From the Program of Developmental Biology, Department of Comparative Biosciences, University of Wisconsin, Madison, Wisconsin 53706
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ABSTRACT |
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Interleukin (IL)-1 is involved in many processes,
including thymic development. However, control of IL-1 expression in
thymic-derived stromal cells (TSC) has not been reported. We found that
IL-1 increased steady-state mRNA levels for IL-1
and IL-1
in TSC-936 and TSC-2C4 cells; stability was not a major determinant of
this effect. To study transcriptional regulation of IL-1
, we
functionally characterized 4 kilobase pairs of the 5'-flanking region
and first intron of the bovine IL-1
gene. The
470/+14 fragment was
sufficient to confer maximal responsiveness to IL-1
upon
transfection into these cell lines. Progressive 5' deletions identified
several IL-1
-responsive regions, including
308 to
226, which we
further characterized. Electrophoretic mobility shift and supershift
analyses showed that IL-1
induced the ability to form multiple
protein complexes with
261/
226 and that one of these contained
nuclear factor Oct-1. A competitor containing a mutated Oct consensus site failed to compete not only for this complex but others as well,
suggesting that this sequence regulates binding of other proteins to
this region. Functional analysis confirmed that this element was
essential for maximal induction of transcription. These findings
document a heretofore undescribed mechanism utilized by TSC for
regulation of IL-1
transcription by IL-1
itself.
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INTRODUCTION |
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The cytokine interleukin 1 (IL-1)1 mediates a wide
variety of inflammatory and hematopoietic processes. Although it is
primarily recognized as an inflammatory agent, it exerts diverse
effects on numerous cell types (for reviews, see Refs. 1-5). Two
distinct agonists, IL-1 and IL-1
, are encoded by distinct but
closely related genes, and are produced by several cell types.
Monocytes are the major sources of IL-1 in response to components
derived from infectious agents such as lipopolysaccharide (LPS), and
much of our knowledge about the control of IL-1 expression has been derived from these cells.
In addition to its well known function in inflammation, IL-1 also plays
an important role in T-cell development (for reviews, see Refs. 6 and
7). In the thymus, thymic stromal cells (TSC) produced a number of
cytokines, including IL-1. IL-1 has been described as a co-mitogen for
some populations of immature thymocytes (8-10) and prevents apoptosis
(11). IL-1 and tumor necrosis factor-
were shown to be required
for early thymocyte commitment and differentiation (12), and IL-1
was shown to be essential for positive selection by thymic nurse cells
(13). In addition to effects on thymocytes, IL-1 also induced DNA
synthesis and morphological changes in TSC (14) and stimulated thymic epithelial cells to produce other cytokines, such as IL-6, IL-8, granulocyte/macrophage-colony stimulating factor, and leukemia inhibitory factor (15-17). These studies suggest that TSC-derived IL-1
serves as an autocrine/paracrine factor to modulate cytokine production
within the thymus. A number of growth factors and hormones have been
shown to regulate IL-1 expression in TSC, including epidermal growth
factor, transforming growth factor-
(18, 19), growth hormone, and
prolactin (20). However, despite its biological significance, the
regulation of IL-1 expression at the transcriptional and
post-transcriptional levels in TSC remains poorly understood.
IL-1 has been shown to induce the expression of its own gene in several cells, including mononuclear cells (21), vascular smooth muscle cells (22), vascular endothelial cells (23), thymoma cells (24), and dermal fibroblasts (25). Whether it exerts a similar autocrine effect in other cells such as TSC has not been examined. Although transcriptional control has been implicated in some studies, little is known about the mechanism(s) of the response.
The regulation of IL-1 transcription has been most extensively studied
in response to LPS in monocytes (for reviews, see Refs. 1, 3, and 26).
