β2-Adrenoceptor Involved in Smoking-Induced Airway Mucus Hypersecretion through β-Arrestin-Dependent Signaling
et al. (2014) b2-Adrenoceptor Involved in Smoking-Induced Airway Mucus Hypersecretion through b-Arrestin-
Dependent Signaling. PLoS ONE 9(6): e97788. doi:10.1371/journal.pone.0097788
b2-Adrenoceptor Involved in Smoking-Induced Airway Mucus Hypersecretion through b-Arrestin-Dependent Signaling
Yujiao Zhou 0
Yuan Zhang 0
Yang Guo 0
Youyi Zhang 0
Ming Xu 0
Bei He 0
Bernhard Ryffel, French National Centre for Scientific Research, France
0 1 Department of Respiratory Medicine and Institute of Vascular Medicine, Peking University Third Hospital; Beijing Key Laboratory of Cardiovascular Receptors Research, Key Laboratory of Cardiovascular Molecular Biology and Regulatory Peptides , Beijing , People's Republic of China, 2 Department of Respiratory Medicine, Changji Renmin Hospital , Changji, Xinjiang , People's Republic of China
Progression of chronic obstructive pulmonary disease is associated with small airway obstruction by accumulation of inflammatory mucous exudates. However, the mechanism of mucin hypersecretion after exposure to cigarette smoke (CS) is still not clear. In this study, we explored the contribution of b2-adrenoceptor (b2-AR) signaling to CS extract (CSE)-induced mucus hypersecretion in vitro and examined the effect of a b-blocker on airway mucin hypersecretion in vivo. NCI-H292 epithelial cell line was used to determine the contribution of b2-AR signaling to CSE-induced MUC5AC production by treatment with b2-AR antagonists propranolol and ICI118551 and b2-AR-targeted small interfering RNA. The effect of propranolol on airway mucus hypersecretion was examined in a rat model exposed to CS. MUC5AC expression was assayed by real-time PCR, immunohistochemistry and ELISA. b2-AR and its downstream signaling were detected by western blot analysis. We found that pretreating NCI-H292 cells with propranolol, ICI118551 for 30 min or b2AR-targeted siRNA for 48 h reduced MUC5AC mRNA and protein levels stimulated by CSE. However,inhibiting the classical b2AR-cAMP-PKA pathway didn't attenuate CSE-induced MUC5AC production, while silencing b-arretin2 expression significantly decreased ERK and p38MAPK phosphorylation, thus reduced the CSE-stimulated MUC5AC production. In vivo, we found that administration of propranolol (25 mg kg21d21) for 28 days significantly attenuated the airway goblet cell metaplasia, mucus hypersecretion and MUC5AC expression of rats exposed to CS. From the study, b2-AR-b-arrestin2-ERK1/2 signaling was required for CSinduced airway MUC5AC expression. Chronic propranolol administration ameliorated airway mucus hypersecretion and MUC5AC expression in smoking rats. The exploration of these mechanisms may contribute to the optimization of b2-AR target therapy in chronic obstructive pulmonary disease.
Funding: This work was supported by a grant from the National Natural Science Foundation of China (No. 81270097; 30910103902; 81121061), the Beijing
Natural Science Foundation (No. 7112745) and Ph.D. Programs Foundation of Ministry of Education of China (No. 20090001110093). The funders had no role in
study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
The normal mammalian airway epithelium produces and is
coated by mucins such as MUC5B and MUC5AC, which protect
the airway epithelia against exogenous insult. However, excessive
epithelial goblet cell metaplasia and mucus hypersecretion
contribute to the pathology of many respiratory diseases such as
chronic obstructive pulmonary disease (COPD), asthma, and
cystic fibrosis. In COPD, mucin hypersecretion contributes to
airway obstruction, accelerated decline of lung function and
increased hospitalization and mortality . MUC5AC is the
predominant mucin produced from goblet cells in human airways;
its expression is evoked by mediators such as neutrophil elastase,
air pollutants and bacterial products [2,3].
Cigarette smoking is the primary cause of COPD and induces
the expression and production of MUC5AC in vitro and in vivo
[4,5]. As the first responders to cigarette smoke (CS), airway
epithelial cells fulfill important roles: they recognize the inhaled
CS components and mount a defense response but also produce
various ligands that cause aberrant activation of some receptors
such as epidermal growth factor receptor, which leads to epithelial
cell hyperplasia and mucous cell metaplasia .
b2-adrenoceptor (b2-AR) is the most common adrenergic
receptor subtype expressed in various types of lung cells, including
airway epithelial cells. However, the role of b2-AR in airway
mucus hypersecretion is still unknown. In an asthma murine
model, the b-agonist formoterol inhibits airway goblet cell
hyperplasia and MUC5AC protein expression, whereas
administration of other b-agonists fails to improve mucus metaplasia [7,8].
