From the Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425
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ABSTRACT |
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Expression of the bradykinin B1
receptor gene is up-regulated in vascular smooth muscle cells (VSMCs)
in response to a variety of inflammatory stimuli. We isolated the
5-flanking region of the human bradykinin B1 receptor gene
and examined its promoter activity by transient transfection analysis.
This region (
2582 to +34) showed promoter activity inducible by
lipopolysaccharide (LPS), tumor necrosis factor
(TNF-
), and
interleukin-1
(IL-1
) in VSMCs. Further deletion analysis revealed
that constructs containing 111 base pairs of 5
-flanking sequence were
sufficient for transcriptional induction. Mutagenesis of a nuclear
factor
B (NF-
B)-like site at
64 to
55 abolished most of the
LPS, TNF-
, and IL-1
inducibility, whereas a mutation of a cyclic
AMP response element at
50 to
43 markedly reduced the basal
promoter activity, and a mutation of the activator protein 1 (AP-1)
site at
78 to
72 had minimal effects. Nuclear extracts from LPS,
TNF-
, and IL-1
-treated VSMCs, IL-1
-treated human hepatoma
HepG2, and human lung fibroblast IMR-90 cells showed strong
inducible binding activity to the NF-
B-like site by gel shift
assays. These results demonstrated that NF-
B-like nuclear factor was
involved in the inducible expression of the human bradykinin
B1 receptor gene during inflammatory processes.
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INTRODUCTION |
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Kinins are biologically active peptides that are formed locally after tissue damage and inflammatory stimuli from precursor kininogens by limited proteolysis (1). Kinins exert a broad spectrum of physiological and pathological effects, including smooth muscle contraction, vasodilation, increased vascular permeability, and pain induction, by binding to their cell surface receptors (2). Two subtypes of kinin receptors, B1 and B2, have been characterized on a pharmacological basis (3). Whereas the B2 receptor is responsive to the intact kinins, bradykinin (BK)1 and Lys-BK (kallidin), the B1 receptor has a higher affinity for the carboxypeptidase metabolites of kinins, des-Arg9-BK and des-Arg10-kallidin (4). Expression cloning of B1 and B2 receptor cDNAs confirms the existence of these two subtypes and reveals that they both belong to the G protein-coupled superfamily of receptors with seven transmembrane domains (5-11). Activation of the kinin receptors leads to generation of inositol phosphate and transient increases in intracellular Ca2+ levels via coupling to phospholipase C, involving, at least, the Gi, Gq/11, and G13 proteins (12-15).
The bradykinin B2 receptor is constitutively expressed in a variety of tissues and cultured cell lines and mediates most of the in vivo effects usually assigned to kinins (16). The bradykinin B1 receptor, however, is not present to any significant extent in normal tissues. It is expressed in a limited number of cultured cell types, such as embryonic lung fibroblasts, vascular smooth muscle cells, and endothelial cells (17-19). B1 receptor-mediated responses are up-regulated in a time and protein synthesis-dependent process (4). Recent studies in animal models of hyperalgesia and in B1 receptor knockout mice with induced peritoneal inflammation suggest that the B1 receptor may play an important role in the pathogenesis of chronic inflammatory diseases (20-22).
The genes encoding B1 receptors have been cloned from the
rat2 and human (23, 24). The
human B1 receptor gene was located close to the
B2 receptor gene within the same chromosomal region at
14q32 (23). The B1 receptor is induced following tissue
injury, following prolonged in vitro incubation of tissues,
or upon exposure in vivo or in vitro to
proinflammatory mediators, such as lipopolysaccharide (LPS) (4).
Whereas cytokines, including IL-1, IL-2, and IL-8 but not TNF-
or
IL-6, induced the B1 receptor, continuous incubation with
the glucocorticoid dexamethasone or the protein synthesis inhibitor
cycloheximide suppressed the induction in vitro (25, 26). To
understand the molecular mechanism underlying the regulated expression
of the B1 receptor gene, we isolated and characterized the
promoter of the human B1 receptor gene. In this study, we present evidence that nuclear factor
B (NF-
B) is involved in the
dynamic regulation of human B1 receptor gene expression.