Several DNA regulatory elements and corresponding nuclear proteins have
been identified, which ultimately participate in the control of IL-1
transcription. These include an upstream induction sequence, which
binds transcription factors NF-IL6, cAMP-response element binding
protein (27), NF-
B (28) and a STAT-like factor (24), and
promoter-proximal regulatory elements containing binding sites for
NF-IL6 (29, 30) and a B-cell and myeloid-specific transcription factor
PU.1 (Spi-1, NF-
A; Refs. 31 and 32). In addition to transcriptional
regulation, the stability of IL-1 mRNA can be selectively modulated
by various stimuli (for review, see Ref. 26). Little is known about how this gene is controlled in other cell types by different stimuli. Moreover, several lines of evidence have suggested that regulation of
IL-1 expression may occur at multiple levels, and the mechanisms may
vary with the particular cell type and stimulus examined (18, 20,
33-37).
In this study, we show that IL-1 is able to increase levels of
mRNAs for both IL-1
and IL-1
in TSC, documenting a positive feedback loop for IL-1 in the thymus. We show that IL-1
regulates its own gene primarily at the level of transcription by inducing Oct-1
binding to a consensus sequence in the 5'-flanking region. These
findings describe a previously unrecognized mechanism for autocrine
regulation of IL-1
in TSC and establish a model system to study IL-1
gene regulation and signal transduction.
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EXPERIMENTAL PROCEDURES |
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Materials--
Recombinant bovine IL-1 was a generous gift
from Dr. Dale Shuster, American Cyanamid Company, Princeton, NJ. LPS
and actinomycin D were purchased from Sigma. Antibodies, anti-Oct-1
(sc-232x), and anti-STAT5b (sc-835x) were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA). Tissue culture plates and flasks were
obtained from Fisher.
Cell Culture and Induction--
Three bovine thymic stromal cell
lines (TSC-936, TSC-2C4, TSC-934) and a bovine endometrial fibroblast
line (End-6.2) were generated by a strategy of temperature-sensitive
conditional transformation as described previously (20, 38). Previous
immunocytochemical studies showed that both TSC-936 and TSC-2C4 cells
stained positively only for vimentin, consistent with a mesenchymal
origin, whereas TSC-934 cells exhibited both vimentin- and
cytokeratin-specific staining (Ref.
20).2 Growth of these cells
resembles tumor cells at the permissive temperature (33 °C) and
reverts to a normal phenotype at the nonpermissive temperature
(39 °C), which is also the normal temperature for bovine cells.
These cells were routinely maintained at 33 °C in Dulbecco's
modified Eagle's medium/F-12 (Life Technologies, Inc.), 100 units/ml
penicillin G, 100 µg/ml streptomycin (Life Technologies, Inc.), and
5% fetal bovine serum (Hyclone; Logan, UT). LB, a bovine dermal
fibroblast line (39), was grown at 39 °C. All experiments were
performed on cells within 20 passages. For stimulation with cytokine,
cells were plated and grown at 39 °C for 3 days to 80-90% confluency. The cells were then washed with Dulbecco's
phosphate-buffered saline (DPBS, Life Technologies, Inc.) twice and
serum-starved for 24 h in Dulbecco's modified Eagle's medium/F12
before treatment. In all experiments, cells were treated with IL-1
at 10 ng/ml (0.5 nM) unless otherwise noted. LPS was used
at 10 µg/ml. For mRNA stability studies, cells were treated with
IL-1
for 3 h followed by the addition of 2 µg/ml actinomycin
D, and incubation was continued for varying times.
RNA Purification and Northern Analysis-- Cells were plated at a density of 4 × 105/T-25 flask and grown at 39 °C for 3 days before serum starvation and cytokine treatment as described above. Total RNA was isolated and analyzed using methods described previously (20). The signals were quantified with a PhosphorImager Storm 840 (Molecular Dynamics, Sunnyvale, CA). All determinations were repeated 2-3 times.
Genomic Library Screening and DNA Sequencing--
A bovine
genomic EMBL3 phage library (40) was screened with an oligonucleotide
probe (5'-GCAATGAAGGTTGGCTGG-3') made from the 5'-untranslated region
of the bovine IL-1 cDNA (41). Nucleotide sequences were
determined on both DNA strands and analyzed using the Genetics Computer
Group programs (42).