In addition, b2-AR signaling has been proven to be required for
the full development of 3 cardinal features of asthma: mucous
hypersecretion, airway hyperresponsiveness and the presence of
inflammatory cells in the lungs and chronic administration of
ICI118551 or nadolol reduced inflammation and mucous
metaplasia, that may contribute to airflow obstruction and airway
hyperresponsiveness of asthma[7,9]. This evidence indicates
b2AR is involved in regulating lung inflammation and mucus
Cardiovascular disease is the most common co-morbidity of
COPD because of the risk of smoking-induced atherosclerosis
. Prior studies have showed that use of b-blockers can reduce
mortality and exacerbation when added to regular therapy for
patients with COPD, independent of cardiovascular disease and
cardiac therapy, without adverse effects on pulmonary function
. We previously showed that chronic exposure to propranolol,
a nonselective b-AR antagonist, had a protective effect on airway
smooth muscle contraction in rats exposed to CS . These
results suggest that b2-AR may be involved in the development of
pathological changes associated with smoking. In the present
study, we hypothesized that b2-AR signaling is required for airway
MUC5AC expression and b-blocker treatment could improve
CSinduced mucus hypersecretion by attenuating MUC5AC
production. We explored the contribution of b2-AR and downstream
signaling to MUC5AC expression in NCI-H292 cells, a
mucinexpressing cell line from human airway epithelia, stimulated by CS
extract (CSE) which is commonly used to mimic the effects of CS.
We further examined the effect of chronic propranolol
administration on MUC5AC production in rats exposed to CS.
Materials and Methods
CSE, used to mimic the effects of CS, was purchased from
Murty Pharmaceuticals (Lexington, KY, USA) and was prepared
using a Phipps-Bird 20-channel smoking machine designed for
FTC testing . The particulate matter from Kentucky standard
cigarettes (1R4F; approximately 11 mg of tar and 0.8 mg of
nicotine/cigarette; University of Kentucky, KY, USA) was
collected on Cambridge glass fibre filters and the amount obtained
determined by weight increase of the filter. The average yield of
CSE was 20.1 mg/cigarette. CSE was prepared by dissolving the
collected smoke particulates in dimethyl sulfoxide (DMSO) to yield
a 4% solution (w/v). The CSE was diluted into DMSO and
aliquots were kept at 280uC. Propronolol (nonselective b-AR
antagonist), ICI118551 (selective b2-AR antagonist), H89 (PKA
inhibitor) PD98059 (ERK inhibitor) and SB203580 (p38MAPKK
inhibitor) were from Sigma-Aldrich (St. Louis, MO, USA).
Rp-8Br-cAMPs and primary antibodies (Abs) for PKA, b-arrestin1,
barrestin2, GAPDH and mouse or rabbit horseradish
peroxidaseconjugated secondary Abs were from Santa Cruz Biotechnology
(Santa Cruz, CA). Primary Abs for phospho-PKA, extracellular
signal-regulated kinase 1/2 (ERK1/2) and phospho-ERK1/2
were from Cell Signaling Technology (CST, Boston, USA).
Primary Abs for MUC5AC and b2-AR were from Abcam
NCI-H292 cells, a mucin-expressing cell line from human
airway epithelia, were obtained from the Cell Resource Center
(IBMS/CAMS/PUMC, China). Cells were maintained in DMEM
supplemented with 10% fetal bovine serum, 1% penicillin/
streptomycin (PS; 100 IU ml21 each), at 37uC in a humidified
5% CO2 atmosphere and subcultured twice weekly. Confluent
cells were incubated in serum-free DMEM for 12 h, then washed
3 times with 10% phosphate buffered saline (PBS), placed under
serum-free conditions and exposed to the indicated concentration
of CSE and inhibitors. Control cells were incubated with medium
alone for the same period. After 24-h stimulation, samples of a
cellconditioned medium and/or cell lysates were collected by brief
centrifugation and stored at 280uC. Most experiments, except for
cell viability assays (96-well microplate), were involved 6-well
plates or 35-mm dishes (Corning, USA) with 16106 cells/well.
