M3 mAChR-mediated IL-8 expression through PKC/NF-B signaling pathways

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  • 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

    Abstract

    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

    Introduction

    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 [37].

    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

    e-mail: hongzhuan_chen@hotmail.com

    Y.-Y. Cui

    e-mail: yongyaocui@hotmail.com

    Inflamm. Res.

    DOI 10.1007/s00011-014-0718-4 Inflammation Research

    123

  • 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

    autoimmune diseases.

    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

    [13]; 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 [1924]. Thus, the elimi-

    nation of interruptions from other cholinergic subtype

    receptors is important to elucidate the specific function of

    M3 mAChR.

    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-

    tadt, Germany).

    Cell culture

    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.

    123

  • 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-

    turers protocol.

    Data analysis

    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).

    Results

    Carbachol induces IL-8 expression via the M3 mAChR

    subtype

    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 [1316]. 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

    subtypes.

    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

    physiological condition.

    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

    123

  • Role of NF-kB activation in M3 mAChR-mediated IL-8

    expression

    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 [29]. 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.

    123

  • M3 mAChR-mediated IL-8 expression

    123

  • 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

    IL-8 expression

    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 [30]. 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

    MAPK activation.

    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.

    Discussion

    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-

    matory diseases.

    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.

    123

  • 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

    disease.

    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

    123

  • Z.-P. Xu et al.

    123

  • 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. [14] 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 [39], 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 [41]. 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. [14] and

    Oenema et al. [5], 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

    123

  • 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

    81273519).

    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

    123

    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

    DiscussionAcknowledgmentsReferences

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