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Vol. 55, Issue 2, 309-316, February 1999
Departments of Medicine and Pharmacology, Duke University Medical Center, Durham, North Carolina
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Summary |
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 Top
 Summary
 Introduction
Materials and methods
Results
Discussion
References
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The expression of the human A1 adenosine receptor gene is controlled by two promoters, promoters A and B, and they are located 600 base pairs apart. The characteristics of the two promoters differ by the activity of expression, tissue specificity, and the potential regulatory elements around them. Promoter A is more active but its expression is observed only in selected tissues, whereas promoter B is constitutively expressed but at much reduced levels. In Chinese hamster ovary (CHO) cells transiently transfected with plasmids containing either promoter linked to a reporter gene, dexamethasone (dex) can stimulate (or enhance) the expression of promoter B much more effectively than that of promoter A. Mutation and deletion studies on plasmids containing promoter B have shown that the stimulation is mediated through multiple regulatory sites, including a serum response element, AP1, and TATA box. However, a single-glucocorticoid response element monomer-binding site between promoters A and B does not have significant contribution to dex-regulated expression. The interactions between glucocorticoid receptor (GR) and some regulatory sites are probably occurring via this protein (GR) interacting with other DNA-binding proteins because there is no GR DNA-binding sequence in the sites studied. The stimulation can be eliminated by mifepristone, an antagonist of GR, indicating the involvement of GR in gene regulation. In addition, dex treatment also stimulated the expression of A1 adenosine receptors in CHO cells transfected with the plasmids containing contiguous genomic sequences of promoter B or promoters A and B linked to the receptor-coding sequence. When promoter A is active and both promoter A and B are present in a construct, dex treatment induced a much smaller percentage of stimulation.
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Introduction |
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Top
Summary
Introduction
Materials and methods
Results
Discussion
References
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The
glucocorticoid family of steroid hormones affects transcriptional
expression of many genes through interaction with its intracellular
receptor, the glucocorticoid receptor (GR). Upon binding to the
receptor, the complex can enter the nucleus and bind to the appropriate
DNA sequence or other factors to regulate gene expression (McEwan et
al., 1997
). GR belongs to a superfamily of nuclear receptor
transcription factors that share a common molecular structure
organization containing a ligand-binding domain at the C terminus and a
DNA-binding domain at the N-terminal side of the ligand-binding domain.
The receptor-binding sequence on the target DNA molecule, known as
glucocorticoid response element (GRE) is a hexamer (TGTTCT) for
monomer-binding or a 15-base pair (bp) sequence for homodimer binding
(Chalepakis et al.,1990). The sequences flanking the GRE, although not
conserved, are also important for GR binding. Besides affecting gene
expression through direct binding to GRE, the receptor (GR) may also
exert its influence through protein-protein interaction with other
transcription factors (McEwan et al., 1997). Depending on the type and
target of the GR interaction, it can up- or down-regulate the affected
gene expression (Diamond et al., 1990).
The expression of human A1 adenosine receptor
(A1AR) gene is controlled by two separate
promoters, promoter A and promoter B, which are about 600 bp apart (Ren
and Stiles, 1995). Promoter B and exon 1B are part of intron 1A when
promoter A is active. Computer analysis of the sequence surrounding
both promoters revealed only one GRE monomer-binding site between
promoter A and B. There is also an AP1 site within promoter B region
that has been shown to be a target of GR interaction (Jonat et al.,
1990; Teurich and Angel, 1995; Garlow and Ciaranello, 1995).
In DDT1 MF-2 smooth muscle cells, treatment with
the synthetic glucocorticoid dexamethasone (dex) caused increased
expression of A1ARs, whereas the adenosine
A2 receptor was down-regulated (Gerwins and
Fredholm, 1991). Similar results were also obtained in rats wherein dex
treatment of adrenalectomized rats showed a marked increase in
A1AR but not A2aAR in brain
tissue (Svenningsson and Fredholm, 1997). In this report, we present
data showing that dex increases the transcriptional expression of
luciferase reporter gene directed by the human
A1AR promoter and that the stimulation can be
eliminated by the GR antagonist mifepristone (RU486), indicating direct
GR involvement. Dex stimulation of gene expression is much more
effective through promoter B than promoter A. Mutation and deletion
studies showed that the dex stimulation involves multiple nuclear-binding sites. We also document that the ability of dex to
stimulate the enhanced production of A1AR is
dependent on whether one or both of the promoters and intervening
sequences are present in the construct transfected into Chinese hamster
ovary (CHO) cells. The A1AR expression was
enhanced when only promoter B was present. But when both promoters were
present and active in the same plasmid, dex was much less effective.
