ORIGINAL RESEARCH PAPER
M3 mAChR-mediated IL-8 expression through PKC/NF-jBsignaling pathways
Zu-Peng Xu Yun Song Kai Yang Wei Zhou Li-Na Hou
Liang Zhu Hong-Zhuan Chen Yong-Yao Cui
Received: 31 May 2013 / Revised: 2 January 2014 / Accepted: 22 January 2014
Springer Basel 2014
Objective M3 muscarinic acetylcholine receptor (mAChR)
plays an important role in the regulation of cytokine pro-
duction in inflammatory diseases. In this study, we
explored the precise role of M3 mAChR under stimulation
with agonist in IL-8 expression and of the signaling path-
way involved in this process.
Materials and methods Recombinant U2OS cells stably
expressing M3 mAChR as a model system were stimulated
by carbachol to evaluate the role of M3 mAChR in the
expression of IL-8.
Results Activation of M3 mAChR with carbachol
increased both IL-8 mRNA and protein expression in a
concentration-dependent manner. Elevated IL-8 expression
was completely antagonized by atropine, 4-DAMP and
tiotropium. M3 mAChR-mediated IL-8 expression was
almost completely inhibited by the NF-jB inhibitorBAY11-7082 and, to a lesser extent, by U0126, SB203580,
and SP600125, which are inhibitors for ERK1/2, p38, and
JNK, respectively. Furthermore, M3 mAChR-mediated
NF-jB activation and IL-8 expression were simultaneouslyattenuated by the PKC inhibitor calphostin C, whereas
PMA, a PKC activator, mimicked the effects of carbachol,
inducing IL-8 expression.
Conclusions Our findings offer insights into the specific
and critical role of M3 mAChR in regulating inflammatory
response and indicate M3 mAChR/PKC/NF-jB signalingaxis driven by endogenous acetylcholine as a potential
therapeutic targets for inflammatory diseases.
Keywords Recombinant U2OS cells M3 mAChR, muscarinic receptor IL-8 Signaling pathways
Muscarinic acetylcholine receptors (mAChRs) are mem-
bers of the G protein-coupled receptors (GPCRs) and are
composed of five receptor subtypes (M1M5), which reg-
ulate the activity of numerous fundamental central and
peripheral functions. Among them, the M1, M3 and M5
mAChR subtypes preferentially couple to the Gq/G11
family of G proteins, whereas the M2 and M4 subtypes
usually interact with the G0/Gi family. Consequently, dif-
ferent second messenger dependent pathways are activated
by each mAChR subtype [1, 2], resulting in the activation
of distinct downstream pathways.
Recent studies suggest that mAChRs mediate several
inflammatory events in a variety of cell types. Functional
mAChRs are expressed in various inflammatory related
cells, and activation of mAChRs triggers the release of a
number of inflammatory mediators and cytokines .
Within the mAChR family, the M3 mAChR subtype
mediates many important physiological functions. M3
mAChR-mediated events are involved in rheumatoid
arthritis and Sjogrens syndrome [8, 9], and the discovery
Responsible Editor: Liwu Li.
Z.-P. Xu and Y. Song are contributed equally to this article.
Z.-P. Xu Y. Song K. Yang W. Zhou L.-N. Hou L. Zhu H.-Z. Chen (&) Y.-Y. Cui (&)Department of Pharmacology, Shanghai Jiao Tong University
School of Medicine, 280 South Chongqing Road,
Shanghai 200025, China
DOI 10.1007/s00011-014-0718-4 Inflammation Research
of the anti-inflammatory effect of tiotropium, a M3-selec-
tive mAChR antagonist, provided new insight for the
inflammatory airway diseases such as chronic obstructive
pulmonary disease (COPD) and severe asthma [6, 1012].