Silencing of b2-AR, b-arrestin1 and b-arrestin2 Expression
The two sets of siRNA oligos were synthesized by GenePharma
Co. (Shanghai) as in Table 1. Target gene knockdown was
achieved by transfection of NCI-H292 cells plated in 6-well dishes
with doses of siRNA (100 pmol) and Lipofectamine2000
(Invitrogen, 5 ml) in 200 ml DMEM without serum; PS. A
nonspecific siRNA was used for NC-siRNA. Lipofectamine2000
alone was a vehicle. For all siRNA transfections, 60% to 70%
confluent monolayers were incubated with the transfection
mixture in 1 ml serum-free medium for 6 to 8 h. Then serum
was replenished and cells were used for various assays after 24 to
This study was carried out in strict accordance with the
recommendations in the Guide for the Care and Use of
Laboratory Animals of the US National Institutes of Health.
The protocol was approved by the Committee on the Ethics of
Animal Experiments of Peking University (permit No.:
LA2010004). We housed 32 male Sprague-Dawley rats (7 weeks old) in
divided cages (4 each) under specific pathogenfree conditions of
the Animal Center of Peking University Health Science Center,
Beijing. Animals were raised at room temperature with a natural
light-dark cycle and had free access to food and water. All rats
were anaesthetized by 20% uratan (0.5 mg/ml intra-peritoneal)
for surgery, overdose of uratan for euthanasia, and all efforts
including comfortable bedding, improved surgical intubation and
standard surgical procedure, were made to minimize suffering.
From weeks 116, rats in Group S and Group S/P were exposed
to whole-body cigarette smoke in inhalation chambers (HOPE,
China) generated from commercial cigarettes (Tar = 10 mg,
cotinine = 0.9 mg, CO = 12 mg per cigarette) at 20 cigarettes
inhalation for 2 h followed by a 4-h recovery, repeated twice a
day. Rats were exposed to CS 6 days per week. The study protocol
was showed in details as Figure 1.
Lung Function Test
Rats were anesthetized by intraperitoneal injection of 20%
urethane (1 mg kg21), then respiration was maintained by a
computer-controlled small-animal ventilator connected to the rat
through a tracheal cannula. Respiratory function was measured as
described . Peak inspiratory flow, peak expiratory flow,
intrapressure (IP) and maximum rising slope of IP (IP slope) were
analyzed by use of Chart 4.1 (AD Instruments, Australia).
Bronchoalveolar Lavage (BAL) and Tissue Extraction
The left lobe and right posterior lobe of the lung were clamped
at the hilus of the lung. In total, 1 ml PBS was infused and drawn
back through the tracheal cannula 3 times. Total leukocytes in
BAL fluid (BALF) were counted by use of a Coulter counter. The
remaining BALF was centrifuged (3000 rpm, 10 min) and
supernatants were stored at 280uC. The left lobe of the lung
was preserved in 4% paraformaldehyde for histology. Tracheal
samples 24 mm in diameter excised from the trachea were
preserved in 3% glutaric dialdehyde for scanning electron
microscopy and transmission electron microscopy. The right
posterior lobe of the lung was preserved in liquid nitrogen for
mRNA and protein analysis.
Tissues were embedded in paraffin and stained with
hematoxylin and eosin or periodic acid-Schiff (PAS). Images were acquired
before any measurements and analyzed by a pathologist blinded to
Antisense -AGAUUG GUAUCUACUGGCGTT
Antisense -UUGUAG GAAACAAUGAUCCTT
treatment. Pathological changes in small airways (#2 mm in
diameter) were assessed by inflammation scores as described .
The maximal luminal area and luminal content were calculated
from images of 5 fields in airways #2 mm in diameter that were
cross-sectioned, captured and viewed under a DM2500 optical
microscope (LEICA, Germany). The content of mucin in the
airway epithelium was measured by use of Image-Pro plus 5.0 as
appropriate fixative as described and sent to the cytology and
pathology labs of Peking University Health Science Center for
conventional electron microscopy examination. Specimens were
coated with gold-palladium in a Hummer II sputter-coating
apparatus and examined under a JEOL JSM-5600LV scanning
electron microscope. For transmission electron microscopy,
mucosal slices were examined under JEOL JEM-1400 electron
Tracheal samples 24 mm in diameter were excised from the
right principal bronchus of 3 rats under urethane anesthesia,
taking care to avoid trachea cannula damage. Samples of
untreated control mucosa were immediately immersed in the
After paraformaldehyde fixation, paraffinized 5-mm sections of
lung tissue underwent immunohistochemical staining. For
MUC5AC, the primary antibody was mouse polyclonal
antiMUC5AC (45M1; 1:200 dilutions). After incubation at 4uC
Primer Sequences (59-39)
Figure 3. Effect of cAMPprotein kinase A (PKA) inhibition on CSE-stimulated MUC5AC expression. Western blot analysis of
phosphoPKA (p-PKA) and total PKA (t-PKA) levels in cells preincubated with (A) Rp-8-Br-cAMPs or (B) H89 for 30 min before stimulation with or without CSE for
15 min. (C, D) RT-PCR analysis of MUC5AC mRNA expression after incubation with or without CSE for 24 h. Data are means 6 SEM from 3 separate
experiments. ***P,0.001 compared to CSE alone. NS, not significant.
overnight, the biotinylated secondary goat anti-mouse antibody
was applied (1:500) for 30 min at room temperature and DAB
chromogen was applied for 1 min.