This has direct implication as to why glucocorticoid-stimulated
A1AR response is tissue-specific.
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Materials and Methods |
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Top
Summary
Introduction
Materials and methods
Results
Discussion
References
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Cell Culture and Transfection. The CHO cells do not express any A1ARs and they were used in our previous study of the receptor promoter activity because the promoter constructs transfected into CHO cells showed higher activity than those transfected into other cells. The transfected CHO cells also showed good response to the dex treatment. Therefore, CHO cells were chosen in this report to study the effect of dex on promoter activity.
CHO cells were grown in Dulbecco's modified Eagle's medium/F-12 medium (catalog no. 11330-032, Gibco Laboratories, Gaithersburg, MD) plus 10% fetal bovine serum in a 37°C incubator containing 5% CO2. For luciferase reporter gene plasmid transfection, cells were transferred to 6-well plates the day before transfection at about 5.2 × 105/well cell density. Transient transfection using lipofectamine (catalog no. 18324-012, Gibco Laboratories) was carried out according to the manufacturer's instruction. For the plasmids with luciferase reporter gene, 10 ng/well plasmid pSV
containing -galactosidase gene was
cotransfected to normalize the transfection efficiency differences
among samples. Since the optimal amount of plasmid DNA used in the
transfection experiment is about 1 µg/well, the plasmid Pmt B/
422
and its base substitution mutants were transfected into cells with this
amount and the equimolar amount of deletion mutants were used. The
total amount of plasmid DNA for deletion mutants was held constant by
adding empty pCMV5 vector which has a similar size. Equimolar amounts
of plasmids containing promoter A were also used in the transfection
experiments. Forty-eight hours after transfection, cells were washed
twice with serum-free medium (2 ml/well) and incubation was continued
in the serum-free medium (Dulbecco's modified Eagle's medium/F-12)
plus 0.1% bovine serum albumin (catalog no. A-8806, Sigma, St. Louis,
MO) with or without 100 nM dex (Sigma). Some samples were treated with both dex and RU486 (500 nM, Sigma).
Luciferase and -Galactosidase Assays.
Twenty-four hours
after the dex treatment, cells were processed for luciferase and
-galactosidase assays according to Promega's protocol for
luciferase assay system with reporter lysis buffer. For each well of
cells, 0.2 ml of 1× lysis buffer was used. Luciferase activity was
measured as luminescence with a BioOrbit 1251 luminometer (Pharmacia
LKB, Piscataway, NJ). The activity of -galactosidase was measured
according to the standard method (Ausubel et al., 1987). The luciferase
activity from different samples was normalized by -galactosidase
activity obtained from the same quantity of sample. Each treatment had
triplicate samples and all experiments were repeated at least three
times. Within each experiment, the promoterless vector pBLPniF (a gift
from Dr. W. E. Kraus, Duke University Medical Center) was also
transfected into CHO cells as a background control. The promoter
activity for each construct was calculated by subtracting the
background activity from the total activity and then dividing by the
activity with the promoterless construct. Therefore, the background
luciferase activity obtained from the promoterless plasmid pBLPniF was
set as one and in this way the variations between experiments were
minimized. The results are presented as fold expression.
[3H]-8-cyclopentyl-1,3-dipropylxanthine
(DPCPX) Binding Assay.
The CHO cells transfected with
plasmids PmtB-A1AR, PmtAB-A1AR, pCMV5/huA1, and
pCMV5/Ex4-6 were processed for receptor-binding assay according to the
protocol described previously (Olah et al., 1992) with the radioligand
[3H]DPCPX (NEN Life Science Products, Boston, MA) at 8 nM.
Total RNA Isolation and RNA Slot-Blot. About 72 h after transfection (24 h after dex treatment), the cell culture medium was removed from the transfected CHO cells and 7 ml of Trizol reagent (Gibco) was added to extract total RNA following the manufacturer's instruction. The pelleted total RNA was resuspended in water and A260 was used to estimate RNA concentration.
