From the Division of Gastroenterology and ¶ New
England Baptist Bone and Joint Institute, Beth Israel Deaconess
Medical Center, Harvard Medical School, Boston, Massachusetts 02215
Received for publication, August 12, 2002, and in revised form, October 23, 2002
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ABSTRACT |
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We have previously shown that colonic epithelial
cells are a major site of MIP-3 The chemokine superfamily of chemoattractant cytokines
comprises small (8-10 kDa), inducible, pro-inflammatory proteins that specialize in mobilizing leukocytes to areas of immune challenge (1-4). Interaction of these molecules with their respective leukocyte receptors induces a characteristic set of responses that are necessary for leukocytes to leave the circulation and infiltrate tissues. These include elevation of intracellular calcium levels, modulation of
adhesion molecule expression, formation of lamellipodia, and migration
of leukocytes along a chemotactic gradient. Thus increased chemokine
production and release is an important mechanism regulating leukocyte
activation and recruitment in response to injury or infection.
To date, over 40 chemokines have been identified, which can be
classified into one of four subfamilies according to the number and
arrangement of conserved cysteine residues (C, CC, CXC, or CX3C) (2, 5-7). Macrophage inflammatory
protein-3 The production of chemokines is largely regulated at the
level of gene transcription (2, 20). Of particular significance is the
NF- Cell Culture--
Caco-2 ileocecal cells (American Type Culture
Collection) were maintained in Dulbecco's modified Eagle's medium
supplemented with 10% fetal calf serum, 100 units/ml penicillin G
sodium, 100 µg/ml streptomycin sulfate, and non-essential amino acids
(Sigma) at 37 °C in an atmosphere of 5% CO2 and 95%
air. For all experiments, confluent Caco-2 cell monolayers were
stimulated by IL-1 Cloning of the Human MIP-3 Construction of MIP-3 Transient Transfection of Caco-2 Colonic Epithelial
Cells--
MIP-3 Electrophoretic Mobility Shift Assay--
To examine the
activation of transcription factors EMSA was performed essentially as
described by Keates et al. (31) Briefly, single-stranded
complementary oligonucleotides bearing either wild-type or mutant
MIP-3 Data Analysis--
Statistical analyses were performed using
SigmaStatTM for WindowsTM version 2.0 (Jandel Scientific Software, San Rafael, CA). Analysis of variance
followed by protected t tests were used for intergroup comparisons.
Cloning and Analysis of the Human MIP-3 Transcription Factor Binding Sites within the First 317 Nucleotides
of the MIP-3
As expected, following stimulation by IL-1 The NF-
To examine NF- Scanning Mutagenesis of the MIP-3
Taken together, these data demonstrate that several regions within the
first 166 nucleotides of the MIP-3 The Binding Element in Site X (CGCCTTC) Interacts with
Constitutively Expressed Zinc-requiring Nuclear Factors in Caco-2
Cells--
As shown in Fig. 4, the MIP-3
To further define the Site X region, we next performed overlapping
scanning mutagenesis between nucleotides
As part of our initial characterization of the unknown factors present
in Caco-2 nuclear extracts that bind the Site X region, EMSAs were
performed in the presence of the zinc-chelating agent EDTA to determine
whether any of the nuclear factors were zinc finger proteins. The
addition of 5 mM EDTA to the EMSA reaction mixture
significantly inhibited the binding of both complexes to the Site X
probe (Fig. 5C). Conversely, addition of 4 mM
ZnSO4 to binding reactions containing 5 mM EDTA
restored binding of each complex to the Site X probe. These data
clearly demonstrate that zinc-dependent Caco-2 nuclear
factors interact with the Site X binding element on the MIP-3 The Zinc Finger Transcription Factors Sp1 and Sp3 Bind to Site X of
the MIP-3 Sp1, but Not Sp3, Up-regulates MIP-3 The Site Y Binding Element (AAGCAGGAAGTT) Binds ESE-1, an
Epithelially Expressed Ets Factor--
The results of the
block-scanning mutagenesis experiment presented in Fig. 4 clearly
demonstrated that both basal and IL-1
To characterize the Site Y region further we next subjected nucleotides
As shown in the cartoon in Fig. 2, our preliminary analysis of the
MIP-3 ESE-1 Up-regulates MIP-3 In this study we demonstrate that at least three separate binding
elements on the MIP-3 In keeping with several recent studies, our findings demonstrate that
binding of p50/p65 NF- Several lines of evidence from the present study indicate that the Site
X region of the MIP-3 The main finding of the present study is that the Site Y region of the
MIP-3 Due to the central role of Ets proteins in hematopoietic cell
development, most previous studies of ESE-1 have focused on the role
played by this nuclear factor in epithelial cell differentiation and
proliferation. In this context, ESE-1 has been shown to transactivate the SPRR2A-cornified envelope precursor protein promoter, the transglutaminase 3 promoter as well as the profilligrin promoter, all
of which are highly expressed during terminal keratinocyte differentiation (34, 57, 58). ESE-1 can also transactivate the Endo
A/keratin 8 gene expressed primarily in simple epithelia (34). In
contrast, Brembeck et al. (59) have reported that keratin 4 promoter activity is repressed by ESE-1 in esophageal squamous
epithelial cells suggesting that this nuclear factor may have dual
effects on transcription depending on the specific promoter involved.
Interestingly, a recent study by Rudders et al. (60)
demonstrated that ESE-1 mRNA production can be up-regulated in
vascular smooth muscle cells, vascular endothelial cells, and THP-1
cells by IL-1 A striking feature of ESE-1 expression in normal non-inflamed human
colon is its predominant expression in epithelial cells (34).
Similarly, recent studies from our laboratory clearly demonstrate that
epithelial cells are a major site of MIP-3 production in human colon and that
enterocyte MIP-3
protein levels are elevated in inflammatory bowel
disease. The aim of this study was to determine the molecular
mechanisms regulating MIP-3
gene transcription in Caco-2 intestinal
epithelial cells. We show that a
B element at nucleotides
82 to
93 of the MIP-3
promoter binds p50/p65 NF-
B heterodimers and is
a major regulator of basal and interleukin-1
(IL-1
)-mediated gene activation. Scanning mutagenesis of the MIP-3
5'-flanking region also identified two additional binding elements: Site X (nucleotides
63 to
69) and Site Y (nucleotides
143 to
154). Site X
(CGCCTTC) bound Sp1 and regulated basal MIP-3
gene transcription.