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EXPERIMENTAL PROCEDURES |
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Cell Culture--
Rat vascular smooth muscle cells (VSMCs) were
explanted from a media layer of the thoracic aorta of male
Sprague-Dawley rats (200-250 g) according to the method of Biro
et al. (27) and maintained in Dulbecco's modified Eagle's
medium (Life Technologies, Inc.) supplemented with 10% fetal bovine
serum (HyClone, Logan, UT), 100 units/ml penicillin, 100 µg/ml
streptomycin sulfate, and 25 mM Hepes, pH 7.4. These cells
exhibit a "hill and valley" growth pattern and are characterized by
positive immunostaining with monoclonal antibodies against smooth
muscle -actin (28). Experiments using VSMCs were performed between
passages 3 and 10. Human hepatoma HepG2 cells were cultured
in Dulbecco's modified Eagle's medium containing 10% fetal bovine
serum and antibiotics. Human diploid lung fibroblasts, IMR-90 cells
obtained from ATCC (Rockville, MD) were cultured as described (7).
Bovine arterial endothelial cells (BAECs) from ATCC were grown in RPMI
1640 medium (Life Technologies, Inc.) supplemented with 20% fetal
bovine serum, 2 mM L-glutamine, and
antibiotics. LPS (Escherichia coli serotype 055:B5), sodium
salicylate, pyrrolidine dithiocarbamate, cycloheximide, and
dexamethasone were purchased from Sigma. Recombinant human IL-1
and
TNF-
were obtained from R&D Systems (Minneapolis, MN).
Cloning of the 5-Flanking Region of the Human B1
Receptor Gene--
A human lambda genomic library (Stratagene, La
Jolla, CA) was screened with an oligonucleotide probe, 5
-AGA AAA CTC
CTC CAA AAG CAG CTC TCA-3
, specific for exon I of the human bradykinin B1 receptor gene (GenBankTM accession no.
U30271) as described previously (11, 23). Positive clones were picked
for further characterization. Lambda phage DNA was digested and
subjected to Southern blotting to identify fragments containing exon I
and possibly the 5
-flanking sequence of the human B1
receptor gene, which were in turn subcloned into pBluescript KS II
vector (Stratagene) and sequenced on both strands using a dsDNA cycle
sequencing system (Life Technologies, Inc.).
Construction of Reporter Plasmids--
A 2.6-kb fragment
spanning nucleotides 2582 to +34 relative to the transcription start
site (29) was amplified by polymerase chain reaction (PCR) and cloned
upstream of a firefly luciferase gene in the promoterless plasmid
pGL2-Basic (Promega, Madison, WI). The resulting construct was named
p-2582Luc. The same fragment as above was also inserted into pGL2-Basic
in the reverse orientation, giving rise to p2582Luc. A series of
deletion constructs containing truncated 5
-flanking fragments of the
human B1 receptor gene was made by PCR. Constructs
containing these 5
-progressively removed fragments were named
p-1473Luc, p-825Luc, p-589Luc, p-251Luc, p-111Luc, and p-43Luc. Mutant
constructs were as follows (lowercase indicates mutation): p-2582mALuc,
with the AP-1 site (
78 to
72), mutated from AGACTCA to AGAgctc;
p-2582mNLuc, with 2 bp in the NF-
B-like site (
64 to
55), mutated
from GGCAATCCCC to ctCAATCCCC; and p-2582mCLuc, with the cAMP response
element (CRE) (
50 to
43), mutated from TGACATCA to TGctAgCA.
Mutants were created using a PCR site-directed mutagenesis using
p-2582Luc as the template (30). A double mutant construct,
p-2582mCNLuc, with both the CRE and NF-
B-like sites mutated as
above, was prepared with the same method using p-2582mNLuc as the PCR
template. Another construct, p1900Luc, with a 1.9-kb PCR-generated
fragment upstream of the translation start codon ATG as described
previously (23), was also produced. All constructs were sequenced to
confirm the product fidelity.