The RNA slot-blot procedure follows the standard protocol (Ausubel et al., 1987). After UV cross-linking, the membrane was prehybridized in
5× SSC (0.75 M NaCl and 0.075 M sodium citrate), 5×
Denhardt's solution, 50% formamide, 1% SDS, and 100 µg/ml yeast tRNA at 60°C for 3 h. The full-length purified RNA probe was
added to the hybridization solution and incubation continued overnight at 60°C. The following day, the membranes were washed once with 2×
SSC, 0.1% SDS at room temperature, twice with 0.2× SSC, 0.1% SDS at
room temperature and twice with 0.1× SSC/0.1% SDS at 60°C. Each
wash lasted about 15 min. The membranes were then sealed in a plastic
bag and exposed to film. The bands on the developed film were scanned
using the densitometer (Bio-Rad model 620 densitometer; Bio-Rad,
Hercules, CA) to estimate the amount of mRNA. The experiments were
repeated three times and the average increase are presented in
Results.
RNA Probe Synthesis.
The DNA template for RNA probe
synthesis was created by polymerase chain reaction (PCR) using a 3' end
primer containing a sequence of T7 promoter which would direct the
synthesis of an antisense RNA. The template for probe B-luc was
amplified based on the plasmid PmtB/422, which contained a fraction
of luciferase coding sequence plus a fraction of promoter B insert in
the plasmid.
Plasmid Construction.
Expression plasmids containing human
A1AR gene promoter A or promoter B or both were constructed
based on similar procedures described previously (Ren and Stiles,
1995). Some plasmids have been used in previous studies such as
PmtA/897 (=pBLPniF/PmtA), PmtA/253, PmtB/422 (=pBLPniF/PmtB),
PmtB/129, PmtB/29, PMTBMUT (Ren and Stiles, 1995), pCMV5/huA1 (Ren
and Stiles, 1994a), and pCMV5/Ex1b3 (=pCMV5/Ex4-6; Ren and Stiles,
1994b). The following list of plasmids were constructed during this study.
1032 nucleotides of the
transcription start site of promoter A and ended at the SmaI
site of the A1 receptor-coding sequence. The
downstream fragment was amplified from the cDNA clone 7A (Ren and
Stiles, 1994) with the 3' primer containing a poly(A) signal at the end
of coding sequence to skip the 3' untranslated sequence. The two
fragments were ligated into the digested plasmid.
The plasmid PmtB-A1AR has the same insert as
PmtAB-A1AR except that the 5' end of the insert
is at 422 of promoter B. Therefore, the promoter A is excluded from
the plasmid.
The plasmids with extended 5' flanking sequence (PmtA/3180) of
promoter A were constructed with PCR fragments amplified from genomic
sequence and replaced the insert of Pmt A/897.
The plasmids containing mutations at GRE, serum response element (SRE),
AP1, and TATA box sites were constructed with PCR fragments according
to the protocol described previously (Ren and Stiles, 1995). The
selected mutants were sequenced to confirm the mutation. The sequence
comparisons between wild type and mutants are illustrated in Table
1.
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Results |
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Top
Summary
Introduction
Materials and methods
Results
Discussion
References
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Dex Stimulates the Expression of a Luciferase Reporter Gene
Controlled by the Human A1AR Promoter.
The promoters
of the human A1AR gene are organized in such a way that
promoter B is about 600 bp downstream of promoter A and that it is part
of intron 1A (Fig. 1). By examining the
available 5' flanking sequences for both promoters, only one GRE
monomer-binding site was discovered about 379 bp upstream from the
promoter B transcription start site and 221 bp downstream from the
promoter A transcription start site. The AP1 site within the promoter B area has been demonstrated to be important for the basal activity of
promoter B (Ren and Stiles, 1995). There are two SREs upstream of
promoter B, SRE-1 at 113/106 and SRE-2 at 69/62. The sequences at the two SREs are almost identical and in the same orientation (Fig.
1).
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422 (=pBLPniF/PmtB)
is at 422 of promoter B and the 5' ends of the inserts for a group of
plasmids containing promoter A are extended a variety of distances up
to 3180 nucleotides of the promoter A transcription start site
(PmtA/253, PmtA/897, and PmtA/3180). When those plasmids were
transfected into CHO cells and treated with 100 nM dex in serum-free
medium for 24 h 2 days after transfection, the luciferase
expression when driven by promoter B alone increased about 88%
compared with the untreated cells. However, the luciferase expression
when driven by promoter A (PmtA/897) had little change when treated
with dex (Fig. 2). When the 5' flanking
sequence of promoter A was extended to about 3 kb (PmtA/'3180),
promoter A expression had only a 10% increase in activity. Although
the absolute increase in activity by dex treatment in promoter A is about the same as that in promoter B, the percentage of change of
activity in promoter B is much greater than that in promoter A because
the basal level of promoter A is much higher than that of promoter B. This suggests that dex regulation of human A1AR gene expression affects the transcriptional activity of promoter B much
more than that of promoter A when the promoters are present separately.