It is reported that the expression of M3 mAChR dramati-
cally increases in pathological conditions and the increased
expression of M3 mAChR is an important incentive for
inflammation. Thus, M3 mAChR has emerged as an
important potential therapeutic target for inflammatory and
The mAChR agonists evoke cytokine production. For
example, bovine bronchial epithelial cells release neutro-
phil, monocyte and eosinophil chemotactic activities after
stimulation with acetylcholine. Acetylcholine also medi-
ates the release of IL-8 and leukotriene B4 in the 16HBE
human bronchial epithelial cell line [6, 14] and leukotri-
ene B4 in the A549 human alveolar epithelial cell line
; however, the effective concentration used in previ-
ous studies is relatively higher than the physiological
concentration [5, 1316]. Thus, it remains unanswered
whether physiological levels of acetylcholine are involved
in the regulation of cytokine production. As the five
mAChRs subtypes share a high degree of sequence
homology, the lack of specific selective compounds con-
sequently hinders the determination of the precise
function and signaling pathways of specific mAChR
subtypes using single traditional pharmacological
approaches [6, 14, 17, 18]. In addition, the reciprocal
interactions between the different mAChR subtypes co-
expressed in cells might strictly limit the definition of M3
mAChRs biological responses . Thus, the elimi-
nation of interruptions from other cholinergic subtype
receptors is important to elucidate the specific function of
IL-8 plays a major role in the inflammatory process by
recruiting neutrophils and T cells into inflammatory sites.
Significantly increased levels of IL-8 have been observed
in a variety of inflammatory and autoimmune diseases [25
28], and multiple signaling pathways, such as PKC, NF-jBand mitogen-activated protein kinase (MAPK) cascades,
are involved in IL-8 expression. Although there are some
insights into the relationship between M3 mAChR and IL-8
expression, the precise role of M3 mAChR under stimu-
lation with physiological levels of agonists in IL-8
expression and of the signaling pathway involved in this
process is unclear.
In this study, using a well-established recombinant cell
model, we investigated the role of M3 mAChR in IL-8
expression and explored whether physiological levels of
mAChR agonists could evoke IL-8 expression. Further-
more, we analyzed the specific signaling pathways
activated by M3 mAChR without the reciprocal interfer-
ence of other cholinergic subtype receptors in this process.
Materials and methods
Regents and antibodies
The following antibodies were used in this study: antibodies
specific for Erk1/2, SAPK/JNK, p38 MARK, phospho-Erk1/
2, phospho-SAPK/JNK, phospho-p38 MARK, IjBa, IKKa,IKKb, phospho-IjBa and phospho-IKKa/b were pur-chased from Cell Signaling Technology (Beverly, MA,
USA). An antibody specific for GAPDH and anti-mouse
and anti-rabbit IgG-conjugated horseradish peroxidase
secondary antibodies were purchased from Santa Cruz
Biotechnology. Carbachol, atropine, 4-DAMP, U0126,
SB203580, SP600125, PMA, and BAY11-7082 were pur-
chased from Sigma Chemical (St Louis, MO, USA).
Tiotropium was purchased from Santa Cruz Biotechnology
(Santa Cruz Biotechnology, Santa Cruz, CA, USA), and
Calphostin C was purchased from Merck (Merck, Darms-
Recombinant U2OS cells (Thermo Fisher Scientific, Wal-
tham, MA, USA) stably expressing human mAChR 3 (M3
mAChR, GenBank Acc. NM 000740) were cultured in
DMEM (Invitrogen, Carlsbad, CA, USA) containing 10 %
fetal bovine serum (Invitrogen, Carlsbad, CA, USA),
100 g/ml penicillin, 100 U/ml streptomycin sulfate (Invit-
rogen, Carlsbad, CA, USA), 0.5 mg/ml Geneticin
(Invitrogen, Carlsbad, CA, USA) and 1 mg/ml Zeocin
(Invitrogen, Carlsbad, CA, USA) in a humidified incubator
at 37 C with 5 % CO2.