Overexpression of Sp1 increased basal luciferase activity, whereas,
substitutions in the Sp1 element significantly reduced reporter
activity. In contrast, Site Y (AAGCAGGAAGTT) regulated both basal
and cytokine-induced gene activation and bound the Ets nuclear factor
ESE-1. Substitutions in the Site Y element markedly reduced inducible
MIP-3
reporter activity. Conversely, overexpression of ESE-1
significantly up-regulated MIP-3
luciferase levels. Taken together,
our findings demonstrate that co-ordinate activation and binding of
ESE-1, Sp1, and NF-
B to the MIP-3
promoter is required for
maximal gene expression by cytokine-stimulated Caco-2 human intestinal
epithelial cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(also known as CCL20 or liver and activation regulated
chemokine) is a recently described CC chemokine that is predominantly
expressed at extralymphoid sites, including the small intestine and
colon and is up-regulated by pro-inflammatory stimuli (8-10). Binding
of MIP-3
to its receptor, CCR6, induces migratory responses in
memory CD4+ T lymphocytes and immature dendritic cells
(11-14). MIP-3
also induces T-lymphocyte adhesion to the
gastrointestinal-specific vascular addressin MAd-CAM-1 (13). In
recent studies we, and others, have shown that enterocytes are a major
site of MIP-3
production in human colon and that epithelial MIP-3
protein levels are elevated in
IBD1 (15, 16). These studies
are complemented by a recent report that MIP-3
mRNA levels were
significantly elevated in chronically inflamed colons from IL-10
knockout mice (17). Interestingly, inhibition of colonic inflammation
in this model by treatment with anti-IL-12 monoclonal antibody resulted
in down-regulation of MIP-3
mRNA levels. Because Crohn's
disease in humans and the IL-10 knockout murine model of colitis are
both characterized by a prominent infiltration of the colonic mucosa by
CD4+ T-lymphocytes (18, 19), these data suggest that
MIP-3
may play an especially important role in the recruitment of
these cells to the epithelial layer during intestinal inflammation.
B/Rel family of transcription factors, which are important regulators of a variety of immune and inflammatory response genes (21-24). Several recent studies have demonstrated that MIP-3
gene expression is NF-
B-dependent (16, 25, 26). In
particular, Fujiie et al. (27) have reported that an NF-
B
binding site in the murine MIP-3
promoter is required for
cytokine-induced gene expression in Caco-2 human colonic epithelial
cells. Whether production of MIP-3
by human intestinal epithelial
cells requires the participation of additional nuclear factors has not
been investigated. In this study we confirmed that the human MIP-3
promoter NF-
B binding site plays a major role in regulating
IL-1
-induced gene transcription in Caco-2 cells. We also
identified two additional 5' regulatory elements: "Site X"
(nucleotides
63 to
69) and "Site Y" (nucleotides
143 to
154). Our findings demonstrate that Sp1 and the epithelium-specific
Ets nuclear factor ESE-1 (binding to Site X and Site Y, respectively)
are important co-regulators of MIP-3
gene expression in Caco-2
colonic epithelial cells.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(5 ng/ml). This stimulus was selected because
colonic levels of this pro-inflammatory cytokine are significantly
elevated in human IBD (28). Furthermore, we have shown previously that
IL-1
can dose-dependently up-regulate MIP-3
mRNA
and protein production in Caco-2 colonic epithelial cells (15).
Promoter Region--
The promoter
region of the human MIP-3
gene was isolated by screening a human
genomic library using a 32P-labeled MIP-3
cDNA
probe. To create the probe, Caco-2 cell RNA (1 µg) was
reversed-transcribed, and cDNAs encoding MIP-3
were amplified by
PCR as previously described (29). PCR primers used to generate the
MIP-3
probe were: sense, 5'-GAG TTT GCT CCT GGC TGC TTT-3';
antisense, 5'-TTT ACT GAG GAG ACG CAC AAT-3'. The genomic library
screening procedure was performed essentially as described by Keates
et al. (30). Briefly, a commercially available
human genomic library in Lambda Fix II (Stratagene) was plated on
Escherichia coli LE 392 at a density of 37500 plaque forming
units/150-mm Petri dish and then transferred to Hybond N+ membranes
(Amersham Biosciences). Plaque filters were then hybridized with a
random primer-labeled human MIP-3
cDNA probe at 42 °C in a
solution containing 25 mM potassium phosphate (pH 7.4), 5×
SSC, 5× Denhardt's solution, 100 µg/ml denatured salmon sperm DNA,
0.1% SDS, 50% (v/v) formamide, and 10% (w/v) dextran sulfate. Final
washes to remove non-hybridized probe were performed in 0.25× SSC at
42 °C. Genomic clones that hybridized with the MIP-3
probe were
replated and rescreened until they were plaque-purified. Lambda DNA was
then isolated from the positive clones and digested with a variety of
restriction endonucleases, and Southern blots of the digests were
probed with the human MIP-3
cDNA probe. Fragments of insert DNA
that hybridized with the probe were then subcloned into pBluescript II
(Stratagene). Genomic DNA 5' to the MIP-3
cDNA probe
(i.e. the MIP-3
promoter) was then sequenced by
"walking" from both ends or from a defined point using
specific oligonucleotide primers.
Luciferase Reporter Genes--
An
849-bp fragment (containing nucleotides
817 to +33) of MIP-3
promoter was prepared by PCR amplification of human genomic DNA using a
sense primer containing a MluI restriction site and an
antisense primer containing a BglII restriction site.
Following digestion with the appropriate restriction enzymes the
MIP-3
promoter fragment was directionally cloned into the pGL3-Basic firefly luciferase expression vector (Promega) to generate a
"full-length" MIP-3
reporter construct. Reporter genes
containing sequentially truncated fragments (
712,
618,
521,
406,
317,
216,
162,
124,
103, and
62 to +33) of the
MIP-3
promoter region were prepared in a similar manner using sense
primers containing MluI restriction sites, and the antisense
primer used to generate the full-length MIP-3
reporter
construct. Mutant reporter constructs containing systematic
scanning substitutions of the MIP-3
promoter or targeted
substitutions in the binding elements of putative regulatory factors
(i.e. NF-
B, C/EBP, Site X, and Site Y; see Table I) were
prepared by oligonucleotide-directed in vitro mutagenesis as
previously described (31).
reporter constructs were transfected into Caco-2
cells using LipofectAMINE 2000 (Invitrogen) according to the
manufacturer's instructions. Briefly, cells were plated 24 h
before transfection at a density of 2 × 105/well on a
twelve-well tissue culture dish (Corning Costar). Three hours prior to
transfection, Caco-2 cells were incubated with fetal calf serum-free,
antibiotic-free media and then transfected with 1.8 µg of MIP-3
reporter gene plasmid DNA or equimolar amounts of MIP-3
reporter
constructs containing truncated promoter sequences. To correct for
variations in DNA uptake by the cells, each test construct was
co-transfected with 0.2 µg of pRL-TK Renilla luciferase control vector (Promega). Transfections using pGL3-Basic vector without
an insert were used as a negative control. For experiments investigating the effect of Sp1, Sp3, and ESE-1 overexpression on
MIP-3
reporter gene activity, Caco-2 cells were co-transfected with
0.5 µg of full-length MIP-3
reporter gene and 1.5 µg of pCMV-Sp1
or pCMV-Sp3 (kindly provided by Dr. G. Suske, Marburg, Germany) or 1.5 µg of pCI-ESE-1. After transfection for 15 h, the media were
removed and replaced with complete growth medium. After a further
24 h, Caco-2 cells were washed twice with sterile phosphate-buffered saline and incubated with serum-free medium for
16 h to reduce background luciferase levels prior to stimulation with IL-1
(5 ng/ml) for 6 h. The firefly and Renilla
luciferase activities of the cells were then measured simultaneously in
each sample using the Dual-Luciferase Reporter Assay System according to the manufacturer's instructions (Promega).