Plasmid Constructions--
The 4-kbp
BamHI/BamHI fragment with or without an
additional 0.6-kbp BamHI/NcoI fragment was
inserted into pGL3-basic (Promega) to generate the pIL-1-luciferase
constructs with or without the first intron (Fig. 4, constructs 1 and
2). Other deletion constructs were generated by digestion with
appropriate restriction enzymes from these plasmids.
278/+14,
255/+14, and -234/+14 constructs (Fig. 5) were derived by polymerase
chain reaction amplification using oligonucleotides corresponding to
the specific regions. Site-directed mutagenesis was carried out using a
mutagenic oligonucleotide (5'-CCAACATATgcTTGCATGATGACAC-3'; sequences
correspond to
247 to
223 of the bovine IL-1
gene, and the
lowercase letters indicate mutated bases) and the MORPHTM
site-specific plasmid DNA mutagenesis kit (5 Prime
3 Prime, Inc.,
Boulder, CO) to introduce an AT to GC mutation in the Oct consensus
site in the
303/+14 and
255/+14 reporter plasmids. All constructs
were confirmed by DNA sequencing. Plasmid DNAs used for transfection
were prepared using the Qiagen plasmid kit (Qiagen, Inc., Chatsworth,
CA).
Transfections and Reporter Gene Assay--
Transfections were
performed by the calcium phosphate method modified from Kingston
et al. (43). For transient transfections, TSC-2C4 cells were
plated at 3.6 × 105/6-cm plate and grown at 39 °C
for 3 days. After transfection, they were washed with Dulbecco's
phosphate-buffered saline three times, incubated in serum free
Dulbecco's modified Eagle's medium/F12 for 12-16 h, and then
stimulated with 0.5 nM IL-1 for an additional 24 h
before harvesting. Cells were lysed with 110 µl of 1× cell culture
lysis reagent (Promega). Luciferase activity was measured by adding 100 µl of luciferase substrate to 50 µl of cell lysate in a Turner
Designs Model 20/20 luminometer (Turner Designs, Sunnyvale, CA).
Relative light units were determined by 10-s integration and normalized
with the co-transfected
-galactosidase activity, as measured by the
Galacto-Light Plus kit (Tropix Inc., Bedford, MA).
Preparation of Nuclear Extracts--
TSC-2C4 and TSC-936 cells
were plated at a density of 1.8-2 × 106/10-cm plate
and grown at 39 °C for 3 days before serum starvation and cytokine
treatment as described above. Nuclear extracts were prepared by a
method modified from Andrews and Faller (44). Cells from two 10-cm
plates were incubated on ice for 10 min in 400 µl of buffer
containing 10 mM HEPES, pH 7.9, 1.5 mM
MgCl2, 10 mM KCl, 0.5 mM
dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, and 2 mM Na3VO4,
vortexed, and then pelleted. A high salt extraction was performed by
resuspending the cells in 25 µl of buffer containing 20 mM HEPES, pH 7.9, 25% glycerol, 420 mM NaCl,
1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl
fluoride, 2 µg/ml aprotinin and incubating on ice for 30 min. The
extraction was repeated, and the supernatants were pooled and stored at
80 °C. Protein concentrations were determined by the BCA protein
assay.
Electrophoretic Mobility Shift Analysis (EMSA)--
Mobility
shift reactions containing 5 µl of nuclear extract (5 µg of total
protein), 3 µg of poly(dI·dC), 4 µl of 5× binding buffer (1×
contains 10 mM HEPES, pH 7.9, 10% glycerol, 50 mM KCl, 2.5 mM MgCl2, 0.1 µg/ml
bovine serum albumin, 1 mM dithiothreitol), and 1 ng of
double-stranded oligonucleotide probe labeled by T4 polynucleotide
kinase purified by Nick Columns (Amersham Pharmacia Biotech) in a final
volume of 20 µl were incubated at room temperature for 20 min. The
reactions were electrophoresed for 1.5-2 h on a 4% polyacrylamide gel
with 2.5% glycerol in 0.25× Tris-buffered EDTA that had been prerun
for at least 1 h. The 308/
226 fragment (Fig. 5D)
was generated by polymerase chain reaction amplification followed by
gel purification. The
308/
274 and
261/
226 oligonucleotides (Fig. 5D) were synthetic double-stranded DNA. For the
competition assays, a 100-fold excess of competitor oligonucleotides
was added to the binding reaction 20 min before the addition of the
radiolabeled probe. Consensus sequences (CS) used as specific
competitors include Oct_CS, 5'-CGTACGTCCATTTGCATGGATCCTCT-3'
(45); GATA_CS, 5'-GAAACAAGATAAGATCAAATT-3' (46); PRE,
5'-AGATTTCTAGGAATTCAAATC-3' (47); and mu
261/
226, 5'-AGTTGTCAGAAAAACCAACATATgcTTGCATGATGA-3' (lowercase letters indicate mutated bases). For supershift assays, extracts were preincubated with 1 µg of antibody for 45 min at room temperature before the addition of the radiolabeled probe. All experiments were
repeated at least two times.