Quantitative real-time RT-PCR
Total RNA was isolated from cells using RNA TRIzol
reagent (Invitrogen, Carlsbad, CA, USA) after administra-
tion of carbachol or PMA for 8 h. First-strand
complementary DNA (cDNA) synthesis was performed
using the RevertAidTM first-strand cDNA synthesis kit
(Fermentas, Burlington, CA, USA). The reverse-transcrip-
tase (RT) mixture was mixed with 2*SYBR Green
polymerase chain reaction (PCR) agent (QIAGEN, Hilden,
Germany), and gene-specific primers were used in a final
volume of 20 ll. Oligonucleotide primers specific for the IL-8 gene were used (50-CTTCTCCACAACCCTCTG-30 and50-ACTCCAAACCTTTCCACC-30) (Invitrogen, Carlsbad,CA, USA). The levels of gene expression were normalized
using GAPDH (50-GTGAAGGTCGGAGTCAACG-30 and50-TGAGGTCAATGAAGGGGTC-30). Quantitative real-time RT-PCR was performed in a model rotor-gene-3000A
(Corbelt Research, Australia). The final results, expressed as
fold differences in target gene expression, relating to both
Z.-P. Xu et al.
endogenous controls gene expression and calibrator, were
determined using the 2DDCT method.High content screening (HCS) for NF-jB translocation
from the cytoplasm to the nucleus.
After fixation and permeabilization, 50 ll of NF-jBprimary antibody solution (Cellomics, Pittsburgh, PA,
USA) was added and incubated for 1 h at 37 C. Afterwashing twice with 200 ll wash buffer, 50 ll DyLight 549Goat Anti-Rabbit/Hoechst solution (Cellomics, Pittsburgh,
PA, USA) was added to each well and incubated at room
temperature for 1 h. Next, the plate was washed twice with
100 ll wash buffer and analyzed using an HCS systemreader (Cellomics, Pittsburgh, PA, USA). The average
intensity ratio between the nucleus and cytoplasm repre-
sented the level of NF-jB activation. Data analysis wasperformed using the Molecular Translocation BioApplica-
tion software (Cellomics, Pittsburgh, PA, USA).
Western blotting analysis
The cells were washed once with cold PBS, and total
proteins were extracted with lysis buffer. The harvested
lysates were centrifuged for 10 min at 4 C to pellet thecellular debris. Equal amounts of protein were loaded
onto a 10 % SDS-PAGE and transferred to polyvinylidene
fluoride membranes. To avoid non-specific binding, the
membranes were blocked with blocking buffer (TrisHCl
50 mM, NaCl 150 mM, 0.1 % TWEEN-20, 5 % non-fat
dried milk powder) for 1 h at room temperature. The
membranes were then incubated with specific primary
antibodies overnight at 4 C. After washing the mem-branes three times with 0.1 % TBS-T (TrisHCl 50 mM,
NaCl 150 mM, 0.1 % TWEEN-20) for 10 min, the
membranes were incubated with a secondary antibody
conjugated to HRP for 1 h at room temperature, followed
by three additional washes with 0.1 % TBS-T. The pro-
tein bands were visualized on film using enhanced
chemiluminescence reagents and analyzed by densitome-
try. All of the protein bands were normalized to GAPDH,
Erk1/2, SAPK/JNK, p38 MARK, IKKa or IKKb.
Measurement of IL-8 in the supernatant
After administration of carbachol or PMA for 24 h, the
release of IL-8 into the cell supernatant was determined
using a commercially available enzyme-linked immuno-
sorbent assay (ELISA) (R&D Systems, Minneapolis, MN,
USA), which was performed according to the manufac-
All of the data are presented as the mean SEM. Differ-
ences between groups were analyzed using ANOVA,
followed by a Dunnetts test for selected pairs, if appro-
priate. Differences between means were considered
significant at a p value of \0.05. All of the statisticalanalyses were performed using Prism version 5.0 (Graph-
Pad Software, San Diego, CA, USA).