promoter binding elements (i.e. NF-
B, Site X,
and Site Y) were prepared by custom oligonucleotide synthesis (Genosys
Biotechnologies). A double-stranded oligonucleotide containing a
consensus binding site for Sp1 was obtained commercially (Santa Cruz
Biotechnology). After annealing, 100 ng of the double-stranded oligonucleotide was labeled using T4 polynucleotide kinase (Promega) in
the presence of [
-32P]ATP (PerkinElmer Life Sciences).
Labeled probes were then purified on a Sephadex G-25 spin column
(Amersham Biosciences). Binding reactions for NF-
B EMSAs were
performed as described by Ferrari et al. (32). Site X EMSAs
were performed according to the method of Wang et al. (33)
except that 100 µM ZnSO4 was added to the binding reactions. Site Y EMSAs were performed according to the protocol of Oettgen et al. (34). Caco-2 cell
nuclear extracts were preincubated for 10 min at room temperature in
the reaction mixture after which the probe DNA was added and the
incubation continued for another 30 min. Probe-protein complexes were
then separated from free probe using 4.5% native polyacrylamide gels. Dried gels were exposed to x-ray film at
80 °C to visualize the probe-protein complexes. To confirm the specificity of the binding reactions supershift assays were performed using antibodies to the Sp
family proteins Sp1, Sp2, Sp3, and Sp4 (Santa Cruz Biotechnology), the
Ets factors ESE-1 (Oncogene Research Products), ESE-2 and Ets-1 (Santa
Cruz Biotechnology), and the NF-
B subunits p50, p52, p65, Rel B, and
c-Rel (Santa Cruz Biotechnology). In some experiments binding
specificity was also determined by competition with excess unlabeled
probe. Antibodies or competing probe were added to the binding
reactions at the start of the 30-min incubation period.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Promoter
Region--
Recent studies from our laboratory have shown that
enterocytes are a major site of MIP-3
production in human colon and
that epithelial MIP-3
protein levels are elevated in IBD (15).
However, the molecular mechanisms that control MIP-3
gene expression
in intestinal epithelial cells are poorly understood. To investigate the nuclear factors that regulate MIP-3
production we first cloned the 5'-flanking region of the gene from a human genomic DNA library using a MIP-3
cDNA probe. Using this approach we identified
three genomic clones, designated G2-1, G6-1, G10-1, that hybridized with the MIP-3
cDNA probe from a screening of ~375,000
plaque-forming units. An 8-kb EcoRI fragment from clone
G6-1 was extensively sequenced and found to contain 878 bp of the
MIP-3
gene 5'-flanking region (Fig.
1). The nucleotide sequence of the
MIP-3
promoter fragment was identical to that reported recently by
Harant et al. (25). Further analysis of this sequence using
the TransFac data base (35), identified putative binding sites for
NF-
B, C/EBP, AP-1, c-Ets, and Sp1/CACCC binding protein within the
first 250 bp of the promoter. A previous study by Hieshima et
al. (8), using 5' rapid amplification of cDNA ends, identified
a transcription initiation point 58-bp upstream from the MIP-3
start
codon.
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Fig. 1.
Nucleotide sequence of the human
MIP-3 promoter region. The putative
transcription start point (nucleotide +1) and translation initiation
codon (nucleotide +59) are capitalized and
shaded. The TATA binding box and binding sites for putative
regulatory transcription factors are underlined with
black bars as indicated.
Promoter Are Sufficient for Responses to IL-1
in
Caco-2 Cells--
Previous studies have shown that the production of
chemokines is largely regulated via up-regulation of gene transcription (2, 20). Moreover, in a recent study we have shown that MIP-3
production by cytokine-stimulated Caco-2 cells requires de
novo mRNA synthesis (15). To begin to characterize the areas
of the 5' promoter region that regulate MIP-3
gene expression, a
series of luciferase reporter constructs containing sequentially
truncated fragments of the MIP-3
promoter were created and
transfected into Caco-2 intestinal epithelial cells (Fig.
2).
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Fig. 2.
The first 317 nucleotides of the
MIP-3 promoter mediate responses to
IL-1
stimulation in Caco-2 cells. Caco-2
cells were co-transfected with full-length or truncated MIP-3
reporter constructs and the pRL-TK control vector. MIP-3
promoter
truncations and putative nuclear factor binding sites are shown
schematically in the cartoon. 48 h after transfection the cells
were incubated with or without IL-1
(5 ng/ml) for 6 h. The
activity of each construct is presented relative to the
IL-1
-stimulated activity of the full-length MIP-3
reporter gene
and corrected for transfection efficiency by the activity of the pRL-TK
control vector. Data are expressed as mean ± S.E.
(n = 6). **, p < 0.01 versus IL-1
-stimulated activity of the preceding shorter
construct.
(5 ng/ml, 6 h) no
significant increase in luciferase reporter gene activity was seen in
Caco-2 cells transfected with empty expression vector or a MIP-3
reporter gene containing the TATA binding site alone (nucleotides
62
to +33) compared with non-treated cells. In contrast, the luciferase
activity of Caco-2 cells transfected with a MIP-3
reporter construct
bearing putative NF-
B & C/EBP binding elements (nucleotides
103 to
+33) was increased 11.9-fold (p < 0.01) by IL-1
stimulation. The basal activity of this construct was elevated ~5.6-fold compared with Caco-2 cells transfected with empty
expression vector. Compared with non-stimulated cells, increases in
IL-1
-inducible reporter activity were also observed when Caco-2
cells were transfected with MIP-3
reporter genes containing the
putative AP-1 binding site (33.6-fold, p < 0.01, nucleotides
124 to +33) and the putative c-Ets binding site
(54.8-fold, p < 0.01, nucleotides
162 to +33). An
additional increase in luciferase activity (~2.5-fold,
p < 0.01) was seen when Caco-2 cells were transfected
with reporter genes containing nucleotides
162 to
317 of the
MIP-3
promoter. However, binding elements in this region had little
effect on IL-1-inducible reporter activity (~30-fold), suggesting
they primarily regulate basal gene expression. Taken together, these
data indicate that nuclear factor binding sites within the first 317 nucleotides of the promoter are both necessary and sufficient for basal
and IL-1
-inducible MIP-3
gene expression in Caco-2 cells.
