IL-4-induced caveolin-1-containing lipid rafts aggregation contributes to MUC5AC synthesis in bronchial epithelial cells
Xia et al. Respiratory Research
IL-4-induced caveolin-1-containing lipid rafts aggregation contributes to MUC5AC synthesis in bronchial epithelial cells
Yu Xia 0 1
Peng-Cheng Cai 0 4
Fan Yu 3
Liang Xiong 3
Xin-Liang He 3
Shan-Shan Rao 1
Feng Chen 6
Xiang-Ping Yang 5
Wan-Li Ma 2 3
Hong Ye 1 2
0 Equal contributors
1 Department of Pathophysiology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology , Wuhan, Hubei 430030 , China
2 Key Laboratory of Pulmonary Diseases, Ministry of Health of China , Wuhan, Hubei , China
3 Department of Respiratory and Critical Care Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology , Wuhan, Hubei , China
4 Department of Clinical Laboratory, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology , Wuhan, Hubei , China
5 Department of Immunology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology , Wuhan, Hubei , China
6 Department of Forensic Medicine, Nanjing Medical University , Nanjing, Jiangsu , China
Background: Mucus overproduction is an important feature of asthma. Interleukin (IL)-4 is required for allergeninduced airway inflammation and mucus production. MUC5AC gene expression is regulated by transcript factors NF-κB. The intracellular Ca2+ ([Ca2+]i) signal is required for activation of NF-κB. The transient receptor potential canonical 1 (TRPC1) channel has been shown to contribute for agonist-stimulated Ca2+ influx in some types of cells. However, the relationships among IL-4, TRPC1 and mucus overproduction in bronchial epithelial cells (BECs) in asthma are poorly understood. Methods: BECs were isolated from large bronchial airway of rats and used as cell model. To present changes of lipid raft, caveolin-1 and TRPC1, immunofluorescence staining and sucrose gradient centrifugation were performed. [Ca2+]i was measured after loading with Fura-2. NF-κB activities were measured by an ELISA-based assay. MUC5AC mRNA and protein levels were detected by real-time quantitative RT-PCR, ELISA analysis and immunofluorescence staining respectively. Results: IL-4 induced Ca2+ influx in BECs, and this was blocked by a Ca2+ influx inhibitor (2-APB). 2-APB also prevented MUC5AC protein synthesis induced by IL-4. Depletion of extracellular Ca2+ resulted in partial decrease in expression of MUC5AC in IL-4 treated cells. NF-κB rather than STAT6 activation mediated IL-4-induced MUC5AC protein synthesis. Then the mechanism of Ca2+ influx was investigated. Immunofluorescence staining and sucrose gradient centrifugation revealed that caveolin-1-containing lipid rafts aggregation was involved in TRPC1 activation and Ca2+ influx in BECs. Lastly, the data revealed that blocking lipid rafts aggregation exactly prevented Ca2+ influx, NF-κB activation and MUC5AC synthesis induced by IL-4. Conclusions: Our results indicate that IL-4-induced caveolin-1-containing lipid rafts aggregation at least partly contributes to MUC5AC synthesis in BECs.
IL-4; Bronchial epithelial cells; Lipid rafts; Intracellular Ca2 +; MUC5AC; Asthma
Mucus overproduction is an important feature of asthma
]. Excessive accumulation of airway mucus leads to the
formation of mucous plugs that reduce the effective
airway diameter and increase airway resistance. Mucins are
glycoproteins that provide mucus with its viscoelasticity.