Carbachol induces IL-8 expression via the M3 mAChR
Although previous studies suggest that M3 mAChR is
closely correlated with the release of IL-8, the effective
concentration of agonist is notably higher than physiolog-
ical concentrations, and the inclusion of interference from
other mAChR subtypes adds an additional layer of com-
plexity to this process . Using recombinant U2OS
cells stably expressing M3 mAChR as a model system, we
explored the specific role of M3 mAChR in IL-8 expres-
sion without the interfering effects of other mAChR
Our present study showed that IL-8 mRNA and protein
expression were significantly induced by the stable ace-
tylcholine analogue carbachol in a concentration-
dependent manner (Fig. 1a, b). When the cells were
exposed to increasing concentrations of carbachol, the IL-8
expression exhibited a greater than 2,000-fold increase at
the mRNA level and a greater than 50-fold increase at the
protein level, which indicated a strong modulatory effect of
M3 mAChR in IL-8 expression. Interestingly, a statistically
significant increase of IL-8 expression occurred at extre-
mely low concentrations of carbachol (0.01 lM formRNA; 0.001 lM for protein; Fig. 1a, b), offering thesupport for the modulatory effect of M3 mAChR under the
To confirm further the effects of M3 mAChR in IL-8
expression, a nonselective mAChR antagonist, atropine,
and the M3 mAChR selective antagonists, 4-DAMP and
tiotropium, were added to the cell culture medium 30 min
prior to carbachol administration. As shown in Fig. 1c and
d, atropine (1 lM), 4-DAMP (1 lM) and tiotropium(1 lM) completely inhibited IL-8 mRNA and proteinexpression induced by carbachol. Taken together, these
results indicate that M3 mAChR is an important receptor
mediating effects of acetylcholine analogue carbachol in
inducing expression of IL-8.
M3 mAChR-mediated IL-8 expression
Role of NF-kB activation in M3 mAChR-mediated IL-8
In addition to IL-8, many types of cytokine release are
regulated by NF-jB. Normally, NF-jB exists in its inactiveform in the cytoplasm, where it binds to the inhibitory
kappa B (IkB) protein. Extracellular stimuli activate the
NF-jB signaling pathway, which leads to the activation ofIkB kinase (IKK), which, in turn, phosphorylates IkB.
Phosphorylated IkB undergoes degradation by proteasomal
release of NF-jB, which subsequently translocates into thenucleus and initiates transcription. Phosphorylation of
IKKa/b and IjBa, degradation of IjBa and the translo-cation of NF-jB are all reliable markers of NF-jBactivation [13, 14, 16].
To explore the role of NF-jB in M3 mAChR-mediatedIL-8 expression, we first explored whether M3 mAChR
could modify the activation of NF-jB . Our resultsshowed that physiological concentrations of carbachol
(1 lM) significantly increased the phosphorylation ofIKKa/b and IjBa, the degradation of IjBa (Fig. 2a) andthe translocation of NF-jB (Fig. 2b). In addition, all of theeffects of carbachol administration were abolished by the
mAChR antagonists, atropine, 4-DAMP and tiotropium
(Fig. 2a, b), indicating that NF-jB activation induced bycarbachol was mediated by the M3 mAChR subtype.
Furthermore, blockade of the NF-jB pathway was per-formed using the NF-jB inhibitor BAY11-7082 to explorewhether NF-jB is required for M3 mAChR-mediated IL-8expression. BAY11-7082 completely inhibited the
Fig. 1 Cells were incubatedwith different concentrations of
carbachol (0.00110 lM). Inthe appropriate group, atropine
(1 lM), 4-DAMP (1 lM) andtiotropium (1 lM) were addedto the cells 60 min prior to the
stimulation with carbachol
(1 lM). Carbachol increased themRNA expression of IL-8
(a) and the IL-8 level insupernatants (b) in a dose-dependent manner. Atropine,
4-DAMP and tiotropium
completely abolished the
carbachol-induced increase in
mRNA (c) and proteinexpression of IL-8 in
supernatants (d). The data areexpressed as the mean SEM
from three independent
experiments. **p \ 0.01 vs.control; ##p \ 0.01 vs.carbachol; $p \ 0.05 vs. controlby t test
cFig. 2 Cells were stimulated with carbachol (1 lM) alone or in thepresence of atropine (1 lM), 4-DAMP (1 lM), tiotropium (1 lM), orNF-jB inhibitor BAY11-7082 (100 lM), which were added to thecultures 60 min prior to the addition of carbachol. Phosphorylation of
IKKa/b and IjBa and degradation of IjBa were detected by westernblotting, and the translocation of NF-jB was detected by HCS afterstimulation with carbachol for 30 min. Phosphorylation of IKKa/b,phosphorylation of IjBa and degradation of IjBa were normalized toIKKa/IKKb, GAPDH and GAPDH, respectively. The averageintensity ratio between the nucleus and cytoplasm represented the
level of NF-jB activation. Carbachol significantly increased thephosphorylation of IKKa/b and IjBa, the degradation of IjBa(a) and the translocation of NF-jB (b) and the effect was completelyabolished by atropine, 4-DAMP and tiotropium (a, b). BAY11-7082(100 lM) completely inhibited the carbachol-induced increase inphosphorylated IjBa, the degradation of IjBa (c) and the translo-cation of NF-jB (d). The carbachol-induced increase of IL-8expression at both the mRNA and protein levels was inhibited by
BAY11-7082 (e, f). The data are expressed as the mean SEM fromthree independent experiments. **p \ 0.01 vs. control; ##p \ 0.01 vs.carbachol
Z.-P. Xu et al.