B, but Not the C/EBP, Binding Element on the
MIP-3
Promoter Is Required for Basal and IL-1
-stimulated MIP-3
Gene Expression--
Previous studies have shown that the NF-
B/Rel
family of transcription factors are key regulators of a variety of
immune and inflammatory response genes, including those of the
chemokine superfamily (21-24). Consistent with these findings, our
data using truncated reporter constructs suggested that the region of
the MIP-3
promoter containing putative NF-
B and C/EBP binding
elements was also likely to be important for the regulation of
inducible gene expression in Caco-2 intestinal epithelial cells. We
therefore examined this region in greater detail using mutated reporter gene constructs and EMSA (see Table I) to
define the role of each nuclear factor binding site. Stimulation of
Caco-2 cells transfected with the wild-type MIP-3
reporter by
IL-1
(5 ng/ml, 6 h) induced an ~40-fold increase in
luciferase activity compared with non-stimulated cells (Fig.
3A). Targeted substitutions
within the putative NF-
B binding site significantly reduced
IL-1-inducible gene expression by ~90% (p < 0.01)
compared with non-stimulated cells. Moreover, in non-stimulated Caco-2
cells, substitution of the putative NF-
B binding site also reduced
basal luciferase activity ~50% (p < 0.01) compared
with cells transfected with the wild-type MIP-3
reporter construct
(Fig 3A). In contrast to these findings, substitution of the
putative C/EBP binding site had no significant effect on either basal
or IL-1
-mediated MIP-3
reporter gene activity. These findings
demonstrate that NF-
B binding site, but not the C/EBP binding site,
is an important regulator of both basal and IL-1
-inducible MIP-3
gene expression in Caco-2 intestinal epithelial cells.
MIP-3 reporter gene deletions and substitutions
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Fig. 3.
The MIP-3
B element binds p50/p50 and p50/p65
NF-
B and is required for basal and
IL-1
-induced gene transcription in Caco-2
cells. A, Caco-2 cells were co-transfected with a
wild-type MIP-3
reporter gene or mutant constructs containing
targeted substitutions in either the NF-
B binding element or C/EBP
binding element and the pRL-TK control vector. 48 h later the
cells were treated with IL-1
(5 ng/ml) for 6 h. The activity of
each construct is presented relative to the non-stimulated activity of
the wild-type MIP-3
reporter gene and corrected for transfection
efficiency. Data are expressed as mean ± S.E. (n = 6). **, p < 0.01 versus non-stimulated
activity of the wild-type construct;
, p < 0.01 versus IL-1
-stimulated activity of the wild-type
construct. B, the time course of complex binding to the
B
element was investigated by EMSA using a MIP-3
-specific probe and
nuclear extracts from Caco-2 cells stimulated with IL-1
(5 ng/ml).
The upper portion of the autoradiogram containing the
probe-protein complexes is presented. Complexes binding to the
MIP-3
-specific
B probe are indicated by arrows.
C, EMSA supershift study of MIP-3
-specific NF-
B
activation in Caco-2 cells. Nuclear extracts from Caco-2 cells treated
with IL-1
(5 ng/ml) for 2 h were incubated with either excess
cold probe or antibodies against the Rel proteins p50, p52, p65, Rel B,
and c-rel then subjected to EMSA. The upper portion of the
autoradiogram containing the probe-protein complexes is presented.
Complexes binding to the MIP-3
-specific
B probe are indicated by
arrows.
B regulation of MIP-3
gene expression in more
detail we next performed EMSAs in non-transfected Caco-2 cells using a
MIP-3
-specific probe. As expected, nuclear extracts from control
Caco-2 cells showed little NF-
B binding (Fig. 3B). Within 30 min of treatment with IL-1
(5 ng/ml) Caco-2 nuclear extracts showed a marked increase in binding of two complexes to the
MIP-3
-specific NF-
B probe (indicated by arrows).
Levels of the faster migrating complex (band 1) appeared to
remain constant over the 6-h time course of the experiment, whereas
levels of the slower migrating complex (band 2) were maximal
after 30 min to 1 h and declined thereafter. To determine which
NF-
B subunits bound to the MIP-3
-specific probe, we next
performed a supershift experiment. Nuclear extracts prepared from
Caco-2 cells treated with IL-1
(5 ng/ml) for 2 h were incubated
with either antibodies against the Rel proteins p50, p52, p65, Rel B,
and c-rel or excess cold probe and then subjected to EMSA (Fig.
3C). The lower complex (band 1) only underwent a
supershift with the p50 antibody indicating the presence of a p50/p50
homodimer. In contrast, the upper complex (band 2) was supershifted by both the p50 antibody and the p65 antibody
demonstrating binding of p50/p65 heterodimers to the MIP-3
NF-
B
binding element.
Promoter Reveals the Presence
of Multiple Binding Elements That Regulate MIP-3
Gene Expression
Caco-2 Cells--
As shown in Fig. 2, truncation of the MIP-3
promoter demonstrated that nuclear factor binding sites within the
first 317 nucleotides of the MIP-3
promoter were essential for
maximal responses to IL-1
in Caco-2 cells. Moreover, these data also indicated that other factors, in addition to NF-
B, which bind to the
promoter between nucleotides
103 and
162 are required for inducible
gene expression. To further characterize the factors that regulate
cytokine-inducible MIP-3
gene expression we next performed scanning
mutagenesis of the MIP-3
promoter between nucleotides
46 to
166
to define specific nuclear factor binding element(s). To accomplish
this a series of 12 full-length MIP-3
reporter constructs containing
sequential 10 bp nucleotide substitutions were prepared and transfected
in Caco-2 cells (Fig. 4). As expected, the luciferase activity of Caco-2 cells transfected with reporter genes
carrying mutations of the NF-
B binding site (constructs 8 and 9;
nucleotides
76 to
96) were substantially reduced (p < 0.01 for both) when compared with cells transfected with the wild-type construct. Caco-2 cells transfected with reporter genes containing substitutions within the putative c-Ets binding element (construct 2; nucleotides
146 to
156) also showed significantly reduced IL-1-inducible luciferase activity (p < 0.01).
Similar findings were observed when nucleotides encompassing the
putative AP-1 site (constructs 5 and 6;
106 to
126) were
substituted. Interestingly, compared with cells transfected with the
wild-type MIP-3
reporter construct, a substantial decrease in
reporter activity (p < 0.01) was also detected when
Caco-2 cells were transfected with a MIP-3
reporter construct in
which nucleotides
66 to
76 (construct 10) were mutated.
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Fig. 4.