Transfection and Luciferase Assays--
Twenty-four hours before
transfection, BAECs, HepG2 cells, or VSMCs were seeded at a
concentration that would achieve 60-70% confluence in 12-well tissue
culture plates. Liposome-mediated cotransfection using a mixture of 400 µl of Opti-MEM, 4.8 µl of Lipofectin (for BAECs) or LipofectAMINE
(for HepG2 and VSMCs), 0.6 µg of the reporter construct,
and 0.2 µg of pCMV-gal (Clontech Laboratories, Inc.) (as a control
for measuring transfection efficiency) per well was carried out as
recommended by the manufacturer (Life Technologies, Inc.). The
transfection mixture was replaced with normal growth medium 6 h
later. Forty-eight hours after transfection, the cells were harvested.
The
-galactosidase activity in cell extracts was determined by an
enzymatic assay with chlorophenol red-
-D-galactopyranoside as a substrate, and the
luciferase activity was measured using a liquid scintillation counter
(Packard Instrument Co.) and the Luciferase Assay system (Promega)
according to the manufacturers' instructions. Luciferase activity in
each sample was normalized to that of
-galactosidase.
Reverse Transcription-PCR Southern Analysis--
Confluent cells
were preincubated with cycloheximide (CHX) and dexamethasone (DEX) for
1 h, and then stimulated for the specified time by IL-1,
TNF-
, or LPS and harvested. Total RNA was extracted with Trizol
reagent (Life Technologies, Inc.) or the method described by Gough (31)
and then treated with RNase-free DNase I for 1 h at 37 °C.
Reverse transcription was performed with 2 µg of total RNA and 100 pmol of random hexamers in a total volume of 20 µl to produce
first-strand cDNA. The quality of RNA samples was evaluated by
reverse transcription-PCR using
-actin-specific primers (sense primer, 5
-GAA CCC TAA GGC CAA CCG TG-3
; antisense primer, 5
-TGG CAT
AGA GGT CTT TAC GG-3
). The levels of BK B1 receptor
cDNAs were evaluated by PCR/Southern blot semiquantitative method.
PCR experiments were performed with 1 µl of the first-strand cDNA in a 50-µl reaction mixture. Rat B1 receptor cDNA was
amplified with the sense primer (5
-AAG ACA GCA GTC ACC ATC-3
) that
bound in the first exon and the antisense primer (5
-GAC AAA CAC CAG ATC GGA-3
) that bound in the second exon (GenBankTM
accession no. U66107).2 Human B1 receptor
cDNA was amplified with the sense primer (5
-AGA AAA CTC CTC CAA
AAG-3
) that bound in the first exon and the antisense primer (5
-GTA
GAT TTC TGC CAC GT-3
) that bound in the third exon (7). Amplification
protocol was as follows: denaturation at 94 °C for 60 s,
annealing at 55 °C for 50 s, and extension at 72 °C for
45 s. All PCRs were linear up to 25-30 cycles. PCR products were
resolved on 0.8% agarose gels and transferred to Hybond nylon filters
(Amersham Corp.). Hybridization conditions were the same as described
(23). Oligonucleotides 5
-AAG ACT GGG ACC TGC TGT AT-3
, 5
-TAC GGC CTG
TGA CAA TG-3
, and 5
-CGC ACG ATT TCC CTC TCA GC-3
were end-labeled
with [
-32P]ATP and T4 polynucleotide kinase (Life
Technologies, Inc.) and used as nested specific probes for rat and
human B1 receptor genes and
-actin, respectively.
Nuclear Extract Preparation--
Confluent cells were washed
once with serum-free medium and then treated with IL-1 (2 ng/ml),
TNF-
(10 ng/ml), or LPS (10 µg/ml) or left untreated as controls.