M3 mAChR-mediated IL-8 expression
carbachol-induced increase of phosphorylated IjBa, thedegradation of IjBa and the translocation of NF-jB(Fig. 2c, d), and after the administration of BAY11-7082,
the increased IL-8 expression induced by carbachol was
decreased by over 99 % at both the mRNA and protein
levels (Fig. 2e, f), suggesting that NF-jB plays a primaryrole in M3 mAChR-mediated events.
Role of MAPK activation in M3 mAChR-mediated
Mitogen-activated protein kinase cascades, including
ERK1/2, p38 and JNK, comprise one of the major signal-
ing systems by which cells transduce and integrate diverse
intracellular signals. Given their important role in IL-8
expression, we examined the role of MAPKs in M3
mAChR-mediated IL-8 expression . First, we deter-
mined whether M3 mAChR might activate MAPK
cascades. Western blotting analysis results showed that
physiological carbachol (1 lM) significantly increased thephosphorylation of ERK1/2, p38 and JNK, and this effect
was completely inhibited by atropine, 4-DAMP and tiot-
ropium (Fig. 3a), suggesting that M3 mAChR mediates
To investigate further whether M3 mAChR-mediated
IL-8 expression required the activation of ERK1/2, p38 and
JNK pathways, the specific MAPK inhibitors U0126,
SB203580, and SP600125, which are inhibitors for ERK1/
2, p38, and JNK, respectively, were added to the cultures
60 min prior to the addition of carbachol. We found that
carbachol-induced MAPK activation was completely
abolished by the addition of the specific MAPK inhibitors
U0126, SB203580, and SP600125 (Fig. 3bd). However,
the M3 mAChR-mediated increase in IL-8 mRNA and
protein expression was partially, but significantly, inhibited
by the specific MAPK inhibitors U0126, SB203580, and
SP600125 (Fig. 3e, f). Interestingly, blocking the MAPKs
pathway resulted in a decrease in IL-8 expression by 46, 39
and 30 % for mRNA and 29, 56 and 50 % for protein,
respectively, although MAPK signaling was completely
abolished by specific MAPK inhibitors. These results
suggested that M3 mAChR-mediated IL-8 expression
induced by carbachol is also modulated by the MAPK
pathways, but to a lesser extent compared to NF-jB.
Involvement of PKC in carbachol-induced NF-KB
activation and IL-8 expression via M3 mAChR subtype
Given the predominant role of NF-jB in M3 mAChR-induced IL-8 expression, we explored whether PKC was
essential for the NF-jB activation induced by carbachol.We found that the carbachol-induced increase of phos-
phorylated IKKa/b and IjBa, the degradation of IjBa and
the translocation of NF-jB were significantly attenuated bythe PKC inhibitor calphostin C (Fig. 4a, b), indicating that
carbachol-induced activation of NF-jB is dependent onPKC. Moreover, calphostin C inhibited the increase of IL-8
expression induced by carbachol both at the mRNA and
protein levels (Fig. 4c, d), and the PKC activator PMA,
which mimics the effects of carbachol, induced a concen-
tration-dependent increase of IL-8 mRNA and protein
expression (Fig. 4e, f). Taken together, we demonstrated
that the M3 mAChR/PKC/NF-jB signaling axis plays akey role in the inflammatory process.