Real-time Quantitative PCR
Total RNA was extracted from frozen lung tissues or cultured
NCI-H292 cells by the Trizol method (Takara Bio Inc. Japan).
Real-time PCR involved use of Lightcycler480 (Eppendorf AG,
Germany) with SYBR Green assay after reverse-transcribing 1 mg
RNA by use of the Reverse Transcription System (Promega,
USA). All data were normalized to mRNA levels of GAPDH. The
primer pairs and expected lengths are in Table 2. The cycling
conditions were 95uC for 30 s, then 40 cycles of 95uC for 5 s, 55uC
for 30 s, and 65uC for 15 s. Relative gene expression was
calculated as 22DDCT . When necessary, 0.8% agarose gel
was used to separate the RT-PCR products.
MUC5AC Mucin Assay
At 24 h after NCI-H292 cells were stimulated with CSE, cell
culture media was collected for determining MUC5AC
concentrations by use of an ELISA kit (R&D, USA). Total mucin and
MUC5AC level in BALF was assayed by use of rat mucin and
MUC5AC ELISA kits (4A Biotech Co., Beijing, China).
Western Blot Analysis
Cells were harvested and lysed with lysis buffer (1% deoxycholic
acid, 0.01 M Na4P2O7, 10% glycerol, 0.1 M NaCl, 5 mM EDTA,
pH 8.0, 0.1% SDS, 0.02 M Tris-HCl, pH 7.4, 0.05 NaF, 1 mM
Na3VO4, 100 mg ml21 phenylmethylsulfonyl fluoride, and 10 mg/
ml aprotinin). Cell lysates underwent protein determination by use
of a Pierce BSA protein assay kit (Pierce, Rockford, IL, USA).
Proteins (50 mg) were loaded onto 10% SDS polyacrylamide gel
and electrophoretically transferred to nitrocellulose membrane
(Pall, Port Washington, USA) that was incubated with antibodies
according to the manufacturers protocol; immunolabelled bands
were visualized by use of the SuperSignal West Pico
chemiluminescence kit (Thermo, USA). Autoradiographs were quantified by
densitometry by use of Image J software. Bands were normalized
to GAPDH expression.
Data are presented as mean 6 SEM. One-way ANOVA and
Newman-Keuls multiple comparison test were used for normally
distributed data as appropriate. A t-test was used to compare two
different groups of treatment to each other. For multiple
comparisons, two-way ANOVA was used. P,0.05 was considered
statistically significant. Statistical calculations were carried out
using Graph Pad Prism 5.0 (San Diego California, USA).
b2-AR was Required for CSE-induced MUC5AC
Production from NCI-H292 Cells
We preincubated NCI-H292 cells with the b2-AR antagonists
propranolol or ICI118551 with or without CSE stimulation to
explore the contribution of b2-AR to the CS-induced airway
MUC5AC production. CSE at 0.1 mg ml21 increased MUC5AC
mRNA level and protein secretion in cells, and preincubation with
antagonists reversed the increase (Figure 2). We further used two
sets of b2-ARtargeted siRNAs to silence b2-AR expression on
NCI-H292 cells, then incubated cells with CSE. b2-AR siRNA
treatment reduced CSE-simulated MUC5AC mRNA and protein
secretion in cells. Thus, b2-AR was required for the CSE-induced
MUC5AC production from NCI-H292 cells. The indicated
Figure 4. Effect of b-arrestin knockdown on CSE-stimulated MUC5AC expression. Cells were transfected with Lipofectamine2000 alone
(vehicle), a nonspecific control siRNA (NC-siRNA, 100 nM), b-arrestin1targeted siRNA (A1-siRNA, 100 nM) or b-arrestin2targeted siRNA (A2-siRNA,
100 nM) for 48 h before incubation with or without CSE for 24 h. (A, B) Western blot analysis of b-arrestin1 and b-arrestin2 protein level to access the
knockdown efficiency. (C, D) Quantitative RT-PCR analysis of MUC5AC mRNA expression. Data are means 6 SEM (n = 3). NS, not significant.
concentration of CSE was determined by a time- and
dosedependent pre-experiment (File S1) and the b2-AR siRNA
transfection and knockdown efficiency is shown in File S2.