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422 showed that the transcript from
promoter B increased by about 45% after dex treatment but the
stimulation was reduced when RU486, a GR antagonist, was included with
dex (Fig. 3). Although the increase of
mRNA is slightly smaller than the increase in luciferase activity after
dex treatment, this is direct evidence of transcriptional regulation of
A1AR gene expression by dex.
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The DNA-Binding Sites SRE, AP1, and TATA Box are Responsible For
Most of the Dex-Stimulated Promoter Activity.
Because there is one
GRE site upstream of promoter B, the previously used deletion mutant
PmtB/129 was used to determine whether it is responsible for the
dex-stimulated expression (Fig. 4A). The
results showed that the basal activity of promoter B doubled,
indicating a possible involvement of an inhibitory element in the
sequence segment and that this sequence including the GRE monomer-binding site accounts for only a small part of the stimulation. In addition, the base substitution mutant PmtB/GRE showed a reduction in basal level expression so the percent increase due to dex-mediated stimulation did not change. Thus, this GRE monomer-binding site probably does not have any significant contribution to the
dex-stimulated promoter activity and other site(s) must be involved in
the mechanism.
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39) significantly reduced the
effect of dex (PmtB/39 in Fig. 4A). Mutations were also made at these
two sites separately to determine which SRE is more effective (Table 1
and Fig. 4B). The base substitution mutation at SRE-1 alone (PmtB/SRE1)
had little effect on the dex-stimulated activity but the same base
substitution mutation at SRE-2 alone caused almost complete elimination
of basal activity (data not shown), indicating that this site is
important for promoter B basal activity. To evaluate the role of SRE-2
on the effect of dex treatment, a different mutant (PmtB/SRE2) was
selected (Table 1). The new mutation was made at nonconserved bases of
SRE and caused a slight increase of basal activity but significantly
reduced the dex-stimulated activity (Fig. 4B). This result indicates
that SRE-2 is partially responsible for GR stimulation. When the AP1 site alone was mutated (PmtB/AP1 MUT in Fig. 4B), the basal activity was significantly reduced and the reduction of dex stimulation also
occurred but percentage-wise not as much as SRE-2 mutant. The double
mutant PmtBMUT has both the TATA box and AP1 sites mutated. In this
case, the basal activity of promoter B was greatly reduced and the
dex-stimulated activity was not significant, indicating the involvement
of TATA box in the process (Fig. 4B). The base substitution mutant
GSAMUT has all three sites GRE, SRE-2, and AP1 mutated and the result
showed that a major part of the dex-stimulated expression was then
eliminated. However, a noticeable amount of dex stimulation was still
present with GSAMUT-transfected cells. In contrast, the 5' deletion
mutant PmtB/24 (Fig. 4A), which had all of the sequences 5' to the
AP1 removed, showed essentially no dex stimulation although it has the
intact AP1 site, again indicating the role of the TATA box in the
mechanism. Finally, when all four sites, GRE, SRE-2, TATA box, and AP1,
were mutated (PmtB/GSTAMUT), no significant level of the dex-mediated
stimulation of promoter B activity was observed even though the mutant
plasmid had the same length insert as the wild-type PmtB/422. This
evidence suggests that dex stimulation of A1AR
gene promoter B expression is through multiple sites and probably
involves general transcription assembly.
Dex Treatment also Stimulates A1AR Gene Expression as Measured by [3H]DPCPX Binding. Because human A1AR gene expression is controlled by two separate promoters, the relationship between the two promoters will determine how mRNA and receptor are expressed. To study the expression pattern of promoters A and B in a more physiological setting, two plasmids PmtAB-A1AR and PmtB-A1AR with inserts containing genomic sequence of either promoter B alone or both promoters A and B together along with the first exons, introns plus the receptor-coding sequence were constructed to transfect CHO cells. The transcripts produced from these plasmids would have to be processed to excise the intervening introns. The results in Fig. 5A showed that the A1 receptor-binding activity was stimulated by dex treatment and that this stimulation was eliminated by RU486, a GR antagonist. When the PmtAB-A1AR-transfected cells were treated with dex, only a small increase of the receptor-binding activity was observed (Fig. 5B) when both promoters were active as determined by reverse transcription-PCR (data not shown). At the same time, the pCMV5/huA1-transfected cells were used as a negative control and showed no response to dex treatment. The results show that dex stimulation of promoter B works on both luciferase reporter gene expression and the expression of A1ARs and that the stimulation is indeed regulated by GR. The results also show that when promoter A is active, the dex treatment is much less effective. In addition, when the CHO cells transfected with the plasmid pCMV5/Ex1b-3 were treated with dex, no stimulation of receptor expression was observed (data not shown) although the transcript produced by this plasmid is about the same as PmtB-A1AR, indicating that the dex stimulation is promoter-dependent but not related to translation.
