states.
This paper is dedicated to the memory of Professor Igor Tamm, a mentor, adviser and friend.