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RESULTS |
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Induction of IL-1 and IL-1
mRNAs by IL-1
in Thymic
Stromal Cells--
To see if there are differences in the ability of
IL-1 to induce IL-1 expression among fibroblast cell lines derived from different tissues, we examined the effects of IL-1
on both IL-1 agonist mRNAs in different cell lines by Northern analysis. To eliminate the possibility that hormones or growth factors present in
serum may induce the expression of IL-1, cells were cultured in
serum-free media for 24 h before treatment. No IL-1 RNA was detected in any unstimulated cells (Fig.
1). IL-1
induced both IL-1
and
IL-1
mRNA accumulation in all three thymic stromal cell lines
(TSC-934, -936, -2C4) to a somewhat different extent. IL-1
increased
only IL-1
mRNA in the endometrial stromal End-6.2 cells.
However, IL-1
had no detectable effect on mRNA levels for either
IL-1 agonist in the LB line, despite the fact that IL-1 receptors have
been described on these cells (48). In contrast to monocytes, the
thymic stromal cell lines TSC-936 and TSC-2C4 were insensitive to LPS
stimulation. This also ruled out the possibility that the IL-1
responses we observed were due to endotoxin contamination in the
recombinant cytokine. These data suggested that the ability of IL-1
to induce IL-1
and IL-1
mRNAs was cell-specific.
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Regulation of IL-1 and IL-1
mRNA
Stability--
Expression of the IL-1
and IL-1
genes is
regulated at multiple levels, including both transcriptional and
post-transcriptional mechanisms. To assess the contribution of mRNA
stability to the IL-1
-induced increases in steady-state levels of
IL-1
and IL-1
mRNAs, we measured the rates of decay of these
transcripts following IL-1
treatment after inhibiting new
transcription with actinomycin D. As shown in Fig. 2, C and
D, both IL-1
and IL-1
mRNA rapidly decayed in both
cell lines, with an estimated half-life of less than 1 h, similar
to that reported for many unstable mRNAs in different cell types
(for review, see Ref. 49). These data suggested a role for
transcriptional regulation in the changes in steady-state levels of
mRNA that we observed.
Cloning of the Bovine IL-1 5'-Flanking Region--
As a first
step toward studying the transcriptional regulation of IL-1
gene by
IL-1
itself, we cloned the bovine IL-1
gene and functionally
characterized the 5'-flanking region. A genomic clone with a 12-kbp
SalI insert was isolated that contained about 7 kbp of
5'-flanking region plus the coding sequences. Primer extension
identified a major transcript with an initiation site 45 base pairs 5'
to the first exon-intron junction; a TATA box was located 25 base pairs
upstream of this site. Multiple other minor start sites were also
observed in TSC-936, TSC-2C4, and peripheral blood mononuclear cells
(data not shown), consistent with the multiple putative TATA boxes
present in the 5'-flanking region of this gene (Fig.
3A). Consensus sequences for a
number of putative transcription factors were present in the
5'-flanking region as well as the first intron. Comparison of the
sequences of the proximal promoter region (
462 to +45) to the human
genomic sequences (50) showed 76% identity, including the regions
indicated in IL-1
responsiveness (Fig. 3B).