Nuclear proteins were then extracted by a modified method of Dignam
et al. (32). Briefly, after being washed with ice-cold
phosphate-buffered saline (PBS), cells were scraped into 1.2 ml of cold
PBS, pelleted, and resuspended in 500 µl of Buffer A (10 mM Hepes-KOH, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 0.2 mM
phenylmethylsulfonyl fluoride) by tapping the tube. The cells were
allowed to swell on ice for 10 min and vortexed vigorously for 10 s after adding 25 µl of 10% Nonidet P-40 (Sigma). The homogenate was
centrifuged for 10 s, and the nuclear pellet was resuspended in
100 µl of cold Buffer C (20 mM Hepes-KOH, pH 7.9, 25%
glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 0.2 mM phenylmethylsulfonyl fluoride) and incubated for 20 min
on ice with shaking. The nuclear extract was centrifuged at 14,000 × g for 5 min at 4 °C, and the supernatant was aliquoted
and stored at
80 °C. Protein concentrations were determined by the
Bradford method (Bio-Rad) (33).
Electrophoretic Mobility Shift Assay--
A double-stranded
oligonucleotide containing a wild type NF-B-like site (
73 to
50,
5
-CAC TTT TGC GGC AAT CCC CAC AAT-3
), was labeled with
[
-32P]ATP as a probe. Ten µg of nuclear extract was
incubated for 20 min at room temperature with 20,000 cpm of the
32P-labeled oligonucleotide probe in a total volume of 25 µl containing 12 mM Hepes, pH 7.9, 60 mM KCl,
1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride,
1 mM DTT, 2 µg poly(dI-dC), and 15% glycerol. For
competition assays, a 100-fold molar excess of unlabeled specific or
mutant competitors was added to the binding reactions and incubated for 10 min at room temperature before the probe was added. Protein-DNA complexes were resolved on a 4% native polyacryamide gel in
Tris/glycine/EDTA buffer at pH 8.5. The sequence of mutant
oligonucleotide competitor had two nucleotide variations (variations
shown in lowercase) in the NF-
B-like site: 5
-CAC TTT TGC ctC AAT
CCC CAC AAT-3
. The sequence of the competitor containing the NF-
B
motif from the promoter of IRF-1 transcription factor was 5
-TTC GGG
CCG GGG AAT CCC GCT AAG-3
(34).
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RESULTS |
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Induction of Bradykinin B1 Receptor Gene
Expression--
It has been shown that stimulation of human lung
fibroblast IMR-90 cells with IL-1 increased both the levels of the
B1 receptor mRNA and the number of B1
receptors (7, 17, 29). We examined whether expression of the
B1 receptor gene was induced in rat VSMCs and human
hepatoma HepG2 cells following IL-1
stimulation. Because
preliminary studies revealed that serum in the medium increased the
B1 receptor message, we washed cells once with serum-free Dulbecco's modified Eagle's medium before incubation with mediators in serum-free medium to eliminate the stimulatory effects of serum. The
B1 receptor mRNA level was low in unstimulated VSMCs,
but it was markedly increased by treatment with IL-1
for 3 h
(Fig. 1). In contrast, the same dose of
IL-1
failed to induce B1 receptor mRNA expression in
HepG2 cells after prolonged incubation (data not shown). We
next examined the levels of the B1 receptor mRNA in
VSMCs incubated with other stimuli and/or CHX and DEX. Proinflammatory agents LPS and TNF-
were also able to up-regulate the B1
receptor mRNA. The protein synthesis inhibitor CHX augmented the
effects of IL-1
and LPS, whereas CHX alone induced an increase in
B1 receptor message. The synthetic glucocorticoid DEX
reduced B1 receptor mRNA levels in unstimulated cells
and suppressed up-regulation by LPS. In contrast to the B1
receptor, the level of
-actin transcript remained the same
irrespective of the presence of various mediators (Fig. 1).
