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INTRODUCTION |
The glucocorticoid receptor
(GR)1 belongs to the family
of intracellular ligand-inducible transcription factors termed the steroid/vitamin D/retinoic acid superfamily (1). All members of this
superfamily share essential structural and functional features, which
are an amino-terminal transactivation domain, a central zinc-finger DNA
binding domain, and a carboxyl-terminal ligand binding domain.
Unliganded GR is present in the cytosol and is associated with a large
multiprotein complex of chaperones, including heat shock proteins
Hsp90, Hsp70, and Hsp56 (1-5). This conformation is essential for
allowing GR to bind steroid ligands with high affinity. Ligand binding
induces a conformational change of the complex leading to dissociation
of the chaperones. Activated GR then translocates to the nucleus and
binds to its specific target DNA sequences termed the glucocorticoid
response elements (GRE) (5, 6). Depending on the structure of the affected gene promoter, glucocorticoids thereby lead to either increased or decreased gene transcription (6-8). Biological effects of
glucocorticoids on most human cell types are generally
anti-inflammatory, characterized by decreased expression of
inflammatory mediators or increased expression of protective mediators
(1-4).
A frequent inflammatory disease that requires the administration of
glucocorticoids is asthma (9, 10). In asthma, treatment regimen
combining glucocorticoids with
2-agonists results in better symptom control and lesser airway inflammation than simply increasing the dose of glucocorticoids (11-13). These clinical observations strongly suggest an interaction of both classes of drugs
at a molecular level. In contrast to GR, a soluble intracellular receptor that resides in the cytosolic compartment when unliganded (1-4), the
2-adrenergic receptor (
2-AR)
constitutes a plasma membrane-anchored, G-protein-coupled receptor with
seven transmembrane spanning domains (14, 15). The
2-AR
is highly expressed in fibroblasts, vascular smooth muscle cells
(USMCs), and epithelial cells of the human lung (14, 16). Signal
transduction by
2-AR occurs upon ligand binding via
activation of adenylate cyclase, which increases the concentration of
intracellular cAMP (14). In addition to the short term effects of
2-AR agonists, such as VSMC relaxation (17),
considerable evidence suggests that
2-AR agonists have
potent anti-inflammatory effects in vitro and in
vivo (11, 18-20). Because activation of GR is essentially responsible for the majority of anti-inflammatory effects, we thus
addressed the question of whether
2-agonists could lead to activation of GR in a ligand-independent manner.
In the present study, cultures of primary human lung fibroblasts and
vascular smooth muscle cells were used to demonstrate that both
glucocorticoids and
2-AR agonists activate the human GR.
We show that the kinetics of GR activation are similar for both
substances, leading to rapid nuclear translocation of functional activated GR after 30 min. Compared with
2-AR agonists,
the endogenous ligands for GR, glucocorticoids, led to complete
depletion of GR from the cytosolic compartment. Activation of GR by
2-AR agonists was mediated by the interaction of the
drugs with the
2-AR because a
2-AR
antagonist, propranolol, abolished the observed activation of GR in a
concentration-dependent manner. Similarly to the effect of
the drugs on GR activation, addition of dibutyryl-cAMP or 8-bromo-cAMP activated the GR, suggesting that the action of
2-AR on
GR activation involves the adenylate cyclase/cAMP pathway. Thus, our
results suggest that ligand-independent activation of GR by
2-AR agonists may be an essential mechanism contributing
to the anti-inflammatory effects evoked by
2-agonists.
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EXPERIMENTAL PROCEDURES |
Materials--
Unless otherwise specified, all reagents were
purchased from Sigma (Buchs, Switzerland). Cell culture media and
additives were purchased from Fakola/Seromed (Basel, Switzerland).