The major finding of this study was that activation of M3
mAChR by the acetylcholine analogue carbachol induced a
significant concentration-dependent increase in IL-8
mRNA and protein expression at physiological concentra-
tions. M3 mAChR-mediated IL-8 mRNA and protein
expression was completely inhibited by an NF-jB inhibitorand, to a lesser extent, by MAPK inhibitors, suggesting
involvement of the NF-jB and MAPK signaling pathwaysin M3 mAChR-mediated IL-8 expression. Moreover, NF-
jB plays a predominant role in M3 mAChR-mediatedsignal transduction events. Furthermore, the M3 mAChR-
mediated NF-jB activation and IL-8 expression was sig-nificantly attenuated by the PKC inhibitor calphostin C and
enhanced by the PKC activator PMA (Fig. 5). Taken
together, these findings offer insights into the specific and
critical role of M3 mAChR plays an important role in
regulating inflammatory response and indicate M3
mAChR/PKC/NF-jB signaling axis driven by endogenousacetylcholine as a potential therapeutic targets for inflam-
In the present study, we demonstrated that the acetyl-
choline analogue carbachol increased IL-8 expression at
both the mRNA and protein levels following M3 mAChR
activation, which was consistent with our previous report
and another report on the effect of muscarinic receptor
agonist on IL-8 production in a 16HBE cell line and den-
dritic cells [14, 31]. In addition, the mAChR antagonist
tiotropium also inhibited IL-8 release induced either by
cigarette smoke extract in 16HBE cells or by lipopoly-
saccharide in BEAS-2B cells [6, 32]. In these two latter
studies, the only source of ligands for the mAChRs was
endogenously released acetylcholine. Although acetylcho-
line is the predominant parasympathetic neurotransmitter,
it is also synthesized by non-neuronal tissues and serves as
an autocrine/paracrine modulator in inflammation and
immune response. Acetylcholine is usually released from
nerve endings or activated cells that are rapidly degraded
by cholinesterase. The physiological concentration in vivo
Z.-P. Xu et al.
is no higher than 1 lM [6, 3335]. However, until now, theeffective concentration of the cholinergic agonist in
reported research was much higher than the physiological
concentration [5, 13, 14, 16]. As the rapid degradation by
cholinesterase, elucidation of the effects of the physiolog-
ical concentration of agonists is particularly important.
Interestingly, our present study demonstrated that the stable
acetylcholine analogue carbachol, when within the physi-
ologically achievable concentration range, could induce
significant IL-8 expression at both the mRNA and protein
levels, although carbachol is completely resistant to
hydrolysis by cholinesterase and exhibits a longer duration
of action compared to acetylcholine. Thus, this finding may
highlight and accentuate an important regulatory role for
endogenous acetylcholine, acting through the activation of
M3 mAChR, in the pathophysiology of inflammatory
In the present study, we found that carbachol-induced
events were antagonized by the nonselective mAChR
antagonist atropine and the M3 mAChR selective
Fig. 3 Cells were stimulated with carbachol (1 lM) alone or in thepresence of atropine (1 lM), 4-DAMP (1 lM), tiotropium (1 lM), orthe specific MAPK inhibitors (U0126, 1 lM; SB203580, 10 lM andSP600125, 100 lM, for ERK, p38 MAPK and JNK, respectively),which were added to the cultures 60 min prior to the addition of
carbachol. Phosphorylation of ERK1/2, p38 and JNK was detected by
western blotting after stimulation with carbachol for 30 min. All of
the protein bands were normalized to total ERK1/2, total p38 or total
JNK for ERK1/2, p38 and JNK, respectively. Carbachol significantly
increased the phosphorylation of ERK1/2, p38 and JNK (a), andatropine, 4-DAMP and tiotropium completely inhibited the phos-
phorylation of ERK1/2, p38 and JNK (a). U0126, SB203580 orSP600125 completely inhibited the activation of ERK1/2, p38 and
JNK (bd). The carbachol-induced increase in IL-8 mRNA (e) andprotein (f) expression was decreased by U0126, SB203580 andSP600125. The data are expressed as mean SEM from three
independent experiments. **p \ 0.01 vs. control; ##p \ 0.01 vs.carbachol
M3 mAChR-mediated IL-8 expression
Z.-P. Xu et al.