Scanning mutagenesis of the
MIP-3 promoter reveals the presence of
multiple binding elements that regulate MIP-3
gene expression in Caco-2 cells. To locate nuclear factor
binding elements that regulate IL-1
-inducible gene expression, the
MIP-3
promoter was subjected to 10-bp block-scanning mutagenesis
between nucleotides
46 and
166. Caco-2 cells were co-transfected
with wild-type or mutant MIP-3
reporter constructs and the pRL-TK
control vector. MIP-3
promoter substitutions and the positions of
various putative nuclear factor binding sites are shown in the
schematic. 48 h after transfection the cells were incubated with
or without IL-1
(5 ng/ml) for 6 h. The activity of each
construct is presented relative to the non-stimulated activity of the
wild-type MIP-3
reporter gene and corrected for transfection
efficiency by the activity of the pRL-TK control vector. Data are
expressed as mean ± S.E. (n = 6). **,
p < 0.01 versus IL-1
-stimulated activity
of the wild-type construct.
promoter appear to be required
for maximal gene expression in Caco-2 cells stimulated by IL-1
.
Moreover, the location of most of these sites corresponds closely to
the position of various putative binding elements identified using the
TransFac data base (35) (see schematic in Fig. 2). Based on
these findings we selected two regions of the MIP-3
promoter for
further study: (i) nucleotides
56 to
81 (designated "Site X"),
which, by analysis using the TransFac data base, contained no consensus
transcription factor binding elements and (ii) nucleotides
136 to
166 (designated "Site Y"), which contained a putative c-Ets
binding motif. The region of the MIP-3
promoter containing a
putative AP-1 binding site (nucleotides
106 to
126) was not studied
further, because regulation of several chemokine genes by AP-1 has been
reported previously (36-38).
promoter construct in
which nucleotides
66 to
76 were substituted showed markedly
diminished basal and inducible reporter gene activity. However, despite
these decreases in overall luciferase activity, the ability of this construct to respond to IL-1
stimulation was similar to the
wild-type construct (~10-fold). This finding suggests that binding
elements located in this region primarily regulate basal MIP-3
gene
expression. To examine whether one or more specific Caco-2 nuclear
factors could interact with this segment of the MIP-3
promoter, a
probe encompassing nucleotides
56 to
81 was synthesized and used
for EMSA. As shown in Fig.
5A, two closely
migrating complexes were detected in Caco-2 cell nuclear extracts using
the Site X probe (designated bands 1 and 2). The
binding of each complex was completely abolished in the presence of a
200-fold molar excess of unlabeled Site X probe indicating specific
binding to the probe (shown in Fig. 5, C and D).
Furthermore, equal levels of each complex were present in both
unstimulated and in IL-1
-stimulated Caco-2 nuclear extracts
consistent with our reporter gene data indicating that this region
regulates basal gene expression.
View larger version (20K):
[in a new window]
Fig. 5.
Zinc finger transcription factors Sp1 and Sp3
bind to the Site X region of the MIP-3
promoter. A, the time course of complex binding
to Site X element was investigated by EMSA using a MIP-3
-specific
probe and nuclear extracts from Caco-2 cells stimulated with IL-1
(5 ng/ml). The upper portion of the autoradiogram containing the probe-protein complexes is presented. Complexes
binding to the MIP-3
-specific Site X probe are indicated by
arrows. B, to define the Site X binding element
the MIP-3
promoter was subjected to scanning mutagenesis between
nucleotides
56 and
81. EMSAs were performed using nuclear extracts
from Caco-2 cells treated with IL-1
(5 ng/ml) for 2 h and a
wild-type MIP-3
Site X probe or one of eight sequentially
substituted Site X gel shift probes. The shaded nucleotide
sequence represents the Site X binding element. C, binding
of complexes to the Site X probe in the presence of EDTA or EDTA in
combination with ZnSO4 was examined by EMSA using nuclear
extracts from Caco-2 cells stimulated with IL-1
(5 ng/ml) for 2 h. D, EMSA supershift analysis of Sp1 binding to the Site X
region of the MIP-3
promoter. Nuclear extracts from Caco-2 cells
treated with IL-1
(5 ng/ml) for 1 h were incubated with
antibodies directed against Sp1, Sp2, Sp3, and Sp4 or with excess
unlabeled Site X probe. E, binding of complexes to the
wild-type Site X probe (lanes 1 and 5) or mutant
probes containing substitutions at nucleotides
67 (lane 4)
or
68 (lane 3) was examined by EMSA using nuclear extracts
from Caco-2 cells stimulated with IL-1
(5 ng/ml) for 4 h. The
ability of excess unlabeled consensus Sp1 oligonucleotide to compete
for binding of complexes to the Site X probe was also investigated
(lane 2). The shaded nucleotide sequence
represents the binding site for Sp1/Sp3.
56 to
81 of the MIP-3
promoter to define a specific binding element for each complex. As
shown in Fig. 5B, binding of complexes 1 and 2 was completely blocked when EMSAs were performed using Site X-scanning mutant probes 4, 5, or 6. In contrast, both complexes were able to
interact (albeit to differing degrees) with Site X-scanning mutant
probes 1, 2, 3, 7, and 8. These findings indicate that complexes 1 and
2 bind to the nucleotide sequence CGCCTTC in the Site X region of the
MIP-3
promoter.
promoter.
Promoter--
To determine whether any known zinc finger
transcription factors could interact with Site X, this region of the
MIP-3
promoter was subjected to further data base analyses using
less stringent search parameters. The results of this examination
suggested that Site X might contain a non-consensus binding element for
the zinc finger protein Sp1. To test whether any of the complexes that bound to the Site X probe contained Sp proteins, a supershift EMSA was
performed using antibodies specific for various Sp family proteins. As
shown in Fig. 5D, an antibody directed against Sp1 (lane 2) was able to supershift both the upper and the lower
complexes, whereas an antibody directed against Sp3 (lane 4)
supershifted the lower complex only. Antibodies directed against Sp2 or
Sp4 were unable to shift either complex (lanes 3 and
5). Binding of all bands to the Site X probe was reduced by
an excess of unlabeled (cold) Site X probe (lane 6)
indicating specific binding. Together, these data indicate that the
upper Site X complex (band 1) contains Sp1, whereas the
lower Site X complex (band 2) contains Sp1 and Sp3. To
confirm these findings we examined the binding of each complex to Site
X EMSA probes containing targeted nucleotide substitutions at positions
67 and
68. These substitutions were chosen because mutations at
each of these positions have been shown previously to prevent the
interaction of Sp1 with its consensus binding element (39). As shown in
Fig. 5E, nucleotide substitution at either of these
positions completely prevented binding of both complexes to the Site X
probe (lanes 3 and 4). Binding of both complexes to the wild-type probe was also substantially reduced in the presence of excess unlabelled consensus Sp1 oligonucleotide (lane
2).