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Potential Regulatory Elements in the 5-Flanking Region of the
Human B1 Receptor Gene--
We previously cloned a
BamHI fragment of the human B1 receptor gene
containing the sole coding exon (23). The fragment of about 1.9 kb,
upstream of the translation start site ATG in the BamHI
fragment, was found to have a significant basal promoter activity in
HepG2 cells (23). In a preliminary study, however, we found
that the fragment was not able to confer IL-1
or LPS inducibility on
the reporter gene in VSMCs and HepG2 cells. As demonstrated
above, B1 receptor gene expression is up-regulated in
response to stimuli by LPS and IL-1
. Thus, the 1.9-kb fragment could
not represent the promoter for the B1 receptor gene, at least in VSMCs under inflammatory stimuli. Later, we and other investigators found that the human B1 receptor mRNA in
IMR-90 cells includes two noncoding exons, indicating that the 1.9-kb fragment is not the 5
-flanking region of the gene (24). In this study,
we isolated the 5
-flanking region of the B1 receptor gene
for elucidating the molecular mechanism of up-regulation of
B1 receptor expression. Sequence determination on both
strands was extended 1500 bp compared with the longest reported
sequence (24). In addition to the consensus TATA box, several other
potential transcription factor binding sites, including Sp1, AP-1,
NF-
B, and CRE, were found in the 5
-flanking region by computer
search (Fig. 2).
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Inducible Promoter Activity of the 5-Flanking Region of the Human
B1 Receptor Gene--
To assess whether the 5
-flanking
region of the B1 receptor gene has inducible promoter
activity, transient transfection was performed in VSMCs and
HepG2 cells. In VSMCs, LPS (10 µg/ml), IL-1
(2 ng/ml), and
TNF-
(10 ng/ml) increased 3-4-fold the luciferase activity of
p-2582Luc, which contained the longest 5
-flanking region (Fig.
3, A and B).
p1900Luc, with a basal promoter activity of about
of that of
p-2582Luc, showed no inducibility of the luciferase activity in the
presence of LPS, IL-1
, or TNF-
. The luciferase activity of
p-2582Luc in HepG2 cells was about
that of VSMCs
(with respect to promoterless pGL2-Basic) and was also inducible by
IL-1
with about 1.8-fold induction (Fig. 3C). On the
contrary, p1900Luc had basal promoter activity more than 8-fold higher
than p-2582Luc in HepG2 cells, although it was not
responsive to IL-1
. The plasmid p2582Luc, containing the 5
-flanking
sequence inserted in the reverse orientation, showed minimal luciferase
activity and was not responsive to IL-1
, TNF-
, and/or LPS in
VSMCs or HepG2 cells (data not shown).
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Cellular Specificity of the Human B1 Receptor
Gene Promoter--
A series of deletion constructs (Fig.
3A), including p1900Luc, were transfected into BAECs,
HepG2 cells, and VSMCs to investigate the basal cellular
specificity of the B1 receptor gene promoter. Due to
variations in transfection efficiency, it is difficult to make an
accurate comparison of the basal activities of the same construct in
different cells. We therefore compared the basal activities of the
series of deletion constructs within each cell line. In VSMCs and
BAECs, which are known to express B1 receptors (19), the
series of deletion constructs exhibited a similar profile of basal
activities (Fig. 5): the 5-flanking
region had higher basal promoter activity than the 1.9-kb fragment, and
the p-111Luc showed a level comparable (about 80%) to that observed with the p-2582Luc, implying that the upstream sequence of 2400 bp in
p-2582Luc has little contribution to its basal activity. However, in
HepG2 cells, the deletion constructs in the 5
-flanking region had a profile of relative basal activity different from those in
VSMCs and BAECs (Fig. 5). Constructs containing truncated 5
-flanking
sequences had significantly lower basal promoter activities than that
of p1900Luc, and the p-111Luc showed a higher basal level than
p-2582Luc, indicating negative regulatory elements residing between
2582 and
111.