antagonists, 4-DAMP and tiotropium, which further dem-
onstrate the specific role of M3 mAChR in mediating IL-8
expression. The efficacy of M3 mAChR activation-induced
IL-8 expression was also substantial, resulting in a 50-fold
increase at the protein level and a 2,000-fold increase at the
mRNA level. These results indicated that, under patho-
logical conditions, M3 mAChR is a potential leading
contributor to inflammation and not just a minor partici-
pant, and this explains the reason for the M3 mAChR
antagonists anti-inflammatory effect in COPD and severe
asthma. This finding is further supported in a report by
Profita et al.  demonstrating that acetylcholine-induced
IL-8 release from 16HBE cells is blocked by tiotropium, an
M3-selective mAChR antagonist. Several studies have
suggested that an adaptive change in mAChR density may
occur under different environmental conditions; for
example, increased expression of M3 mAChR and changes
in M3 mAChR expression can influence disease severity or
the efficacy of therapeutic agents [3, 6, 14, 3638].
Increased expression of M3 mAChR is an important
incentive for inflammation. Up-regulation and/or aug-
mented expression of mAChRs could enhance the
sensitivity of endogenous ligands at the same concentra-
tion, and this enhancement contributes to the effectiveness
of physiological concentrations of the agonist [5, 14, 16].
Changes in mAChR density may also affect the reciprocal
interactions between different mAChR subtypes co-
expressed in the same cells , resulting in an imbalance
of the orchestral state of the organism [1924, 40]. The
enhanced expression of M3 mAChR may have a substantial
effect on the potency of its pro-inflammatory role in dis-
ease under physiological concentration stimulation.
However, further studies are needed to clarify this point.
The last portion of our study was devoted to the
examination of signaling pathways, which coordinately
regulate IL-8 transcription following the activation of M3
mAChR. IL-8 expression is closely related to the activation
of the NF-jB and MAPK pathways . In the presentstudy, we demonstrated that M3 mAChR activation by
physiological carbachol induced IL-8 expression, which
was associated with the activation of downstream signaling
events, including activation of the NF-jB and MAPK(ERK, p38 and JNK) signaling pathways. This demon-
stration was initially based on evidence that carbachol
increased the phosphorylation of IKKa/b and IjBa, thedegradation of IjBa and the translocation of NF-jB, aswell as the phosphorylation of ERK1/2, p38 and JNK.
Furthermore, direct inhibition of the IjB/NF-jB, ERK1/2,p38 and JNK signaling pathways with specific inhibitors
also abrogated carbachol-induced IL-8 expression. These
findings are consistent with those of Profita et al.  and
Oenema et al. , who reported that mAChR stimulation
mediated the release of IL-8 via activation of the NF-jBand ERK1/2 pathways in the 16HBE cell line or in human
airway smooth muscle cells and confirmed the involvement
of the JNK pathway in M3 mAChR-induced IL-8 expres-
sion. Our findings and others further confirmed that both
the NF-jB and MAPK signaling pathways are involved inIL-8 expression through M3 mAChR activated by musca-
rinic receptor agonists. Interestingly, the present study
demonstrated that the carbachol-induced IL-8 expression
by an NF-jB-mediated mechanism was more potentcompared to the MAPK-mediated mechanism, suggesting
that M3 mAChR-mediated activation of the NF-jB path-way is the predominant signaling pathway in carbachol that
induces IL-8 expression. This result differs from other
reports, which demonstrated an identical role of NF-jB andMAPKs in mAChR activation-induced cytokine expression
[5, 6, 14]. However, it still remains unclear whether this
discrepancy is related to the reciprocal effect of other
mAChR subtypes, and thus, further studies are needed to
clarify these mechanisms.