Gene Expression in Caco-2
Cells--
To determine whether the Sp1/Sp3 binding element located in
Site X could functionally regulate MIP-3
gene expression, we next
synthesized reporter constructs carrying nucleotide substitutions at
positions
67 and
68 and transfected them into Caco-2 cells. As
shown in Fig. 6A, mutation of
the MIP-3
promoter at either position reduced IL-1
-mediated
luciferase activity by ~75% (p < 0.01) compared
with the wild-type control. The basal activity of each of the mutant
constructs was also substantially reduced (by ~50%,
p < 0.01). These findings are consistent with our EMSA studies presented in Fig. 5E and demonstrate that increased
MIP-3
gene expression in Caco-2 intestinal epithelial cells appears to correlate with binding of Sp1 and/or Sp3 to Site X of the promoter. To examine Sp regulation of MIP-3
gene expression further, we next
investigated whether overexpression of Sp1 and/or Sp3 in Caco-2 cells
could transactivate MIP-3
promoter activity. As shown in Fig.
6B, transfection of Caco-2 cells with the Sp1 expression vector (pCMV-Sp1) increased basal and IL-1
-inducible MIP-3
luciferase reporter gene activity ~2-fold compared with cells
transfected with the empty vector (p < 0.001 for
both). In contrast, transfection of Caco-2 cells with the Sp3
expression construct (pCMV-Sp3) had little effect on basal or
IL-1
-mediated MIP-3
reporter activity.
View larger version (18K):
[in a new window]
Fig. 6.
Sp1, but not Sp3, up-regulates
MIP-3 gene expression in Caco-2 cells.
A, to determine whether the Sp1/Sp3 binding element in Site
X functionally regulates MIP-3
gene expression, Caco-2 cells were
transfected with a wild-type reporter gene or mutant constructs
containing targeted substitutions at positions
67 or
68 of the
promoter. Caco-2 cells were also co-transfected with the pRL-TK control
vector. 48 h later the cells were treated with IL-1
(5 ng/ml)
for 6 h. The activity of each construct is presented relative to
the non-stimulated activity of the wild-type MIP-3
reporter gene and
corrected for transfection efficiency. Data are expressed as mean ± S.E. (n = 8).
, p < 0.01 versus non-stimulated activity of the wild-type construct;
**, p < 0.01 versus IL-1
-stimulated
activity of the wild-type construct. B, Caco-2 cells were
co-transfected with the wild-type MIP-3
reporter gene and either a
pCMV-Sp-1 expression vector, a pCMV-Sp3 expression vector, or control
vector (pcDNA3.1). 48 h later the cells were treated with
IL-1
(5 ng/ml) for 6 h. Luciferase activities are presented
relative to the non-stimulated control activity of the MIP-3
reporter gene and corrected for extract protein concentration. Data are
expressed as mean ± S.E. (n = 12).
,
p < 0.001 versus non-stimulated activity of
the wild-type construct; ***, p < 0.001 versus IL-1
-stimulated activity of the wild-type
construct.
-inducible reporter activity
are significantly reduced when nucleotides
146 to
156 in the
MIP-3
promoter are mutated. Moreover, in contrast to our findings
with Site X, the level of luciferase activity following IL-1
stimulation (~5-fold) was significantly reduced compared with that
seen with the wild-type construct (~10-fold). This finding suggested
that Caco-2 nuclear factors interacting with Site Y regulate both basal
and IL-1
-inducible MIP-3
gene expression. To characterize Site Y
we next performed an EMSA experiment to determine whether any Caco-2
nuclear factors could interact with this segment of the MIP-3
promoter. As shown in Fig. 7A, one major complex (designated band 1) was identified using
the Site Y probe in Caco-2 cell nuclear extracts. Moreover, a 200-fold molar excess of unlabeled probe was able to completely prevent the
formation of this complex with the Site Y probe illustrating the
specificity of the interaction (shown in Fig. 7C, lane
6). Similar to our findings with Site X, equal binding of this
complex was seen in non-stimulated as well as IL-1
-stimulated Caco-2 nuclear extracts suggesting that this region regulates constitutive MIP-3
gene expression.
View larger version (44K):
[in a new window]
Fig. 7.
ESE-1, an epithelially expressed member of
the Ets transcription factor family, binds to the Site Y region of the
MIP-3 promoter in Caco-2 cells. A, the time course
of complex binding to Site Y element was investigated by EMSA using a
MIP-3
-specific probe and nuclear extracts from Caco-2 cells
stimulated with IL-1
(5 ng/ml). The upper portion of the
autoradiogram containing the probe-protein complexes is presented.
Complex binding to the MIP-3
-specific Site Y probe is indicated by
an arrow. B, to define the Site Y binding element
the MIP-3
promoter was subjected to scanning mutagenesis between
nucleotides
137 and
163. EMSAs were performed using nuclear
extracts from Caco-2 cells treated with IL-1
(5 ng/ml) for 1 h
and a wild-type MIP-3
Site Y probe or one of eight sequentially
substituted Site Y gel shift probes. The shaded nucleotide
sequence represents the Site Y binding element. C, EMSA
supershift analysis of ESE-1 binding to the Site Y region of the
MIP-3
promoter. Nuclear extracts from Caco-2 cells treated with
IL-1
(5 ng/ml) for 4 h were incubated with antibodies directed
against ESE-1 (lane 2) or Ets-1 (lane 3) or with
excess unlabeled oligonucleotides bearing: the ESE-1 binding site from
the SPRR2A promoter (lane 4), a mutant SPRR2A ESE-1 binding
element (lane 5), the wild-type Site Y sequence (lane
6), and a mutant Site Y sequence substituted at positions
148
and
149 (lane 7). The shaded nucleotide
sequence represents the binding site for ESE-1.
138 to
163 of the MIP-3
promoter to overlapping scanning
mutagenesis to identify a specific binding element. As shown in Fig.
7B, binding of Caco-2 cell nuclear factors to Site Y was
unaffected when EMSAs were performed using scanning mutant probes 1, 2, 3, or 8. In contrast, complex formation was clearly disrupted when Site
Y scanning mutant oligonucleotides 4, 5, 6, or 7 were used as probes.
These data demonstrate that the nucleotide sequence AAGCAGGAAGTT
comprises the MIP-3
promoter Site Y binding element.
promoter sequence using the TransFac data base (35) indicated
a consensus binding motif for the nuclear factor c-Ets was located
within the Site Y region at nucleotides
143 to
150. To determine
whether the Caco-2 nuclear protein that bound Site Y was an Ets factor
supershift EMSAs were performed. No supershifts were observed when
antibodies directed against the ubiquitously expressed proteins Ets-1
(Fig. 7C, lane 3), PEA-3 and Elk-1 (data not
shown) were tested. In view of these findings, we next tested antibodies directed against three epithelially expressed members of
this family: ESE-1, -2, and -3. An antibody directed against ESE-1
clearly prevented the formation of complex 1 and was accompanied by a
broad supershift in the upper portion of the gel (Fig. 7C, lane 2). Antibodies directed against ESE-2 or ESE-3,
however, were without effect (data not shown). To confirm this finding competition, EMSA was performed using a wild-type SPRR2A probe (which
contains a previously characterized ESE-1 binding site from the SPRR2A
promoter) and a mutant SPRR2A probe (which is unable bind ESE-1) (34).