CSE-induced MUC5AC Expression in NCI-H292 Cells was
Independent of b2ARcAMPPKA
b2-AR activation normally leads to increased intracellular
cAMP levels, and cAMP production normally leads to activation
of the catalytic subunit of PKA, which in turn phosphorylates
cAMP-responsive element binding protein (CREB), allowing it to
bind to CRE sites. To determine whether CSE-induced
MUC5AC expression in NCI-H292 cells operated through a
b2ARcAMPPKA pathway, we pretreated cells with the cAMP
analogue Rp-8-Br-cAMPs and PKA inhibitor H89. Inhibiting the
cAMPPKA pathway did not decrease the CSE-induced
MUC5AC production (Figure 3). Therefore, CSE-induced MUC5AC
expression was b2ARcAMPPKA independent.
b2ARb-arrestin2ERK/p38MAPK Mediated CSE-induced
MUC5AC Expression in NCI-H292 Cells
b2-AR is a well-known G proteincoupled receptor (GPCR) and
has many potential sites of phosphorylation. b-arrestin can
uncouple the receptor from G protein, thus terminating
desensitization and internalization of the receptor [19,20]. In addition,
barrestin can serve as multi-functional adaptors and signal
transducers, directing the recruitment, activation, and scaffolding
of cytoplasmic signaling molecules, including mitogen-activated
protein kinase (MAPK) and the Ser-Thr kinase Akt [21,22].
MUC5AC gene expression is critically regulated by MAPK (ERK,
p38MAPK and JNK) [23,24]. We sought to determine whether
b2-AR activation induced airway epithelial MUC5AC production
by CSE via a b-arrestinMAPK pathway. We pretreated cells with
2 sets of b-arrestin-targeted siRNA, b-arrestin1 siRNA and
barrestin2 siRNA. CSE-stimulated MUC5AC expression was
significantly decreased in cells pretreated with b-arrestin2 siRNA
but not b-arrestin1 siRNA (Figure 4).
Because b-arrestin2 is associated with MAPK signaling , we
observed intracellular ERK1/2 and p38MAPK activation after
barrestin2 siRNA pretreatment. We further pretreated cells with the
ERK inhibitor PD98059 or p38MAPK inhibitor SB203580 and
examined MUC5AC expression. Consistently, ERK1/2 and
p38MAPK phosphorylation was significantly suppressed in cells
pretreated with b-arrestin2 siRNA, and cultures pretreated with
PD98059 or SB203580 showed decreased CSE-induced
MUC5AC production, but their combination failed to further reduce
the MUC5AC production (Figure 5). Therefore, b-arrestin2
ERK1/2 and p38MAPK is required for b2-AR mediated
MUC5AC expression induced by CSE.
Much clinical evidence has shown that b-blockers are safe and
beneficial for patients with COPD and cardiovascular disease.
However, we need solid data before recommending regular use of
b-blockers to reduce airway or lung inflammation and mucus
hypersecretion in COPD, because of concerns that b-blockers
might induce bronchospasm and worsen lung function [26,27].
However, these concerns have been challenged by recent
Figure 5. Effect of b-arrestin knockdown on CSE-stimulated MAPK activation. Western blot analysis of phospho-ERK1/2 (p-ERK1/2), total
ERK1/2 (t-ERK1/2), phospho-p38MAPK (p- p38MAPK) and total p38MAPK (t-p38MAPK) in cells treated with Lipofectamine2000 (vehicle), nonspecific
control siRNA (NC-siRNA, 100 nM) or b-arrestin2targeted siRNA (A2-siRNA, 100 nM) before CSE stimulation as in Figure 4. (A, B) Representative
western blot is from 3 independent experiments. (C, D) Data are the means 6 SEM of 3 separate experiments. Cells were pretreated with ERK1/2
inhibitor PD98059 (50 mM) or p38MAPK inhibitor (5 mM) for 60 min before CSE treatment. (E) Quantitative RT-PCR analysis of MUC5AC mRNA
expression. Data are means 6 SEM (n = 3). NS, not significant.
evidence: the risk of bronchospasm can be reduced by starting
bblockers at a lower dose and slowly titrating up . Therefore, we
used long-term and low-dose propranolol as a conservative
treatment and observed its effect on lung function, airway/lung
inflammation and mucus hypersecretion in rats exposed to CS to
verify the findings of b2-AR in vitro. The weight change as well as
the survival rate of rats during the study was showed in File S3.