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Identification of LPS-Responsive Elements--
To delineate
LPS-responsive elements within the 5-flanking region of the human
B1 receptor gene, transient transfection of VSMCs was
performed with the series of deletion constructs in the presence or
absence of LPS. Fig. 6 shows that the
induction of promoter activity upon stimulation with LPS was evident in all constructs except for p-47Luc. The level of induction by LPS in
p-111Luc was similar to all other constructs containing additional upstream sequences up to 2582 bp. The promoter activity of p-47Luc was
about 4.5% that of p-2582Luc and showed no induction with LPS
treatment. The data do not indicate that upstream elements are
LPS-responsive but rather that an LPS-responsive region located at
111 to
47 in the 5
-flanking portion mediates the LPS induction of
the promoter activity.
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NF-B-like Site Mediating the Induction by LPS and
IL-1
--
Fig. 2 shows the presence of a CRE site, an NF-
B-like
site and an AP-1 site in the LPS-responsive region. In addition, CHX superinduced B1 receptor mRNA, whereas PDTC, an NF-
B
activation inhibitor, reduced the LPS inducibility, suggesting the
involvement of NF-
B in the induction of B1 receptor gene
expression. To test this hypothesis, we performed electrophoretic
mobility shift assays. A probe spanning the promoter from
73 to
50
containing the wild type NF-
B-like site was used. A faint binding
activity was detectable in unstimulated VSMCs, and it was increased
upon stimulation by LPS, IL-1
, and TNF-
(Fig.
7A). The binding complexes
were entirely competed by cold probes and by an oligonucleotide
containing a canonical NF-
B site, but not by the mutant
oligonucleotide (Fig. 7B) or oligonucleotides containing the
C/EBP, OCT-1, NF/CTF, or AP-1 binding site (Stratagene) (data not
shown), indicating the specificity of the NF-
B-like binding. With
nuclear extracts from IMR-90 and HepG2 cells, the same
specific binding complexes induced by IL-1
were also observed,
indicating that the NF-
B-like site is also functional in IMR-90 and
HepG2 cells (Fig. 7A). To further determine the
functional importance of the NF-
B-like site, we mutagenized this
site in construct p-2582mNLuc. The sequence was changed from 5
-GGC AAT
CCC C-3
to 5
-CTC AAT CCC C-3
, identical to the mutant
oligonucleotide competitor used in the gel shift assay. When
p-2582mNLuc was transfected into VSMCs, mutation of the NF-
B-like
site almost abolished the induction of promoter activity by LPS,
IL-1
, and TNF-
(Fig. 8). A similar
result was obtained with HepG2 cells: the observed IL-1
inducibility of p-2582Luc was completely abrogated by the mutation
(data not shown), indicating that the NF-
B-like site appears to be
necessary for the induction by IL-1
. To support the key role of the
NF-
B-like site in the induction of B1 receptor gene
expression, we also mutagenized the CRE and AP-1 sites. The
p-2582m ALuc exhibited almost the same promoter activity as the
wild type p-2582Luc. On the other hand, the CRE mutant p-2582mCLuc was
still responsive, but with a diminished induction and at a lower basal
level. As expected, the double mutant p-2582mCNLuc was not responsive
to LPS and IL-1
and demonstrated a much lower basal activity (Fig. 8).
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DISCUSSION |
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Kinin receptors were classified into two subtypes, B1
and B2 (3). Whereas the B2 receptor is
constitutively expressed, the B1 receptor is highly induced
following tissue injury or inflammatory stimuli (4). However, the
molecular mechanism of this induction is not understood. In the present
study, we characterized the promoter of the human bradykinin
B1 receptor gene. We first showed that expression of the
bradykinin B1 receptor gene is inducible in primary
cultured rat vascular smooth muscle cells but not in human hepatoma
HepG2 cells. We then demonstrated that the 5-flanking region of the human B1 receptor gene, extending from
2582
to +34, conferred full responsiveness to LPS and IL-1
by transient transfection of the fragment-driven luciferase reporter gene. Functional analysis showed that a 111-bp sequence from the
transcription initiation site, which contained the NF-
B-like binding
site, was sufficient for the induction of transcription in VSMCs in response to LPS, IL-1
, and TNF-
. Exposure to LPS, IL-1
, and TNF-
increased the formation of NF-
B-like complexes with this element, as demonstrated by gel shift assays. In contrast, mutation of
this NF-
B-like site abolished most of the LPS, IL-1
, and TNF-
inducibility of the B1 receptor promoter construct in the same cells. Taken together, these data for the first time suggest that
the NF-
B-like nuclear factor is involved in the bradykinin B1 receptor gene induction process.