A 200-fold molar excess of unlabeled wild-type SPRR2A substantially
reduced binding of complex 1 to the Site Y probe (lane 4).
In contrast, binding this complex to the Site Y probe was unchanged in
the presence of excess cold mutant SPRR2A probe. Finally, excess
unlabeled mutant Site Y probe substituted at positions
148 and
149
(i.e. in an analogous fashion to the mutant SPRR2A probe)
was also unable to compete for binding of complex 1 to the wild-type
Site Y probe.
Gene Transcription in Caco-2
Cells--
As shown in Fig. 7C, our EMSA studies indicate
that the Site Y region of the MIP-3
promoter interacts with ESE-1,
an epithelially expressed Ets transcription factor, in nuclear extracts
prepared from Caco-2 cells. To elucidate the role ESE-1 may play in the functional regulation of MIP-3
gene expression we next synthesized a
mutant reporter construct carrying targeted substitutions at positions
148 and
149, which, by EMSA analysis, was unable to bind ESE-1. As
shown in Fig. 8A, in Caco-2
cells transfected with the mutant construct there was significant
reduction in both basal and IL-1
-stimulated luciferase activity
compared with cells transfected with the wild-type reporter gene
(p < 0.001 for both). Moreover, the level of induction
(i.e. -fold increase) observed with mutant construct
following IL-1 stimulation (~15-fold) was substantially decreased in
comparison to Caco-2 cells transfected with the control construct
(~40-fold). These findings indicate binding of ESE-1 to Site Y is
required for both basal and inducible MIP-3
gene expression in
intestinal epithelial cells. To confirm these data, we also examined
MIP-3
reporter gene activity in Caco-2 cells co-transfected with
either a pCI-ESE-1 expression vector or an empty control (pCI) vector.
In keeping with our other findings, overexpression of ESE-1 in Caco-2
cells significantly up-regulated basal and IL-1
-induced MIP-3
reporter activity ~3-fold (p < 0.001 for both)
compared with cells transfected with the pCI control construct (Fig.
8B).
View larger version (15K):
[in a new window]
Fig. 8.
The interaction of ESE-1 with its Site Y
binding element up-regulates MIP-3 gene
expression in Caco-2 cells. A, to determine whether the
ESE-1 binding element in Site Y can functionally regulate MIP-3
gene
expression, Caco-2 cells were transfected with a wild-type reporter
gene or a mutant construct carrying targeted substitutions at positions
148 and
149 of the promoter. Caco-2 cells were also co-transfected
with the pRL-TK control vector. 48 h later the cells were treated
with IL-1
(5 ng/ml) for 6 h. The activity of each construct is
presented relative to the non-stimulated activity of the wild-type
MIP-3
reporter gene and corrected for transfection efficiency. Data
are expressed as mean ± S.E. (n = 8).
,
p < 0.001 versus non-stimulated activity of
the wild-type construct; ***, p < 0.001 versus IL-1
-stimulated activity of the wild-type
construct. The shaded nucleotide sequence represents the
binding site for ESE-1. B, Caco-2 cells were co-transfected
with the wild-type MIP-3
reporter gene and either a pCI-ESE-1
expression vector or control vector (pCI). 48 h later the cells
were treated with IL-1
(5 ng/ml) for 6 h. Luciferase activities
are presented relative to the non-stimulated control activity of the
MIP-3
reporter gene and corrected for extract protein concentration.
Data are expressed as mean ± S.E. (n = 16).
, p < 0.001 versus non-stimulated
activity of the wild-type construct; ***, p < 0.001 versus IL-1
-stimulated activity of the wild-type
construct.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
promoter are required to regulate MIP-3
gene expression in IL-1
-treated Caco-2 human colonic epithelial cells: an NF-
B site (nucleotides
82 to
93); an Sp1 site
(nucleotides
63 to
69); and an ESE-1 site (nucleotides
143 to
154). Using wild-type and mutant MIP-3
reporter constructs we show
that the NF-
B binding element is a major regulator of MIP-3
gene
activation, because substitutions in this site substantially reduced
luciferase reporter gene activity and, importantly, blocked responses
to IL-1
. EMSA analysis indicated binding of p50/p65 NF-
B
heterodimers to the MIP-3
B site 30 min to 6 h after IL-1
treatment, consistent with previous reports of the kinetics of MIP-3
production by Caco-2 cells. In contrast, our reporter studies indicated
the Sp1 binding site appeared to primarily regulate basal MIP-3
gene transcription in Caco-2 cells. Overexpression of Sp1 increased basal
luciferase activity levels, whereas, site-directed substitutions in the
Sp1 element, CGCCTTC, significantly reduced reporter activity. Similar
to our findings with the NF-
B site, our reporter data suggest the
ESE-1 site is required for both basal and cytokine-induced gene
activation in Caco-2 cells. Overlapping scanning mutagenesis of the
Site Y region demonstrated the nucleotide sequence AAGCAGGAAGTT comprised the ESE-1 binding site. Specific mutations in this sequence markedly reduced MIP-3
gene expression. Conversely, overexpression of ESE-1 in Caco-2 cells up-regulated MIP-3
luciferase activity. Taken together, our data indicate that activation and binding of ESE-1,
Sp1, and NF-
B to the MIP-3
promoter is required for maximal gene
expression in cytokine-stimulated Caco-2 intestinal cells.
B to the MIP-3
promoter is requisite for
gene up-regulation. Izadpanah et al. (16) have reported that
infection of HT-29 cells with an adenoviral vector encoding a mutant
(non-degradable) I
B
protein can markedly inhibit cytokine-induced MIP-3
protein production. Moreover, MIP-3
gene expression in TNF
-stimulated G-361 human melanoma cells has been shown by Harant et al. (25) to require an intact NF-
B binding element.