Dexamethasone, fluticasone, salbutamol, salmeterol, and
propranolol were provided by Glaxo Wellcome (Middlesex, UK). Antibodies
to human GR and
2-AR were purchased from Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA). Reagents for avidin/biotin
immunoperoxidase staining were purchased from Vector Laboratories
(Burlingame, CA). Premixed proteinase inhibitors
(CompleteTM) were from Boehringer Mannheim AG (Rotkreuz,
Switzerland). Reagents used for luciferase reporter gene assay were
obtained from Promega (Wallisellen, Switzerland). Gradient SDS gels
were purchased from Bio-Rad (Glattbrugg, Switzerland). Polyvinylidene
diflouride protein transfer membranes were obtained from Millipore
(Bedford, MA). [
-32P]ATP and ECL visualization liquid
was purchased from Amersham (Zürich, Switzerland).
Cell Culture--
Primary cell lines of fibroblasts
(n = 3) or VSMC (n = 3) were
established from human lung tissue biopsies obtained from patients undergoing lobectomy or pneumectomy, as described previously (21). Fibroblasts were cultivated in RPMI 1640 supplemented with 10% FCS, 8 mM L-glutamine, and 20 mM HEPES.
VSMC were cultivated in minimal essential medium, supplemented with 5%
FCS, 8 mM L-glutamine, 1% minimal essential
medium vitamin mix, and 20 mM HEPES. For experiments, cells
were grown on 150-mm cell culture dishes until 80% confluent. Cells
were then growth-arrested by serum starvation in low serum medium
(0.1% FCS) for 48 h prior to stimulation with the indicated drugs
(dexamethasone, formeterol, salmeterol, salbutamol, propranolol,
8-bromo-cAMP, or dibutyryl-cAMP). Low serum medium was replaced every
12 h. No antibiotics or antimycotics were added to the culture
conditions at any time.
Immunohistochemistry--
Expression of GR and
2-AR by fibroblasts or VSMC was assessed by
immunohistochemistry, as described previously (22). Cells were seeded
onto 8-well chamber slides at 80% confluency and growth-arrested for
24 h. After washing three times with ice-cold phosphate-buffered saline (PBS), cells were fixed in PBS containing 4% formaldehyde. Cells were washed three times in PBS, and endogenous peroxidase activity was blocked by incubating the cells for 1 h in 80%
methanol with 0.6% hydrogen peroxide. After washing three times in
PBS, cells were incubated with whole goat serum to prevent unspecific binding of primary antibodies. Primary antibodies (1:50 each, in PBS)
were added to the cells for 1 h. Cells were washed three times in
PBS and incubated with the secondary, biotinylated antibody for 30 min.
After washing the cells three times in PBS, avidin and biotinylated
horseradish peroxidase was added and specific binding was visualized by
staining with 3-amino-9-ethyl carbazole. Unspecific binding of the
secondary antibody was examined by following the outlined protocol,
without the addition of primary antibodies. Cells were counterstained
with Mayer's hemalum (22).
Preparation of Cytosolic and Nuclear Extracts--
Nuclear and
cytosolic extracts from stimulated or unstimulated control cells were
prepared at the indicated time points as originally described by Dignam
et al. (23). Cells were washed twice in ice-cold PBS and
harvested in 1 ml of PBS. The samples were centrifuged for 30 s at
10,000 × g, and cell pellets were resuspended in 50 µl of low salt buffer (20 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM NaVO4, 1 mM EDTA, 1 mM EGTA, 0.2% Nonidet P-40, 10%
glycerol, supplemented with a set of proteinase inhibitors, CompleteTM). After 10 min of incubation on ice, the samples
were centrifuged at 13,000 × g for 2 min (4 °C),
and the supernatants were taken as cytosolic extracts. Nuclei were
resuspended in high salt buffer (20 mM HEPES, pH 7.9, 420 mM NaCl, 10 mM KCl, 0.1 mM
NaVO4, 1 mM EDTA, 1 mM EGTA, 20%
glycerol, supplemented with CompleteTM), and nuclear
proteins were extracted by shaking on ice for 30 min. Samples were then
centrifuged at 13,000 × g for 10 min (4 °C), and
the supernatants were taken as nuclear extracts.