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Identification of IL-1-responsive Elements in the IL-1
5'-Flanking Region--
To determine the role of transcription in the
IL-1
-induced increases in the steady-state levels of mRNA, a
fragment containing 4 kbp of the 5'-flanking region plus the first
intron was linked to the luciferase reporter gene, and this construct
was transiently transfected into TSC-2C4 cells. Stimulation of the
cells with IL-1
increased luciferase activity 3.7-fold (Fig.
4, C and D, lane 1). To further localize sequences necessary for IL-1
autoregulation, we generated a series of 5'-deletion constructs
containing different amounts of the 5'-flanking region with or without
the first intron (Fig. 4A) and examined their responsiveness
in both TSC-2C4 and TSC-936 cells. Because TSC-936 cells were difficult
to transiently transfect, we generated stably transfected cells. To
normalize for different copy numbers of the foreign gene in different
stable cell lines, data are presented as -fold induction by IL-1
relative to the medium control. In both stably transfected TSC-936 and transiently transfected TSC-2C4, the
470/+14
EcoRI-BamHI fragment was sufficient to confer
maximal responsiveness to IL-1
(Fig. 4, B-D, lane
6). Basal levels varied with different constructs in transiently
transfected TSC-2C4 (Fig. 4C), suggesting multiple positive
and negative regulatory elements. The stimulatory effect of IL-1
was
dose-dependent; increases in luciferase activity were
observed at concentrations as low as 20 pM, reaching
maximal levels at 0.5 nM in both cell lines (data not
shown).
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Characterization of Protein Complexes That Bind to the 308/
226
Region--
To visualize protein-DNA interactions within the
303/
234 region, EMSA was performed. Using a probe spanning
308 to
226 (Fig. 5D), which included the
303/
234 fragment and
some surrounding sequences, we examined complex formation in nuclear
extracts prepared from TSC-2C4 cells stimulated with IL-1
for
increasing times. As shown in Fig.
6A, extracts from the
IL-1
-stimulated cells were able to form at least five complexes.
Among these, complex 2 was not detected in the untreated cells and was
strongly induced by IL-1
. Complexes 1 and 3 were present at low
levels before treatment and further increased by IL-1
. These
complexes were induced by IL-1
as early as 30 min and remained
intact up to 9 h. Competition assays showed that all five
complexes were specifically competed by excess unlabeled
308/
226
oligonucleotide (Fig. 6B, lane 2). The
oligonucleotide, PRE, containing the consensus site for transcription
factor STAT5 (47) was used as a nonspecific control because it had no
sequence homology to the
308/
226 fragment. Only complex 4 was
competed by PRE, suggesting that this complex is nonspecific (Fig.
6B, lane 5). Nuclear extracts from
IL-1
-treated TSC-936 cells yielded a very similar pattern (data not
shown). To determine which region of the DNA was responsible for the
specific binding, oligonucleotides
308/
274 and
261/
226,
containing the two IL-1
-inducible regions described in Fig. 5, were
used as competitors. We found that oligonucleotide
261/
226
effectively competed for binding in complexes 1, 2, and 3 (Fig.
6B, lane 4), whereas a 100-fold excess of the
308/
274 fragment only weakly competed for binding in complexes 1 and 3 (Fig. 6B, lane 3). Based on these results,
we pursued identification of the proteins binding to the
261/
226
region.
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IL-1-induced Nuclear Factor Oct-1 Binding to the
261/
226
Region--
EMSA with the
261/
226 oligonucleotide probe identified
multiple complexes formed after IL-1
treatment (Fig.
7). Similar to the pattern of complexes
observed with the
308/
226 probe, binding was detected after 30 min
of IL-1
treatment and remained elevated after 9 h of cytokine
exposure (data not shown). IL-1
specifically induced the ability to
form complexes 1, 2, 3, and 5 at all the time points we examined. A
faint band of lower mobility was detectable in some experiments.
Complex 5 may contain some nonspecific proteins because a 100-fold
excess of the
261/
226 oligonucleotide itself was not able to
completely abolish the signal (Fig. 7A, lane 3).