These investigators also showed increased MIP-3
reporter gene
activity following overexpression of p65 NF-
B. In another study
Imaizumi et al. (26) reported that Tax, a 40-kDa protein
encoded by human T cell leukemia virus type-1, was capable of inducing
NF-
B-dependent MIP-3
gene expression in the human T
cell line JPX-9. Overexpression of Tax in JPX-9 cells was also found to
induce binding of p50/p65 NF-
B to the human MIP-3
promoter as
determined by EMSA. Finally, Fujiie et al. (27) have
reported that an NF-
B binding site in the murine MIP-3
promoter
is required for IL-1
or TNF
-induced gene expression in Caco-2 and
293T cells and that this element binds p50/p65 NF-
B heterodimers. In
previous studies from our laboratory we have shown that in active
inflammatory bowel disease primary colonic epithelial cell MIP-3
protein levels are significantly elevated compared with non-inflamed
enterocytes (15). Furthermore, increased p65 NF-
B nuclear
translocation has been reported in epithelial cells and lamina propria
macrophages from patients with active inflammatory bowel disease (40,
41). p65 antisense treatment can also ameliorate intestinal
inflammation in mice (42). Thus, p65-containing NF-
B is likely to be
an important regulator of the observed increases in enterocyte MIP-3
production in human IBD.
promoter contains a binding site for the zinc
finger nuclear factor Sp1, and that Sp1 can functionally regulate
MIP-3
gene expression in Caco-2 cells. First, the EMSA supershift
experiment shown in Fig. 5D clearly demonstrates that Sp1
and Sp3 in Caco-2 cell nuclear extracts can interact with a Site
X-specific probe in vitro. Second, site-directed mutations
in the Site X binding element that prevent binding of Sp1 and Sp3 by
EMSA are associated with markedly reduced MIP-3
luciferase activity
in Caco-2 cells. Finally, basal MIP-3
reporter gene activity is
significantly up-regulated in Caco-2 cells overexpressing Sp1, but not
Sp3. Functional Sp1 binding elements that primarily regulate basal gene
expression have been identified in the promoters of several other
chemokine genes, including Gro
, MCP-1, and RANTES (regulated on
activation normal T cell expressed and secreted) (39, 43, 44). In
addition, a previous study from our laboratory has shown that Sp1 and
ZBP-89 bind, in a mutually exclusive manner, to overlapping elements on
the ENA-78 promoter to regulate gene expression in IL-1-treated Caco-2
cells (31). Exactly how Sp1 regulates MIP-3
gene expression at the
molecular level cannot be elucidated from the present study. Several
reports indicate that post-translational modifications to Sp1
(e.g. phosphorylation and O-glycosylation)
regulate both its abundance and binding activity in cells (45). Indeed,
previous studies have shown increased Sp1 DNA binding activity,
phosphorylation, and protein levels in IL-1-stimulated synovial
fibroblasts and HepG2 cells (46, 47). Sp1 may also participate in
regulating MIP-3
gene expression through its demonstrated
interactions with a variety of other transcription factors including
NF-
B or co-activator proteins such as CRSP (48, 49). Moreover, Sp1
also appears to be able to directly interact with several elements of
the general transcription apparatus, including TATA box binding protein
(TBP) (50) and the TBP-associated factors hTAFII130 and hTAFII55 (51,
52).
promoter contains a binding element for the epithelially
expressed Ets nuclear factor ESE-1 and that ESE-1 can transactivate
MIP-3
promoter activity in Caco-2 cells. Ets-like binding elements
have been previously implicated in the regulation of other chemokine
genes, including PF4, PBP, RANTES, MIP-1
, and MCP-3 (44, 53-56).
The ubiquitously expressed factor Ets-1 was found to transactivate the
PF4 promoter, whereas, PBP gene expression was regulated by PU.1, an
Ets factor restricted primarily to cells of the immune system. For
RANTES, MIP-1
, and MCP-3 the specific Ets factor regulating gene
expression was not identified. Thus, to our knowledge, this is the
first demonstration that ESE-1 can regulate chemokine gene expression
in colonic epithelial cells or, indeed, in any other cell type.
, TNF
, and lipopolysaccharide. These authors also
showed that ESE-1 can interact directly with the p50 subunit of NF-
B
to synergistically transactivate the inducible nitric-oxide synthase gene promoter. This interaction with p50 NF-
B was
dependent on the presence of the ETS and A/T hook domains in ESE-1. The A/T hook domain, which is also found in the high mobility group proteins (e.g. HMG-I(Y)) (61, 62), is thought to alter
chromatin structure through interactions with A/T-rich DNA sequences.
HMG proteins are also known to facilitate gene expression via
interactions with other nuclear proteins, including NF-
B and ATF-2
(63). Thus, ESE-1 may contribute to MIP-3
gene expression via A/T
hook-mediated modifications to chromatin structure, the recruitment of
co-activator proteins such as p50 NF-
B, or both.
production in normal
colon and IBD (15). These findings suggest that expression of ESE-1 may
be required for epithelial MIP-3
gene expression in vivo.
Recently, the effect of targeted deletion of the ESE-1 (also designated
Elf-3) in mice has been reported by Ng et al. (64). These
authors found that ESE-1 deficiency was characterized by marked
alterations to the architecture of the small intestine. In particular,
there was poor villus formation and abnormal morphogenesis and
differentiation of absorptive enterocytes and goblet cells. Surprisingly, histologic examination of colonic tissues failed to
detect any gross abnormalities between wild-type and ESE-1-deficient mice. A potential explanation for this finding may be that the function
of ESE-1 is compensated for by other Ets family members (e.g. ESE-3, also known as Ehf) in the colon. In this study,
however, we found no evidence that ESE-3 interacts with the Site Y
region of the MIP-3
promoter. This is in agreement with a prior
study indicating that ESE-1 and ESE-3 differentially transactivate a variety of epithelial-specific gene promoters suggesting each of these
factors has distinct target gene specificities (65). In view of the
emerging role of ESE-1 as a novel transcriptional mediator of immune
and inflammatory response gene expression, it will be important to
characterize further the interactions between ESE-1, Sp1, and NF-
B
in human colonic enterocytes and to determine whether expression of
ESE-1 directs the predominant epithelial expression of MIP-3
in
normal and inflamed human colon.
![]() |
ACKNOWLEDGEMENT |
---|
We thank Dr. Timothy C. Wang for helpful comments and suggestions.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants DK-54920 and CA-76323 and by the Massachusetts General Hospital/Center for the Study of Inflammatory Bowel Diseases (Grant DK-43551).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/EBI Data Bank with accession number(s) AY150053.
§ Recipient of a Research Fellowship Award from the Crohn's and Colitis Foundation of America.
Recipient of a Career Development Award from the Crohn's and
Colitis Foundation of America. To whom correspondence should be
addressed: Division of Gastroenterology, Dana 501, Beth Israel Deaconess Medical Center, 330 Brookline Ave., Boston, MA 02215. Tel.:
617-667-1266; Fax: 617-667-2767; E-mail:
akeates@caregroup.harvard.edu.
Published, JBC Papers in Press, October 31, 2002, DOI 10.1074/jbc.M208241200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
IBD, inflammatory bowel disease;
IL, interleukin;
EMSA, electrophoretic
mobility shift assay;
TNF, tumor necrosis factor
;
RANTES, regulated on activation normal T cell expressed and secreted.
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REFERENCES |
---|
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