Western Blot Analysis--
For Western blotting, cells were
seeded onto 150-mm cell culture dishes and allowed to reach 80%
confluence. After 48 h of serum starvation, cultures were
stimulated with the indicated concentrations of dexamethasone,
fluticasone, salbutamol, or salmeterol and harvested at the indicated
time points. Expression and localization of glucocorticoid receptor was
determined in cytosolic and nuclear extracts of the cells by Western
blot analysis using gradient SDS-polyacrylamide gels (4-15%) as
described earlier (24). Aliquots of cytosolic and/or nuclear extracts
along with prestained molecular weight markers were applied to the gels
and run at 25 mA constant current for 4 h at room temperature.
After electrophoresis, proteins were electroblotted on Immobilon-P
transfer membranes for 90 min with 1 mA/cm2 at room
temperature. Membranes were blocked in 5% skimmed milk in TBS-Tween
(10 mM Tris, 150 mM NaCl, 0.05% Tween 20, pH
8.0) for 1 h at room temperature. After blocking, membranes were
incubated with an antibody specific to GR at 4 °C overnight. The
following day, membranes were washed three times with TBS-Tween and
then incubated with the secondary, peroxidase-coupled antibody at a dilution of 1:5000 for 1 h at room temperature. Membranes were then washed three times in TBS-Tween, and specific bands were visualized using an ECL system according to the manufacturer's instructions.
Electrophoretic Mobility Shift Assays--
DNA mobility shift
assays were performed as originally described by Sen and Baltimore
(25). Oligonucleotides comprising the consensus sequences for GR
(5'-AAG ATT CAG GTC ATG ACC TGA GGA GA-3') (GRE) were end-labeled with
[
-32P]ATP using T4-polynucleotide-kinase. Aliquots of
nuclear extracts (1 µg) were incubated with the labeled consensus
oligonucleotides under binding conditions (4% glycerol, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM dithiotreitol, 50 mM NaCl, 10 mM
Tris-HCl, pH 7.5, 50 µg/ml poly(dI-dC)) in a total volume of 10 µl.
Reactions were carried out at room temperature for 30 min, and
protein-DNA complexes were analyzed on a 4% polyacrylamide gel. The
identity of GR was confirmed by addition of competitive unlabeled
consensus sequence oligonucleotides or by addition of monoclonal
antibody specific for GR.
Luciferase Reporter Gene Assay--
Two days before
transfection, cells were seeded into 12-well plates (1 × 104 cells/well) and were serum-deprived for 24 h.
Cells were then subjected to liposomal transfection using the cationic
lipid Tfx-50 at a DNA to lipid ratio of 1:3 (0.5 µg of plasmid/well).
A plasmid containing a GR-driven promoter fragment of the human
p21(WAF1/CIP1) gene, WWP-Luc subcloned in front of a
Luciferase gene was kindly provided by Prof. Dr. B. Vogelstein (Johns
Hopkins University, Baltimore, MD) (26). Transfection was carried out
in the absence of FCS for 2 h at 37 °C in a 100% humidified
atmosphere. Cells were then incubated with either salmeterol,
salbutamol, or dexamethasone. After 36 h cells were washed twice
with ice-cold PBS and lysed, and equal amounts of lysates were analyzed
for Firefly luciferase expression. In brief, 10-µl aliquots of cell
lysates were mixed with 50 µl of luciferase reagent buffer, and
luminescence of the samples was integrated over a time period of
10 s in a LUMAC Biocounter M1500P (Landgraaf, Netherlands).
Statistical Analysis--
For statistical analysis Student's
t test and ANOVA analysis were performed. A p
value <0.05 was estimated significant.
Ethic Committee Approval--
The protocol for establishing
primary human cell cultures from biopsies obtained during surgery was
approved by the ethical committee of the Faculty of Medicine,
University Hospital Basel (approval number M75/97).