This may also be due to phosphatases present in the nuclear extracts
that may interact only with phosphorylated probes but not cold
competitors (51). Complex 4 appeared to be nonspecific because it was
competed by PRE (Fig. 7A, lane 7).
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DISCUSSION |
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We have demonstrated that IL-1 increases mRNAs for both
IL-1 agonists in TSC, providing a potential autocrine-paracrine
positive feedback loop for IL-1 activity in the thymic
microenvironment. This effect appears to be cell-specific, since the
expression patterns we observed differed in stromal cell lines derived
from different tissues as well as from those described in other cell types. The mechanism by which IL-1 exerts this effect appears to be
primarily at the level of transcription, despite the major role for
modification of mRNA stability in control of gene expression for
these cytokines in other cells (26, 49). Our investigation of the
IL-1-induced increase in transcription of the IL-1
gene in TSC
revealed a different mechanism than has been described for IL-1 action
on other target genes in other cells types and required distinct
sequences in the 5'-flanking region of the IL-1
gene than have been
described in the response to other agents. In TSC, this involves a
member of the pit, oct, unc (POU) domain family of transcription
factors, Oct-1, binding to an Oct consensus element positionally
conserved in the IL-1
gene across species.
Oct-1 is a widely expressed member of the POU domain family of transcription factors. POU factors contain two highly conserved domains, a POU-specific domain and a POU homeodomain, which mediate binding to DNA as well as interactions with other proteins that contribute to regulation of transcription (for reviews, see Refs. 52 and 53). Oct-1 has been shown to regulate transcription of a number of housekeeping as well as some tissue-specific and developmentally regulated genes, such as small nuclear RNA, H2B, Pit-1 (56), immunoglobulin genes (57), and several cytokine genes, including IL-2 (58), IL-3, IL-5, granulocyte/macrophage-colony stimulating factor (59), and IL-8 (60). It may inhibit (Pit-1, IL-8) or stimulate (the others) transcription of these target genes; accumulating evidence indicates this is dependent on sequence context, allowing interactions with other transcription factors or regulators.
In TSC, we found that the binding activity of Oct-1 to sequences in the
5'-flanking region of the IL-1 gene was specifically induced by
IL-1
, and our functional analysis confirmed an essential role of
this mechanism in mediating the response. Oct-1 did not appear to play
a role in determining basal transcription of IL-1
in TSC, in
contrast to the role of Oct-1 in regulation of some of the cytokine
genes noted above (59, 60). The role of Oct-1 in IL-1
autocrine
regulation and other IL-1 actions in other cell types remains to be
determined. Factors not implicated in the present studies appear to be
involved in IL-1-stimulated IL-1
gene transcription in some cells.
Tsukada et al. (24) have shown that in the murine EL4
thymoma cell line, IL-1
induced binding of a STAT-like factor to the
LPS and IL-1-responsive element located at position
2863 to
2841 of
the human IL-1
gene. The
4- to
0.5-kbp region was not implicated
in the IL-1 response in our cells, nor were we able to detect any
IL-1
-induced complex formation in TSC using EMSA with consensus
sequences for STAT 1, 3, or 5.2 In addition, IL-1 does not
stimulate Oct-1 activity in all systems. It has been reported that
IL-1-
in combination with cycloheximide or actinomycin D could not
superinduce the binding activity of Oct-1 in A549 cells (61), and in
Caco-2 and HepG2 epithelial cell lines, IL-1
treatment resulted in
removal of Oct-1 from the IL-8 promoter, where it functions as a
repressor (60). Taken together, our findings and these reports are
consistent with different mediators of IL-1 action depending on the
target cell as well as complex regulation of the activity of Oct-1 by
IL-1
, which is dependent on promoter sequence and cell context.