MUC2, MUC4, MUC5AC, and MUC5B proteins are
considered to be airway mucins. In asthma, there is
over-expression of the major mucin glycoprotein,
Allergic pulmonary inflammation and airway
hyperreactivity in most cases of asthma is closely related in T
helper type 2 cells (Th2) responses [
]. Th2 cells
predominantly secrete cytokines interleukin (IL)-4, IL-5,
IL9 and IL-13, which play a central role in the
pathophysiology of asthma. These type 2 cytokines are targets for
pharmaceutical intervention of asthma. Studies showed
that IL-4 is required for allergen-induced airway
inflammation and mucus production [
expression is increased in mice over-expressing IL-4 compared
to transgenic-negative control [
]. IL-4 binds to IL-4
receptor in airway cells and can trigger STAT6 and NF-κB
signal pathway which is likely to be involved in the
regulation of MUC5AC expression.
MUC5AC gene expression is regulated by transcript
factors NF-κB and STAT6 [
]. NF-κB is involved in
cytokine-induced MUC5AC expression. IL-17 promoted
NF-κB translocation which subsequently binds to the
NFκB-binding sequences of the MUC5AC promoter, and
leads to up-regulation of MUC5AC expression in human
bronchial epithelial cells [
]. The intracellular Ca2+
([Ca2+]i) signal is required for activation of NF-κB [
and subsequently regulates downstream gene expression.
But how Ca2+ signal is triggered under pathophysiological
conditions remains poorly understood.
Transient receptor potential canonical 1 (TRPC1) is a
transmembrane protein expressed in a range of vertebrate
cells. TRPC1 channel has been shown to contribute to
agonist-stimulated Ca2+ influx in salivary cells and
pancreatic acinar cells [
]. Extensive studies have confirmed
the contribution of TRPC1 to store-operated Ca2+ entry
(SOCE). The interaction between TRPC1 and the key
components of SOCE, STIM1, and Orai1 determines the
activation of TRPC1 [
]. TRPC1 is sub-cellular
compartmentalised, at least in part in cholesterol-rich caveolae.
Plasma membrane lipid rafts domains (LRD), which
contain high concentrations of cholesterol and
sphingolipids, are known to function as centers for the assembly
of signaling complexes. Such assembly is known to
regulate cellular functions such as transcytosis, protein
sorting, cell adhesion and migration as so on. Lipid rafts
aggregation facilitate the formation of the
STIM1-Orai1TRPC1 complex and the activation of SOCE [
Caveolin-1, a cholesterol-binding protein is involved in
the generation of caveolar lipid rafts. The
agoniststimulated Ca2+ signals have been shown to originate at
caveolin-1 enriched plasma membrane regions [
In asthma, there are increases in the number of
mucusproducing goblet cells in airway epithelium . The
bronchial epithelial cell (BEC) is also one of important
cell types that produce mucus production [
the role of lipid rafts/TRPC1 and Ca2+ influx in BECs of
asthma is unknown.
We hypothesized here that IL-4 increased BECs
MUC5AC gene expression through lipid rafts
aggregation and TRPC1 channel activation. In the current study,
we found that IL-4 caused caveolin-1 containing lipid
rafts aggregation and TPRC1 colocalization, and
disruption of lipid rafts prevented IL-4 induced NF-κB
activation and MUC5AC expression.
Reagents and materials
Methyl-β-cyclodextrin (M-βCD), 2-Aminoethyl
diphenylborinate (2-APB), ethylene glycol tetraacetic acid
(EGTA) and Pronase E were purchased from
SigmaAldrich (St. Louis, MO, USA). Epithelial cell medium
was obtained from Cell Biologics (Chicago, IL, USA).
IL4 was obtained from Pepro Tech (Rocky Hill, NJ, USA).
Anti-caveolin-1 antibody was purchased from BD
Transduction Laboratories (Lexington, KY, USA), rabbit
antiTRPC1 polyclonal antibody was purchased from Abcam
(Cambridge, UK), anti-P65 antibody was purchased from
Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Cholera toxin subunit B-Alexa Fluor 488 and Trizol Reagent
were obtained from Life Technology (Grand Island, NY,
USA). Fura-2-AM was purchased from Dojindo
laboratories (Kumamoto, Japan). MUC5AC ELISA kit was obtained
from Cloud-Clone Corp (Wuhan, China). Anti-MUC5AC
antibody was purchased from Absin (Shanghai, China).