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RESULTS |
Fibroblasts and VSMC Express the Glucocorticoid and
2-Adrenergic Receptors--
To analyze whether primary
human lung fibroblasts or VSMC, in culture, express
2-AR
or GR, we performed immunohistochemistry of the cells with antibodies
specific for
2-AR or GR We found that GR was expressed
by both cell types. Immunohistochemical staining for GR in
serum-deprived cells was predominantly cytoplasmic with no staining of
nuclei (Fig. 1A) and was most
prominent in a perinuclear compartment of the cells (arrows,
Fig. 1A), indicating that unliganded GR resides in close
proximity to the nucleus. No signal was obtained when the secondary
antibody alone was applied (Fig. 1C).

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Fig. 1.
Expression of 2-AR and GR in
primary human lung fibroblasts. Human lung fibroblasts were
cultivated from human tissue biopsies and seeded onto 8-well chamber
slides. Cells were fixed and subsequently incubated with rabbit
polyclonal antibody specific for human GR (Fig. 1A) or human
2-AR (Fig. 1B). Background staining was
assessed by incubation with secondary antibody alone (Fig.
1C). Immunoreactivity was visualized using an avidin-biotin
peroxidase stain. Pictures are representative for cell lines of primary
human fibroblasts (n = 3) and vascular smooth muscle
cells (n = 3).
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As demonstrated in Fig. 1B, primary human fibroblasts
expressed the
2-AR. Clear expression of the
2-AR is shown with the staining being localized at the
plasma membrane (arrows, Fig. 1B), in contrast to
the lack of signal when the secondary antibody alone was applied (Fig.
1C). Staining for
2-AR was not exclusively found at the plasma membrane, an observation that may indicate the
cytoplasmic recircularization of
2-AR as recently
described (14). Similar observations were made using VSMC.
2-Agonists Activate the Glucocorticoid
Receptor--
Activated GR rapidly disappears from the cytoplasmic
compartment and translocates to the nucleus (1-5). To analyze
activation of GR, we assessed the cytosolic depletion and nuclear
translocation of GR by immunohistochemistry and Western blotting of
cytosolic and nuclear extracts. We determined the effects of two
2-AR agonists, salbutamol and salmeterol, and compared
their effects to two glucocorticoids, fluticasone and dexamethasone,
serving as positive controls.
Immunohistochemical analysis revealed that incubation of cells with
dexamethasone (Fig. 2C) or
fluticasone (Fig. 2D) for 4 h, with both
glucocorticoids at a concentration of 10
8 M,
induced complete translocation of the GR from the cytosol into the
nucleus. Interestingly, incubation of cells with the
2-AR agonists, salmeterol (Fig. 2E) or
salbutamol (Fig. 2F), both at a concentration of
10
8 M, also resulted in translocation of the
GR to the nucleus, 4 h after addition of the drugs. However, the
effect of
2-AR agonists seemed to be not as complete as
observed in the presence of glucocorticoids These results suggest a
ligand-independent activation of GR by
2-AR agonists
mediated via
2-AR.

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Fig. 2.
Immunostaining analysis of the cellular
distribution of GR in human primary fibroblasts. Serum-deprived
human primary fibroblasts expressed GR in a nucleus-associated
compartment but not in the nucleus (Fig. 2A). Unspecific
staining of the second antibody was excluded when cells were only
incubated with the second antibody (Fig. 2B). When cells
were cultivated in the presence of either dexamethasone (Fig.
2C) or fluticasone (Fig. 2D), the GR translocated
into the nucleus. GR was also translocated to the nucleus when cells
were treated with salmeterol (Fig. 2E) or salbutamol (Fig.
2F). All drugs were used at a concentration of
10 8 M, and experiments were repeated in three
different fibroblast and two VSMC lines.
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Fig. 3A gives additional
evidence that dexamethasone and fluticasone rapidly induced depletion
of GR from the cytosolic, as shown by Western blotting. Depletion was
rapid occurring 30 min after treatment of the cells and completed with
no GR left in the cytosolic compartment between 30 min and 4 h
after addition of the glucocorticoids.