NF-B is a pleiotropic transcription factor involved in the
regulation of many genes implicated in the immune response and inflammatory processes (35). In resting cells, NF-
B is sequestered in the cytosol by association with inhibitory proteins of the I-
B
family. Stimulation by agents such as TNF-
, LPS, and IL-1
initiates a phosphorylation-dependent proteolytic
degradation of I-
B, allowing active NF-
B to translocate into the
nucleus and inducing transcription by binding to defined promoter
elements (35). The B1 receptor was suggested to play an
important role in the actions of kinins in chronic inflammation (4).
The involvement of NF-
B-like nuclear factor in the transcriptional
induction of the B1 receptor gene is consistent with the
role for NF-
B in the activation of genes mediating inflammation.
As noted, NF-B is critical for the inducible expression of genes
involved in inflammation, and we therefore tested the effects of
pyrrolidine dithiocarbamate and sodium salicylate, inhibitors of
NF-
B activation (34, 36). We did not observe apparent inhibitory
effects of both agents on the basal transcriptional activity of the
human B1 receptor gene promoter, but we did observe some
inhibitory effects on induction by LPS. This lack of effect might be
attributed to an NF-
B-like activity constitutively expressed or
induced by serum in VSMCs (37, 38), because PDTC and sodium salicylate
seemed to block activation of NF-
B only before stimulation, and they
did not alter the NF-
B binding activity (34, 36).
Glucocorticoids have been used as anti-inflammatory drugs (39). It was reported that the synthetic glucocorticoid dexamethasone inhibited the stimulant effect of LPS on the responsiveness to des-Arg9-BK in isolated rabbit aortic strips (25, 26). We examined the effect of dexamethasone on the induction of the B1 receptor in VSMCs. Dexamethasone decreased the expression of the B1 receptor mRNA under basal conditions, which is in agreement with the observation that continuous exposure to dexamethasone inhibited the development of the contractile response to B1 agonists in rabbit aortic strips and human umbilical vein rings (25, 40) and suppressed the LPS inducibility (see Fig. 1). Our transient transfection experiments demonstrated that dexamethasone down-regulated the basal transcriptional activity of the human B1 receptor gene promoter and inhibited the induction by LPS in VSMCs. Thus, dexamethasone inhibited, at least in part, B1 receptor expression at the transcriptional level.
Both in vitro and in vivo studies have suggested
that cytokines play an important role in the B1 receptor
induction process (4, 25, 26). Our study has shown that the protein
synthesis inhibitor cycloheximide enhanced B1 induction by
LPS and IL-1, indicating that the induction of the B1
receptor by LPS and IL-1
does not require new protein synthesis and
that IL-1 is not essential for the LPS induction, although VSMCs
produce IL-1 in response to endotoxin (41). The enhanced effect by
cycloheximide is generally attributed to superinduced NF-
B activity
due to blocking of the synthesis of the inhibitory protein I-
B (42).
In addition, our results showing that recombinant human TNF-
up-regulates B1 receptor gene expression and increases
NF-
B-like binding activity in VSMCs are in contradiction with the
finding that TNF-
had no effect on the spontaneous development of
the response to kinins in vitro (26). In addition to
potential species and experimental system differences, one hypothesis
that might explain the discrepancy is that TNF-
may have inhibitory
effects in the posttranscription process.