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Discussion |
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Top
Summary
Introduction
Materials and methods
Results
Discussion
References
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The expression of the human A1AR gene is
controlled by two separate promoters and the transcripts they produce
differ only by the first nontranslated exons. When promoter A is
active, promoter B and exon 1B are part of intron 1A and are spliced
out after transcription. In addition, the transcriptional activity of
promoter A is much higher than that of promoter B when they were tested separately (Ren and Stiles, 1995) and the reason for that is not clear.
Because the sequences surrounding the two promoters are quite
different, their regulation patterns may also be different. For
example, a purine-rich sequence at the transcription start site of
promoter A is very important for the promoter activity and binds to
nuclear protein(s) preferentially in single-stranded form (Ren and
Stiles, 1998). This sequence is not present in promoter B. Instead an
AP1 site next to the transcription start site of promoter B and an SRE
at 69/62 are important for the promoter B activity. Moreover, both
promoters A and B have a nuclear factor 
B (NFB) site located
upstream of the promoters. The NFB site upstream of promoter A is
involved in the stress-related stimulation of
A1AR expression, whereas the NFB site upstream
of promoter B has no effect (Nie et al., 1998).
Because glucocorticoid (dex) stimulation of A1AR
expression has been reported in DDT1 MF-2 smooth
muscle cells (Gerwins and Fredholm, 1991) and rat brain (Svenningsson
and Fredholm, 1997), it was important to learn how the human
A1AR gene responds to dex treatment at the
molecular level. A computer search of the 5' flanking sequences for
both promoters revealed only one GR monomer-binding site between
promoters A and B. Although the homodimer of GR seems to be the most
efficient binding format for GR function (Chalepakis et al., 1990), the
GR monomer binding can also affect gene expression. Our results show
that although the deletion of a sequence segment including the GRE at
379/372 of promoter B reduced the level of dex stimulation more
than the base substitution mutation at GRE (Fig. 4), this GRE
monomer-binding site does not contribute a significant amount of
GR-regulated activity increase. Since a major part of the dex
stimulation was still present after GRE mutation or deletion, other
transcription factors and/or sites may interact with GR without GRE
involvement. This type of protein-protein interaction has played a
major role in hormone-mediated transcriptional regulation involving
nuclear receptor transcription factors including GR (McEwan et al.,
1997). A primary target of GR interaction in promoter B is the AP1
site, an interaction that has been reported in many studies (Diamond et
al., 1990; Jonat et al., 1990; Garlow and Ciaranello, 1995; Teurich and
Angel, 1995). When the AP1 site alone was mutated (PmtB/AP1 MUT in Fig.
4B), a small percentage of reduction of dex stimulation was observed
after a drop of basal activity (Fig. 4B). This result may indicate a
partial role for the AP1 site in the GR-mediated increase in
transcriptional activity. When both the TATA box and AP1 site, which
are in close proximity and are the major regulatory components of
promoter B, were mutated (PmtBMUT in Fig. 4B), dex stimulation was
further reduced with a similar level of basal activity as that of
PmtB/AP1 MUT. This result may suggest the interaction of GR with the
general transcription initiation complex associated with the TATA box.
In human osteocalcin gene promoter, the GR-binding sequence and TATA
box sequence overlap. Thus, the competitive binding of GR at the TATA
box caused negative regulation of expression (Meyer et al., 1997). The
interaction between GR and TATA box in the human
A1AR promoter B could be protein-to-protein
because there is no GR-binding site involved.