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Fig. 3.
Western analysis of GR activation.
Primary human lung fibroblasts were grown onto 150-mm cell culture
dishes until 80% confluence. Cells were growth-arrested by serum
starvation (0.1% FCS) for 48 h and treated with 1 nM
fluticasone or 1 nM dexamethasone (A) and 10 nM salmeterol or 10 nM salbutamol
(B) for the indicated time periods. Cytosolic and nuclear
extracts were prepared, and equal aliquots were separated on gradient
4-15% SDS-polyacrylamide gel electrophoresis gels and transferred on
nitrocellulose membranes. GR (92 kDa) was detected after incubation
with rabbit polyclonal anti-GR antibody (Santa Cruz) following standard
protocol for ECL Western detection (Amersham). In C,
activation of GR is shown by parallel detection of GR in cytosolic
(lanes 1-3) and nuclear extracts (lanes 4-6).
Extracts were prepared at 30 min as described for A and
B. Extracts from untreated cells (lanes 1 and
4) were applied together with extracts from
salmeterol-treated (10 nM) (lanes 2 and
5) and fluticasone-treated cells (lanes 3 and
6).
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Interestingly, the two
2-AR agonists, salmeterol and
salbutamol, induced depletion of GR from the cytosolic compartment when added to the cell cultures (Fig. 3B) in a
time-dependent manner. Activation of GR by the
2-AR agonists, salmeterol and salbutamol, began at 30 min and continued over a time period of 4 h. Compared with
glucocorticoid-induced translocation of GR, the
2-AR
agonist-mediated depletion of GR from the cytosolic fraction was not
complete. Low amounts of GR were still detected in the cytosol of the
cells after 4 h. Thus,
2-AR agonists seemed to
provoke a more sustained signal that leads to prolonged activation of
GR, as compared with the rapid onset and complete signal provided by glucocorticoids.
As shown in Fig. 3C, salmeterol (Fig. 3C,
lane 2) and fluticasone (Fig. 3C, lane
3), at 1 h, led to significant depletion of GR from the
cytosolic compartment (Fig. 3C, lane 1). The
glucocorticoid fluticasone was clearly more potent than the
2-AR agonist salmeterol. The depletion of GR from the
cytosol coincided with an increase in nuclear GR of the respective
samples (Fig. 3C, lanes 4-6). Compared with
untreated cells (Fig. 3C, lane 4),
salmeterol-treated (Fig. 3C, lane 5) and
fluticasone-treated (Fig. 3C, lane 6) cells exhibit a significantly higher content of GR in nuclear extracts.
Translocated Glucocorticoid Receptor Binds to Its Consensus
Sequence--
To assess whether GR that is translocated into the
nucleus in response to both classes of drugs was capable of DNA
binding, we performed electrophoretic mobility shift assays (EMSA) of
nuclear cell extracts using an oligonucleotide comprising the GR
consensus sequence, the GRE. Fig. 4
depicts characteristic EMSA demonstrating GRE binding activity within
nuclear extracts from glucocorticoid-treated cells (Fig. 4A)
as well as from
2-AR agonist-treated cells (Fig. 4B). Untreated cells (0 h) served as a control and exhibited
weak binding activity for the 32P-labeled GRE
oligonucleotide, suggesting small amounts of active GR being present in
the nuclei of serum-starved cells. This observation was in accordance
with immunohistochemistry and Western blots, both demonstrating that
low amounts of GR were present within the nucleus of fibroblasts and
VSMC.

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Fig. 4.