Antibodies against β-actin, p-STAT6 and p-P65 were
purchased from Cell Signaling Technology (Danvers, MA,
USA). Specific STAT6 inhibitor AS1517499 and selective
IκB kinase (IKK) inhibitor IKK16 was purchased from
MedChem Express (Monmouth Junction, NJ, USA).
Isolation and primary culture of rat BECs
BECs were isolated from large bronchial airway of rat as
described in the literature [
]. The experiments were
performed in accordance with the Guide for the Care
and Use of Laboratory Animals and approved by the
Institutional Animal Care and Use Committee (IACUC) of
the Tongji Medical College, Huazhong University of
Science and Technology. In brief, airway was isolated from
rat under sterile conditions, rinsed with ice D-Hanks
twice, then digested with 1% Pronase E in DMEM/F-12
at 4°C for 14 h. Then BECs were harvested with ice
Dhanks contain 5% newborn bovine serum (NBS) and
centrifuged at 1000 rpm for 5 min. The spun down cells
were resuspended with epithelial cell medium (Cell
Biologics, Chicago, IL, USA) and incubated in a 5% CO2
incubator (Thermo Fisher Forma, Waltham, MA, USA) at
37 °C. These cells were used in experiments without
subculture. In most experiments, BECs were treated by
the indicated factors in serum-free medium when the
cells were 60–70% confluence. For MUC5AC
immunostaining, the isolated cells digested with protease were
cultured on cover slip with serum-containing medium
for 24 h in the Petri dish. Some digested cells were
presented as small clusters, but not completely separated
cells. IL-4 containing serum-free medium was
administered when the cells reached about 30% confluence.
BECs [Ca2+]i were measured after loading with 10 μM of
the acetoxymethyl ester form of Fura-2 for 30 min at
room temperature. Then, the fluorescence of Fura-2 was
recorded with a stimulation of 20 ng/ml IL-4, in the
presence or absence of 10 mM M-βCD or 50 μM 2-APB after
excitation at 340 ± 10 and 380 ± 10 nm using a xenon
short-arc lamp (Ushio). Bandpass interference filters
(Omega Optical, Brattleboro, VT 05301) selected
wavelength bands of emitted fluorescence at 510 ± 10 nm. In
the process of [Ca2+]i measurements, we firstly got stable
[Ca2+]i signal, 250 s later, we treated cells with 2-APB or
IL-4. M-βCD was used before [Ca2+]i measurements. In
some experiments, we firstly treated cells with 2-APB or
M-βCD, then added IL-4 to the cells at 1800s. Emitted
Fura-2 fluorescence was collected and measured using a
spectrofluorometer (PTI, Deltascan).
To acquire the final [Ca2+]i with the ratios from Fura-2
fluorescence, we had detected the minimum and
maximum Fura-2 fluorescence ratio (Rmin and Rmax) with Ca2
+-free buffer without calcium (8.182 g NaCl, 0.335 g KCl,
0.385 g EGTA, 2 g Glucose, 1.2 g HEPES, 0.2 g MgCl2, 1 L
H2O, PH7.4) and Ca2+-high buffer with high
concentration of Ca2+ (8.182 g NaCl, 0.335 g KCl, 0.555 g CaCl2, 2 g
Glucose, 1.2 g HEPES, 0.2 g MgCl2, 1 L H2O, PH7.4). The
Rmin was determined using Ca2+-free buffer and
stimulated by 3 μM ionomycin (Sigma). The same to Rmin, the
Rmax was measured with Ca2+-high buffer and stimulated
by 3 μM ionomycin. The final [Ca2+]i was calculated with
Rmin and Rmax follow this equation: [Ca2+]i = Kd*β*(R –
Rmin)/(Rmax – R), Kd is a constant which is 224 for BECs,
and the β values is the fluorescence intensity emitted by
380 ± 10 nm within Ca2+-free buffer. Ca2+ influx duration
was calculated from the beginning of Ca2+ level rise to the
time of Ca2+ level return to the base.