Early studies showed that the 1.9-kb fragment upstream of the
translation start codon ATG in exon III (which covers intron II, exon
II, and part of intron I) had a high basal promoter activity in
HepG2 cells based on CAT assays (23). On the basis of this finding, we inferred that this fragment might be the promoter for the
human B1 receptor gene. Based on their finding in
transformed IMR-90 cells, Yang and Polgar (29) thought that intron II
of the human B1 receptor gene might function as an
alternative promoter. We included this 1.9-kb fragment in the current
study, inserted upstream of the luciferase reporter instead of the CAT
reporter that was used previously (23). This fragment exhibited a
strong basal promoter activity in HepG2 cells, which was
6-8-fold higher than that of the 2582-bp 5-flanking promoter. It is
of interest to note that the basal promoter activity of this fragment
in VSMCs is 6-fold lower than that of the 2582-bp 5
-flanking promoter. Because the 5
-flanking promoter was able to confer IL-1
inducibility in both VSMCs and HepG2 cells and the 1.9-kb
fragment was not, it is not likely that the 1.9-kb fragment serves as
the promoter in VSMCs and HepG2 cells under inflammatory
conditions. Whether and under what conditions this 1.9-kb fragment or
part of it may function as an alternative promoter still needs further
investigation.
The NF-B-like site is functional in VSMCs as well as in IMR-90 and
HepG2 cells. IMR-90 cells are known to express endogenous B1 receptors under basal conditions (17). Because of the
low transfection efficiency, we did not use IMR-90 cells for the
transient transfection study. Whether the identified NF-
B-like site
mediates IL-1
inducibility of B1 receptor expression in
IMR-90 cells is not clear. Because we could not detect B1
receptor expression in HepG2 cells even when NF-
B-like
nuclear factor was activated, activation of NF-
B-like activity
seemed to be insufficient for B1 receptor expression, at
least in HepG2 cells. Some other (tissue-specific) factors
must be involved, especially under basal conditions. The participation
of serum in the medium in the regulation of B1 receptor expression makes the search more complicated. We identified an AP-1
site and a CRE in close proximity to the NF-
B-like site (Fig. 2).
The AP-1 site and the CRE seem to play a minor role in the inducibility
of the B1 receptor gene. Although the AP-1 site is less
functional, at least in VSMCs, the CRE seems to have an important role
in the basal transcriptional activity of the B1 receptor
gene promoter according to site-directed mutagenesis. However, we did
not observe any stimulatory effects of the cAMP analog, dibutyryl-cAMP,
on B1 receptor mRNA induction and on human B1 receptor gene promoter activity (data not shown). We
also failed to observe any complex formation using the CRE site as a
probe. Even so, we can not exclude the possibility that NF-
B may
interact with the AP-1 or CRE binding proteins as reported by
other investigators (43, 44).
In conclusion, we have described the role of NF-B-like nuclear
factor in the regulation of the inducible expression of the B1 receptor gene. Understanding the regulatory mechanisms
of the B1 receptor gene by LPS and IL-1
should provide
important insight for developing therapies to antagonize B1
receptor-mediated detrimental effects in inflammatory diseases.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants HL 29397 and HL 52196.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) AF032889.
To whom correspondence should be addressed: Dept. of
Biochemistry and Molecular Biology, Medical University of
South Carolina, 171 Ashley Ave., Charleston, SC 29425. Tel.:
803-792-4321; Fax: 803-792-1627.
1
The abbreviations used are: BK, bradykinin;
VSMC, vascular smooth muscle cell; IL, interleukin; TNF, tumor necrosis
factor; PCR, polymerase chain reaction; bp, base pair; kb, kilobase
pair; PDTC, pyrrolidine dithiocarbamate; LPS, lipopolysaccharide; BAEC, bovine arterial endothelial cell; NF-B, nuclear factor
B; CRE, cAMP response element; CHX, cycloheximide; DEX, dexamethasone.
2 K. X. Chai, A. Ni, J. Chao, and L. Chao, unpublished data.
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