Increased DNA binding to GRE probe
oligonucleotide in nuclear extracts of fibroblasts treated with
fluticasone, dexamethasone, salbutamol, or salmeterol. Fibroblasts
were cultured on 150-mm cell culture dishes, treated with
10 9 µM fluticasone or 10 9
M dexamethasone (A) and 10 8
M salbutamol or 10 8 M salmeterol
(B). Nuclear extracts were prepared at the indicated time
points as described under "Experimental Procedures." Equal aliquots
of nuclear extracts were incubated with [ -32P]ATP
end-labeled GRE oligonucleotides, and the formation of specific
GR·GRE complexes was analyzed on 4% polyacrylamide gels. Free probe
without nuclear extracts was applied as negative control.
FR, fragment.
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When cells were stimulated with fluticasone or dexamethasone, GR was
rapidly activated as observed by its increased binding to the GRE
oligonucleotides. This observation supports the nuclear translocation
of GR as seen in immunohistochemistry and Western blot analyses. GR
activation was observed as early as 30 min after stimulation with
glucocorticoids (Fig. 4A). A similar effect was noted with
salmeterol- or salbutamol-treated cells (Fig. 4B). Both
2-AR agonists induced rapid activation of GR after 30 min with a subsequent decline of GRE binding activity thereafter.
The identity of GRE binding activity in nuclear extracts of the cells
was confirmed by adding unlabeled competitor GRE oligonucleotides or
antibodies specific for GR to the samples (Fig.
5). Addition of unlabeled competitor GRE
oligonucleotides led to complete disappearance of the GR·GRE complex
(Fig. 5, A, lane 3, and C, lane
3). When the samples were incubated with monoclonal antibodies to
GR, specific bands diminished in a concentration-dependent
manner (Fig. 5A, lanes 3-6). Thus, these
observations together identified GRE binding activity within nuclear
extracts of the cells as GR.

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Fig. 5.
GR activation by 2-agonists is
dependent upon their interaction with the 2-AR.
A, identity of the GRE band observed in nuclear extracts
from salmeterol (10 8 M)-treated fibroblasts
(lane 2) was confirmed by the addition of unlabeled
competitor GRE oligonucleotides in 10-fold excess (lane 3)
or the addition of decreasing amounts of the 2-AR
antagonist propranolol (was blocked by preincubation of the cells with
the 2-AR antagonist propranolol (lane 4,
10 7 M; lane 5, 10 8
M; lane 6, 10 9 M;
lane 7, 10 10 M). Lane 1 represents free probe without addition of extracts. Nuclear extracts
were prepared 30 min after addition of salmeterol. In B,
nuclear cell extracts from salmeterol (10 8 M)
cells were incubated with various concentrations of a rabbit polyclonal
anti-GR antibody (lane 3, antibody dilution of 1:5;
lane 4, antibody dilution of 1:10; lane 5,
antibody dilution of 1:20). To visualize supershifts the gels had to be
overexposed to x-ray films. The role of cAMP in 2-AR
agonist-mediated GR activation is depicted in C. Lane
1, GRE fragment alone; lane 2, nuclear extracts of
cells stimulated with dexamethasone 10 8 M;
lane 3, as lane 2 but in the presence of
nonlabeled competitive GRE oligonucleotide (10-fold); lane
4, serum-deprived cells; lane 5, in the presence of
salmeterol (10 8 M); lane 6, in the
presence of dibutyryl cAMP (10 6 M);
lane 7, cells treated with a combination of salmeterol
(10 8 M) and a PKA inhibiting peptide (3 × 10 6 M). Specific GR·GRE complexes were
detected as described for Fig. 4.
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Similar to the results obtained with
2-agonists both
8-bromo-cAMP or dibutyryl cAMP induced a DNA mobility shift for GRE (data not shown), indicating that the mechanism for GR activation by
2-agonists may be due to an increase of intracellular
cAMP levels (Fig. 5C, lane 6). The stimulatory
effect of cAMP on GR activation was dose-dependent and
occurred at physiologically relevant concentrations (data not shown).
As shown in Fig. 5C (lane 7) a protein kinase A
(PKA) inhibiting peptide partly reduced the salmeterol-induced
activation of GR. This may further suggests the involvement of the
cAMP/PKA pathway.