Chronic Propranolol Administration Attenuated Airway
Mucus Hypersecretion in Rats Exposed to CS
We first evaluated the general histopathological changes and
lung function in pure air-exposed rats, CS-exposed rats and
CSexposed rats administrated propranolol. Chronic propranolol
administration ameliorated a number of CS-induced pathological
alterations such as airway occlusion, fibrosis, and smooth muscle
Figure 6. Chronic administration of propranolol attenuates airway mucus obstruction in rats exposed to CS. (A) Representative rat
lung sections stained with periodic acid-Schiff exposed to air and vehicle (Group C), CS-exposed rats with vehicle (Group S) or prapronolol for 4 weeks
(Group S/P). Scale bar, 200 mm. (B) Quantification of the area of airway lumen and luminal content of airways #2 mm in diameter. (C) ELISA of total
mucins in bronchoalveolar fluid (BALF). Data are mean 6 SEM. ***P,0.001, compared with Group C; ###P,0.001 compared with Group S.
proliferation, especially goblet cell metaplasia (File S4). Among
the 4 lung function indexes, propranolol decreased the peak
expiratory flow, intra-airway pressure and intra-airway pressure
slope (File S5). As compared with air-exposed rats, CS-exposed
rats showed increased airway mucus, and propranolol treatment
significantly decreased airway luminal mucus accumulation and
total mucins in BALF (Figure 6). Mucins secreted from goblet cells
are the primary components maintaining mucus viscosity. In rats
Figure 7. Chronic administration of propranolol inhibits airway goblet cell metaplasia and mucin hypersecretionin in rats exposed
to CS. (A) Scanning electron microscopy of the luminal surface shows the apices of ciliated and goblet cells (arrow). Bar = 10 mm. (B) Transmission
electron microscopy of airway epithelia shows massive mucin-containing secretory granules in goblet cells in response to CS. Bar = 5 mm.
Figure 8. Chronic administration of propranolol decreases airway epithelial MUC5AC expression in rats exposed to CS. (A) MUC5AC
glycoprotein expression in lung tissue by immunohistochemical staining. Bar = 200 mm and 100 mm in the upper and lower images, respectively. (B)
Quantitative RT-PCR analysis of mRNA level of MUC5AC in lung homogenate. (C) ELISA of MUC5AC protein in BALF. Data are means 6 SEM in each
exposed to CS, airways showed massive goblet cell metaplasia,
mucus hyperplasia and collapsed cilia between the dense,
overlying mucus and the epithelial cell surface; treatment with
propranolol significantly decreased the production of secretory
granules induced by CS (Figure 7).
Chronic Propranolol Administration Decreased MUC5AC
Expression in Lungs of Rats Exposed to CS
MUC5AC is the main component of airway mucus. We further
examined the effect of propranolol on MUC5AC expression and
secretion. Chronic administration with low-dose propranolol
significantly decreased CS-stimulated MUC5AC expression in
airway epithelia, MUC5AC mRNA level in lung homogenates
and protein level in BALF (Figure 8). Thus, b-AR was involved in
regulating the airway MUC5AC production. The detailed
mechanism of b2-AR involved in cigarette smoke -induced
MUC5AC production was shown in Figure 9.
Cigarette smoking is the primary risk factor of COPD
associated with airway mucus hypersecretion. MUC5AC is the
major inducible mucus associated with COPD progression .
We found that blocking b2-AR with propranolol, ICI118551 or
siRNA significantly decreased CSE-induced MUC5AC expression
in NCI-H292 cells and chronic administration of propranolol, a
nonselective b-AR antagonist, significantly decreased the airway
epithelial mucus hyperproduction in rats exposed to CS. These
effects may depend inhibition of CS/CSE-simulated
b2-ARbarrestinERK1/2 and p38MAPK phosphorylation.
b2-AR signaling shows tissue specificity and diverse bioeffects. A
ligand may simultaneously activate more than one G protein
coupled receptor signaling pathway and certain ligands may be
able to preferentially direct receptor signaling to a specific
pathway. In airway smooth muscle cells, cAMPPKACREB is
considered the conventional b2-ARactivated signal pathway,
which mainly causes bronchodilatation. In macrophages, b2-AR
activation has anti-inflammatory properties, inhibiting NF-kB
Figure 9. Schematic of the b2-AR signaling pathway involved in CS-induced upregulation of MUC5AC production in NCI-H292 cells,
a mucin-expressin cell line from human airway epithelia. Wtih CSE stimulation, b2-AR is activated, which increases cAMP and recruits many
barrestins. Increased cAMP binds and activates PKA but does not stimulate MUC5AC transcription and production. b-arrestin2 phosphorylates ERK1/2
and p38MAPK, the key players, which activate transcription factors to stimulate MUC5AC transcription and production. b-AR antagonists, kinase
inhibitors and molecular biological methods of interfering with signaling molecules are also shown.