In the mobility shift assays, we found that the IL-1-induced
complexes were formed as early as 30 min. This rapid response suggested
that IL-1
may activate the participating protein factors through a
post-translational mechanism, such as phosphorylation or
dephosphorylation. Phosphorylation has been shown to increase or
decrease the activity of POU domain transcription factors in a
site-specific manner (52, 53). Several of the multiple kinases and
phosphatases that have been demonstrated to be activated by IL-1 in
different systems (for reviews, see Refs. 2 and 62) have been
implicated in control of the activity of Oct-1, including protein
kinase A (63, 64) and protein kinase C (63). Phosphorylation of Oct-1
at S-385 in the POU homeodomain inhibited DNA binding to the H2B
promoter (64), and inhibition of phosphatases resulted in decreased
Oct-1 binding to DNA by EMSA using extracts from the B-cell Daudi cell
line (63). Of note, pretreatment with the phosphatase inhibitor sodium
orthovanadate blocked IL-1 induction of IL-1
transcription in
TSC.2 However, our observed recruitment of Oct-1 to the
DNA-protein complex in response to IL-1 may not necessarily be a
result of modification of Oct-1 itself but rather an accessory protein
that then facilitates Oct-1 binding. Additional studies to examine IL-1-induced modifications of Oct-1 and the IL-1 signaling cascade in
TSC as well as other IL-1 target cells are necessary to clarify the
mechanism and cell/target gene specificity.
The POU domain also provides an interface for interactions with other
proteins. Many cellular as well as viral factors have been shown to
associate with Oct-1 in response to different stimuli (52, 53). In our
studies in TSC, we found that an oligonucleotide containing a mutation
in the Oct consensus site not only failed to abolish formation of the
Oct-1-containing complex but also failed to compete for the other
complexes formed with the 261/
226 probe, suggesting that binding of
Oct-1 was required for binding of additional proteins to this DNA.
Involvement of the GATA family of transcription factors suggested by
our data provides candidates to study these interactions. The
308/
274 region, adjacent to the region containing the Oct consensus
site, contains three additional GATA-like response elements. These
sequences were also able to compete for some protein binding to the
261/
226 probe, suggesting that GATA proteins could be common
factors shared by these two regions. GATA factors have been most
studied for their roles in regulation of hematopoiesis (54, 65), but
this growing family of transcription factors has recently been found to
play tissue-specific roles in multiple other cell types (66-68). Like
the POU family of transcription factors, recent evidence indicates that
interactions with other cell-specific proteins may modulate their
activities (69).
The demonstration in this study that IL-1 increased levels of both
IL-1
and IL-1
mRNAs in TSC implies a role for IL-1
in
regulating cytokine production in the thymus, contributing to T-cell
development. We used this system to study IL-1 regulation of IL-1
transcription and identified several responsive regions in the
5'-flanking region of the bovine IL-1
gene. Our data demonstrated that IL-1
stimulated transcription in part through the nuclear factor Oct-1 binding to an Oct consensus element in these cells. Identification of additional regulatory elements and factors binding to
these sequences may reveal how these factors interact in IL-1
control of transcription of its own gene, increase our knowledge of
IL-1 signal transduction, and improve our understanding of control of
thymic development and function.
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ACKNOWLEDGEMENTS |
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The authors are grateful to Drs. C. J. Czuprynski (Department of Pathobiological Sciences, University of Wisconsin, Madison, WI) and T. G. Golos (Wisconsin Regional Primate Center, University of Wisconsin, Madison, WI) for reagents and thoughtful advice. We would like to thank Dr. H.-T. Chen (currently at Cornell University Medical Center, New York, NY) for providing bovine genomic library, and Erin Klaffky for her technical assistance.
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FOOTNOTES |
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* This work was supported by United States Department of Agriculture Grant 96-35204-3664.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF026543
Present address: Joslin Diabetes Center, One Joslin Place, Boston,
MA 02215.
§ To whom correspondence should be addressed: Dept. of Comparative Biosciences, 2015 Linden Drive West, Madison, WI 53706. Tel.: 608-263-9825; Fax: 608-263-3926.
1 The abbreviations used are: IL-1, interleukin 1; TSC, thymic stromal cells; LPS, lipopolysaccharide; EMSA, electrophoretic mobility shift assay(s); CS, consensus sequences; kbp, kilobase pair(s); POU, pit, oct, unc.
2 Y.-H. Tseng and L. A. Schuler, unpublished data.
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REFERENCES |
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