GR Activation by
2 Agonists Depends on Their
Interaction with the
2-Adrenergic Receptor--
We
analyzed whether the described effects of
2-AR agonists
were dependent upon binding of the drugs to the
2-AR by
preincubating the cells with the
2-AR antagonist,
propranolol. When propranolol was administered to the cells 30 min
prior to stimulation with salmeterol (10
8 M),
GR activation was dose-dependently inhibited as shown by EMSA (Fig. 5A). Propranolol at a concentration of
10
7 M (Fig. 5A, lanes
4-7) clearly inhibited GR activation by salmeterol (Fig.
5A, lane 2), whereas propranolol at
10
9 M (Fig. 5A, lane 4)
had no effect on salmeterol-induced GR activation. The data thus
demonstrate that functional interaction of
2-agonists with their respective receptor, the
2-AR, is responsible
for
2-AR agonist-induced activation of GR.
Translocated Nuclear GR Is Functional--
To assess whether GR
activated by glucocorticoids or
2-AR antagonist was
functional, we assessed whether the drugs affected a glucocorticoid
driven p21(WAF1/CIP1) promoter/Luciferase construct (26).
At concentrations higher than 10
9 M
dexamethasone activated the reporter gene p21(WAF1/CIP1).
Although p21(WAF1/CIP1) activation was inconsistent at a
dexamethasone concentration of 10
8 M, it was
constantly expressed at a concentration of either 10
7
M or 10
6 M (data not shown). The
achieved expression of p21(WAF1/CIP1) with dexamethasone at
10
7 M was about 197 ± 29% of control
(Fig. 6). Similar to the
glucocorticosteroid the two
2-AR antagonists, salmeterol
(136 ± 6%) and salbutamol (149 ± 21%), induced the
expression of Firefly luciferase in a concentration range of
10
8-10
6 M. In accordance to
the above described results on GR activation determined by Western
blotting and EMSA compared with the effect of glucocorticoid treatment
the activation of p21(WAF1/CIP1) was less prominent in the
presence of both salmeterol or salbutamol (Fig. 6). Thus, although not
as potent as the glucocorticoids, the
2-AR agonists
clearly activated the p21(WAF1/CIP1) promoter, suggesting
that GR activation by
2-AR agonists also leads to
altered gene transcription.

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Fig. 6.
2-AR agonists activate
p21(WAF1/CIP1) expression, a glucocorticoid inducible
gene. Cells were transiently transfected with a WWP-Luc luciferase
construct and stimulated with the indicated drugs at a concentration of
10 8 M for both 2-AR agonists
and at 10 7 M for dexamethasone.
Bars present the mean ± S.E. of at least three
independent experiments, obtained in three different cell lines.
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 |
DISCUSSION |
The mechanism of GR activation in a ligand-independent manner has
been the subject of several recent investigations (27, 28). In this
study, we demonstrate ligand-independent activation of GR by two
2-agonists, salmeterol and salbutamol. Both drugs were
potent activators of GR in the absence of agonistic ligands as
demonstrated by immunohistochemistry, Western blotting, EMSA, and
reporter gene assays. GR was rapidly depleted from the cytosol of
primary human lung fibroblasts and VSMC and translocated into the
nucleus. Translocated GR was functional as it bound to its specific DNA
recognition sequence, GRE, and also activated a GR-inducible luciferase
reporter gene assay, p21(WAF1/CIP1). Finally, GR activation
by
2-agonists was dependent upon their binding to
2-AR, reasoning that downstream signaling initiated by
2-AR agonist/
2-AR interaction is
responsible for the observed GR activation.