activation and cytokine production induced by pro-inflammatory
stimuli while increasing IL-1b and IL-6 protein production in the
absence of pro-inflammatory stimuli . Collectively, these
findings suggest that b2-AR produces different signals in different
stimuli and the signals may have a different role in different cells.
Our study of ICI118551, a selective b2-AR antagonist, and
b2ARtargeted siRNA administered to NCI-H292 cells, a cell line
from human airway epithelia, revealed that b2-AR participated in
MUC5AC production induced by CSE. b2-AR blockade
decreased the induction of respiratory MUC5AC mucin by CSE
both at the mRNA and protein levels (Figure 2).
We first observed the effect of the conventional b2-ARcAMP
PKA pathway on MUC5AC production; however, the results
suggested that CSE-stimulated MUC5AC production was
independent of PKA (Figure 3). Our results suggested that the
MUC5AC expression was dependent on MAPK activation, but
independent of PKA activation, which agreed with study of the
anti-inflammatory role of the cAMPPKA pathway .
Rp-8-BrcAMP is the most potent cAMP antagonists. Its role is to occupy
cAMP binding sites and prevents dissociation and thus activation
of the kinase holoenzyme. Rp-8-Br-cAMP discriminates between
the two isozymes of protein kinase A and prefers type I .
Thus,different doses of Br-8-Rp-cAMPs application showed no
dose-response of MUC5AC expression, which indicated that the
MUC5AC expression was independent of PKA activation. A
similar result was showed after pretreating cells with PKA
inhibitor H89. Other studies have shown that long-term tobacco
smoke resulted in desensitization of the cough receptors within the
airway epithelium and internalization of toll-like receptor 4 on
human macrophages and cystic fibrosis transmembrane
conductance regulator on pulmonary epithelia [33,34,35].
As mentioned previously, b-arrestin could desensitize and
internalize activated b2-AR [36,37]. Moreover, b-arrestin, as
non-classical b2-ARdependent signal binding to the endocytic
machinery, use additional docking sites to link various MAPKs.
ERK1/2, p38-MAPK and JNK are the main members of the
MAPK family involved in regulating MUC5AC expression .
Mucus overproduction induced by smoke inhalation was found
associated with increased epithelial MUC5AC protein expression
and depended on activation of the JNK pathway . A second
PKA-independent and MAPK-dependent pathway of b2-AR
activation has been reported in neurons .
b-arrestin has 2 isoforms, b-arrestin1 and b-arrestin 2. We used
siRNA to silence the expression of b-arrestin-1 and b-arrrestin-2 in
NCI-H292 cells. Silencing b-arrrestin2 expression suppressed
CSE-stimulated ERK1/2 activation, thus inhibiting MUC5AC
production (Figures 4 and 5). CSE exposure may have induced
b2AR internalization, because both isoforms of b-arrestin can
promote desensitization of b2-AR, but only b-arrestin2 promotes
internalization of b2-AR [37,41]. We demonstrated a potent
stimulatory effect of CSE on MUC5AC mucin secretion from
NCI-H292 cells, and activation of b2-AR with b-arrestin2 but not
PKA was involved in MUC5AC gene expression. We proposed a
detailed mechanism of b2-AR involved in CSE-induced
MUC5AC production: wtih CSE stimulation, b2-AR is activated,
which increases cAMP and recruits many b-arrestins. Increased
cAMP binds and activates PKA but does not stimulate MUC5AC
transcription and production. b-arrestin2 phosphorylates ERK1/2
and p38MAPK, the key players, which activate transcription
factors to stimulate MUC5AC transcription and production
We further established a rat model with a prominent feature of
airway inflammation and mucus secretion by CS exposure and
evaluated after chronic low-dose propranolol intervention.
Propranolol, one of non-selective b-blockers, is the most commonly
prescribed classes of cardiovascular medication. Despite increasing
evidence that BBs are safe and can actually be beneficial in
patients with COPD, their use in this population is limited .