The results from deletion mutants (PmtB/129 and PmtB/39) in Fig. 4A
indicate that the sequence between 39 and 29 of promoter B caused a
reduction of dex stimulation. Within this area, there are two serum
response elements SRE-1 (113/106) and SRE-2 (69/62). When they
were individually mutated to test which one caused the reduction of dex
stimulation, mutations in SRE-1 resulted in a slight increase of basal
activity but little change in dex-mediated stimulation (Fig. 4B),
whereas the same sequence mutation in SRE-2 caused a complete
elimination of basal activity of promoter B (data not shown). This SRE
site is important to the basal activity of promoter B as we indicated
in our previous study (Ren and Stiles, 1995). To study the effect of
dex on gene expression, a reasonable level of basal activity is needed.
Thus, we changed the sequence mutation in SRE-2 in such a way that only
the variable positions in the consensus sequence were mutated. A
mutant, PmtB/SRE2, so selected showed a close to 50% decrease in dex
stimulation. Glucocorticoid regulation of gene expression through
direct binding on SRE has been reported in c-fos oncogene
promoter (Karagianni and Tsawdaroglou, 1994), in which GR binding
repressed SRE-dependent gene activation. The results in our study
showed that the two almost identical SREs upstream of promoter B
responded to dex treatment differently. SRE-1 has little effect but
SRE-2 affects both basal and dex-stimulated activity. Whether GR
affects A1AR expression through direct binding to
SRE-2 or through interaction with other transcription factors such as a
serum response factor is not yet known.
According to the deletion and mutation studies, the SRE-2 and TATA box seem to be responsible for most of increase in the dex-stimulated activity, whereas the GRE monomer-binding site does not have much influence. The AP1 site is important for the promoter activity and is probably involved in the dex-mediated stimulation due to its location between the transcription start site and TATA box.
Because the transcription of promoters A and B seems to be regulated by
different mechanisms, the relationship between the two promoters is
important to the expression of the A1AR gene. In
our previous study we have found that the transcript from promoter A is
only present in certain tissues, whereas the transcript from promoter B
is present in all of the tissues that express
A1AR (Ren and Stiles, 1994a). Most of our work
had tested the promoters in separate plasmids linked to a reporter
gene. Therefore, it would be important to learn the effect of dex on
the expression of A1AR when the "complete"
gene with its natural genomic sequence including introns was expressed.
The inserts of PmtAB-A1AR and PmtB-A1AR contain the continuous genomic sequence
of the A1AR gene except intron 2, which disrupts
the coding sequence; also, the 3' untranslated sequence was
removed. The CHO cells transfected with these plasmids express
A1AR as measured by the radioligand-binding assay
(Fig. 5). The results clearly demonstrated the stimulatory effect of
dex on the expression of A1AR and the involvement
of GR by RU486 reversal of the effect. In addition, the results also showed that dex treatment was most effective on the expression of
promoter B when promoter A was not present. Therefore, in the tissues
where promoter A is not active, dex treatment may be more effective.
The effect of GR regulation can be positive, such as that seen in the
human A1AR and rat serotonin-2 receptor
(Garlow and Ciaranello, 1995) or negative (Goodman et al., 1996; Pei,
1996) depending on the binding sites and cell types. In addition,
multiple binding sites are involved in GR-regulated expression in
certain cases such as the human A1AR promoter B
and rat insulin-like growth factor binding protein-1 promoter (Suh et
al., 1996).
In summary, the results of this study show that dex can stimulate the human A1AR promoter B expression through multiple regulatory elements. The effect is mediated by GR and probably by protein-protein interaction (Fig. 6). We, of course, can not rule out that the dex also influences other genes that consequently have an effect on A1AR expression. The expression from promoter A appears to be much less affected by the dex treatment. It appears that glucocorticoid responsiveness is complex and requires multiple DNA sites and will be tissue-specific depending on whether or not promoter A is expressed.
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Footnotes |
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Received July 6, 1998; Accepted November 10, 1998
This study was supported by National Heart, Lung and Blood Institute Specialized Center of Research Grant 5P50HL54314 in Ischemic Disease (G.L.S.).
Send reprint requests to: Gary L. Stiles, M.D., Duke University Medical Center, Box 3444, Durham, NC 27710. E-mail: glsmd{at}duke.edu
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Abbreviations |
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A1AR, A1 adenosine receptor; dex, dexamethasone; GR, glucocorticoid receptor; DPCPX, 8-cyclopentyl-1,3-dipropylxanthine; RU486, mifepristone; SRE, serum response element; GRE, glucocorticoid response element; CHO, Chinese hamster ovary; PCR, polymerase chain reaction.
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References |
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Top
Summary
Introduction
Materials and methods
Results
Discussion
References
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B.
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3).