Activation of GR results in altered transcription of several cytokine
genes involved in the inflammatory process leading to their repression
or activation (6-8). Our findings are especially important to
understand the underlying molecular mechanism of drug action in the
treatment of asthma. Here, glucocorticoids and
2-AR
agonists are the most effective drugs in the treatment of this disease
(9, 10). Clinical studies on asthmatic individuals suggested that the
administration of salmeterol, in combination with glucocorticoids,
resulted in an improved symptom control being more effective than
increasing the dose of the glucocorticoid alone (11-13). We therefore
assumed that an interaction between both substances might occur at the
level of GR activation, intensifying the anti-inflammatory potency of
glucocorticoids. It is known that glucocorticoids induce transcription
of the
2-AR gene (29, 30), preventing agonist-induced
desensitization of the
2-AR itself and improving the
therapeutic efficacy of
2-AR agonists. It is unknown,
however, whether
2-agonists are capable of affecting GR activity.
An earlier report (31) analyzing the cellular effects of
2-AR agonists in rat lung tissue cubes demonstrated an
inhibitory effect of unphysiologically high concentrations
(10
6 M) of salbutamol on the binding of GR
from crude tissue homogenates to their recognition sequence, GRE. This
report failed to show direct activation and/or inhibition of GR itself.
In our study, we compared GR activation by
2-AR agonists
with GR activation by specific ligands, two glucocorticoids, in a
definite cell population, either primary human lung fibroblasts or
VSMC. The comparison between ligand-dependent (induced by
glucocorticoids) and ligand-independent (induced by
2-AR
agonists) GR activation demonstrated that both mechanisms occurred in a
similar time frame. Ligand-dependent activation of GR was
apparently more potent and resulted in total depletion of GR of the
cytosolic compartment. In contrast, ligand-independent activation of GR
was sustained over a longer period of time. The cellular function of
ligand-independent activation of GR was illustrated by a luciferase
reporter gene, WWP-Luc (21). The WWP-Luc construct contains the
promoter region (
2.4 kilobases to +1 base pair) of the
p21(WAF1/CIP1) gene. This construct was shown to be
activated by glucocorticoids (32). p21(WAF1/CIP1) is a cell
cycle kinase inhibitor and accounts for some of the antiproliferative effects of glucocorticoids in fibroblasts (33).
The biochemical modulation of GR is suggested to be achieved by
phosphorylation (activation) and dephosphorylation (inactivation) at
seven different phosphorylation sites (34-39). How can activation of
GR by
2-AR agonists be explained at the molecular level?
In general, signal transduction upon interaction of
2-AR
agonists with the
2-AR results in the activation of
G-proteins that are coupled to the intracellular domain of the receptor
(14, 40). Although we could not demonstrate the immediate upstream
event preceding GR activation in response to
2-AR
agonists, we assume an event in the
2-AR agonist signal
transduction pathway to be responsible for the observed GR activation.
This is evidenced by the fact that the
2-AR antagonist,
propranolol, prevented
2-AR agonist-induced GR
activation.
2-AR activation leads to an increase of
intracellular cAMP (40), PKA (14, 40), and calmodulin (CaM) (41). We
assessed the effect of two cAMP mimetics, which also activated GR.
These findings suggested a possible involvement of the known
2-AR agonist-mediated cAMP pathway, which was further supported by the finding that a PKA inhibiting peptide abolished the
salmeterol-induced activation of GR. To the contrary, CaM, a ubiquitous
intracellular signaling molecule located to plasma membrane receptors
and ion channels (42), directly activated GR in a ligand-independent
manner (43). The mechanism responsible for GR activation by CaM was
suggested to be a phosphorylation of specific tyrosine residues of GR
(43). Analogously, CaM-dependent phosphorylation of the
estrogen receptor, another member of the steroid/vitamin D/retinoic
acid superfamily, has been reported to correlate with the activation of
the estrogen receptor itself (44). It is therefore possible that
2-AR agonists activate GR involving the action of CaM in
a similar way.
In conclusion, our study demonstrates, for the first time,
ligand-independent activation of GR by two different
2-agonists using primary human cell lines of lung
fibroblasts and VSMC. GR activation by
2-agonists
explains, at least in part, the as yet unresolved anti-inflammatory
potency of
2-agonists seen in vivo and
in vitro.