Because of concerns that BBs might induce bronchospasm and
worsen lung function, there has been uncertainty with regard to
using b-blockers in COPD patients. Such concerns, however, have
been challenged by recent evidence: cardio-selective b-blockers are
less likely to cause bronchospasm, and, additionally, the risk of
bronchospasm can be reduced by starting b-blocker treatment at a
lower dose and slowly titrating up [28,43]. Moreover, treatment
with BBs may reduce the risk of exacerbations and improve
survival in patients with COPD but without overt cardiovascular
disease . In this study, echocardiography was performed on
the rats at baseline and repeated after smoking for 4 months to
evaluate the function of heart, but no overt cardiovascular disease
was found in the rat model (data not shown). Comparatively,
goblet cell hyperplasia and mucin hypersecretion occurred in
airway epithelia with CS exposure and could be inhibited by
chronic administration of propranolol (Figures 7 and 8). This
result suggested that the benefit from using propranolol in CS
induced animals did not necessarily correlate with reduced
cardiovascular side effects. The possible explanations for this
result include 1) propranolol inhibiting the activation of the
sympathetic nerve. Our previous study showed elevated serum
levels of norepinephrine in CS-exposed rats, and propranolol
significantly decreased the serum norepinephrine levels .
However, in airways of all species studied, particularly humans,
cholinergic mechanisms predominate and the sympathetic nervous
system influences the volume secretion and mucus content
probably by activation of alpha-adrenergic pathways . 2)
Propranolol increases b2-AR level in airway epithelia. In a murine
asthma model, chronic administration of b-blockers attenuating
mucus production was associated with upregulated b2-AR level in
airway epithelia, and transgenic overexpression of b2-AR in
airway epithelial cells decreased bronchoconstriction and airway
hyperresponsiveness [7,47]. However, we did not find a significant
altered b2-AR level in airway epithelia after propranolol treatment
(data not shown). 3) Propranolol inhibits b2-AR downstream
signaling molecules. b2-AR signaling was found required for the
full development of mucous metaplasia, and inhibition of all
b2AR signaling but not biased agonism was responsible for the
beneficial effects of chronic inverse-agonist treatment on mucous
metaplasia . Moreover, b2-AR is the most common b-AR
subtype expressed in various cell types in the lung including airway
epithelial cells. b2-AR level has been found increasing along the
human airway, and the density in the subsegmental bronchus and
lung parenchyma was approximately two-fold higher than that of
muscarinic acetylcholine receptors in the same region. b1-AR was
also detected in lung parenchyma but not in the bronchus, which
suggests the inhibition was mainly mediated through b2-AR .
Our study contains some limitations. 1) Mainstream CS is a
complex mixture inhaled into the respiratory system.
Approximately 4,700 substances have been identified in fresh tobacco
smoke. Many factors influence CS-stimulated phosphorylation of
ERK1/2, such as epidermal growth factor receptor, toll-like
receptor or NADPH oxidase, and interactions between these
factors and b2-AR [49,50]. 2) Propranolol is a nonselective b-AR
antagonist, but the contribution of b1-AR to MUC5AC
production is not involved here. However, our previous study found that
treatment with metoprolol, a b1-AR selective antagonist, did not
improve airway mucus hypersecretion in a rat model of CS
exposure (data not shown). The protective effect of propranolol in
the rat model exposed to CS may be mainly achieved by inhibiting
b2-AR signals. 3) In vitro systems usually lack complex interactions
with other cell types and the disease process can be affected by the
complex cytokine milieu established by inflammatory cells in
addition to CS exposure. Thus, the findings in vitro may not exactly
reflect responses in vivo.
In conclusion, our findings indicate a detrimental effect of
b2AR activation on airway mucus hypersecretion. Some pathological
signals of b2-AR are initiated under CS exposure and proper
application of b-blockers may, to a point, benefit airway disease.
The exploration of these mechanisms may contribute to the
optimization of b2-AR target therapy in smoking associated airway
disease, like COPD.
Transfection and knockdown efficiency of
Effect of propranolol on lung function.
We thank pathologist Pei Fei (Beijing, China) for the lung section
histopathology score and Laura Smales (BioMed Editing, Toronto,
Canada) for critical reading of the manuscript.
Conceived and designed the experiments: BH MX Youyi Zhang.
Performed the experiments: Y. Zhou Yuan Zhang YG. Analyzed the
data: Y. Zhou Yuan Zhang. Contributed reagents/materials/analysis tools:
Y. Zhou Yuan Zhang. Wrote the paper: Y. Zhou. Developed the research
ideas and experimental design: BH MX Youyi Zhang. Performed animal
experiments: Y. Zhou Yuan Zhang YG. Cell cultures, statistical analysis
and wrote manuscript: Y. Zhou. Manuscript writing and editing: BH MX
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