M3 mAChR is a G-protein-mediated receptor, and PKC
plays an important role in most G-protein-mediated
receptor mediating biological effects and often acts
upstream of NF-jB. In the present study, we found that thecarbachol-induced increase in phosphorylated IKKa/b andIjBa, the degradation of IjBa and the translocation of NF-jB were significantly attenuated by the PKC inhibitorcalphostin C and that the activation of PKC with PMA,
which mimics the effects of carbachol, could significantly
enhance IL-8 expression. These results confirmed that the
activation of NF-jB induced by carbachol via M3 mAChRis PKC-dependent. This result is consistent with a previous
report, which demonstrated that PKC, which is strongly
activated by cholinergic receptor agonists, stimulates sev-
eral downstream pathways, including the IKKa/b, IjBa,NF-jB and Raf-1/MEK/ERK1/2 signaling pathways invarious cell types, and thus mediates cell survival and pro-
inflammatory signaling [14, 4245].
bFig. 4 Cells were stimulated with carbachol (1 lM) or PMA(0.0011 lM) alone or in the presence of calphostin C (5 lM), whichwas added to the cultures 60 min prior to the addition of carbachol.
Phosphorylation of IKKa/b and IjBa and the degradation of IjBawere detected by western blotting, and translocation of NF-jB wasdetected by HCS after stimulation with carbachol for 30 min.
Phosphorylation of IKKa/b, phosphorylation of IjBa and degradationof IjBa were normalized to IKKa/b, GAPDH and GAPDH,respectively. The average intensity ratio between the nucleus and
cytoplasm represented the level of NF-jB activation. Calphostin Ccompletely inhibited the increase in the phosphorylation of IKKa/band IjBa, the degradation of IjBa (a) and the translocation of NF-jBinduced by carbachol (b). The carbachol-induced IL-8 expressionboth at the mRNA and protein levels was inhibited with calphostin C
by 99 % (c) and 97 % (d), respectively. PMA also increased themRNA (e) and protein (f) levels of IL-8 in a dose-dependent manner.The data are expressed as the mean SEM from three independent
experiments. **p \ 0.01 vs. control; ##p \ 0.01 vs. carbachol
M3 mAChR-mediated IL-8 expression
These results constitute the first evidence regarding the
precise role of M3 mAChR in IL-8 expression and explo-
ration of the signaling pathways related to this biological
process using a well-established recombinant cell model.
Our results indicate that the acetylcholine analogue car-
bachol stimulates IL-8 expression both at the mRNA and
protein levels following M3 mAChR activation at the
physiological concentration, and M3 mAChR-mediated IL-
8 expression is predominantly mediated by PKCNF-jB-dependent pathways. Our findings offer insights into the
specific and critical role of M3 mAChR in regulating
inflammatory response and indicate M3 mAChR/PKC/NF-
jB signaling axis as a potential therapeutic targets forinflammatory diseases.
Acknowledgments This work was funded by the National NaturalScience Foundation of China (No. 30873109, 81173084, and
Conflict of interest The authors have no competing interests(financial or otherwise) with respect to this article.
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M3 mAChR-mediated IL-8 expression
M3 mAChR-mediated IL-8 expression through PKC/NF- kappa B signaling pathwaysAbstractObjectiveMaterials and methodsResultsConclusions
IntroductionMaterials and methodsRegents and antibodiesCell cultureQuantitative real-time RT-PCRWestern blotting analysisMeasurement of IL-8 in the supernatantData analysis
ResultsCarbachol induces IL-8 expression via the M3 mAChR subtypeRole of NF-kB activation in M3 mAChR-mediated IL-8 expressionRole of MAPK activation in M3 mAChR-mediated IL-8 expressionInvolvement of PKC in carbachol-induced NF-KB activation and IL-8 expression via M3 mAChR subtype