NF-κB activity assay
An ELISA-based assay was performed to measure
endogenous NF-κB activities as described previously [
after BECs were treated with 20 ng/ml IL-4 for 24 h in the
presence or absence of M-βCD or 2-APB (M-βCD or
2APB was administrated half an hour before IL-4 using), the
cells were lysed with lysis buffer (20 mM HEPES pH 7.5,
0.35 M NaCl, 20% glycerol, 1% NP-40, 1 mM MgCl2·6H2O,
0.5 mM EDTA, 0.1 mM EGTA) containing a protease
inhibitor cocktail (Calbiochem, La Jolla, CA, USA) on ice for
10 min. The supernatant obtained after centrifugation at
14,000 rpm for 30 min at 4 °C was recovered. The
doublestranded probe with single-stranded-linker was generated
by 1:1 mix of the following two oligonucleotide with the
5′-AGTTGAGGGGACTTTCCCAGGCC(C)34-C-3′, the 3′ end biotinylated and 5′-GCCT
GGGAAAGTCCCCTCAACT-3′, respectively. The probe
was denatured at 94 °C for 10 min, annealed at room
temperature overnight and then linked to
streptavidincoated 96-well plates (Roche, Mannheim, Germany) by
incubating 2 pmol of probe per well for 1 h at 37 °C in 50 μl
PBS. After wash, 20 μl of cell extract were mixed with
30 μl of binding buffer (4 mM HEPES pH 7.5, 100 mM
KCl, 8% glycerol, 5 mM DTT, 0.2% BSA, 40 μg/ml salmon
sperm DNA) in the above microwells incubated at room
temperature with mild agitation (200 rpm) for 1 h. After
wash, mouse anti-NF-κB p65 monoclonal antibody
(1:1000 diluted) was incubated for 1 h at room
temperature. After wash, peroxidase-conjugated goat
antimouse IgG were incubated at room temperature for 1 h.
After wash, 100 μl tetramethylbenzidine was incubated
at room temperature for 10 min before adding 100 μl
of stopping solution (2 M H2SO4). Optical density was
then read at 450 nm under a microplate reader (Biotek
Instruments, Winooski, VT, USA) using a 655-nm
reference wavelength. Backgrounds are determined in lysis
buffer and subtracted before data analysis.
Real-time quantitative RT-PCR (qRT-PCR) analysis for
MUC5AC mRNA levels
Total RNA was extracted from BECs using Trizol
Reagent. cDNAs were synthesized from total RNA by
reverse transcription according to the manufacturer’s
instruction. Then cDNAs were used for amplification by
qRT-PCR in a 25 μl reaction using SYBR® Premix EX
Taq™ II (TaKaRa) with a Mini Opticon Real-time PCR
Systerm (BIORAD). MUC5AC mRNA expression was
normalized with β-actin. The MUC5AC primers were F:
3-GCTCATCCTAA GCGACGTCT-5, R: 3-GGGGGCA
TAACTTCTCTTGG-5, and the β-actin primers were F:
3-CGGCATTGTCACCAACTG-5, R: 3-CGCTCGGTCA
ELISA analysis for MUC5AC protein levels
Intracellular MUC5AC protein levels were measure
using ELISA kits following the manufacturer’s
instructions (Cloud-Clone Corp, Wuhan, China).
Immunofluorescence staining and confocal microscopy
To determine the protein level of MUC5AC, BECs
were cultured on cover slips, after pretreated with
EGTA (0.5 mM, 2 mM or 5 mM), AS1517499 or
LKK16 for 30 min, cells were treated with IL-4
(20 ng/ml). 24 h later, BECs were stained with rabbit
anti-MUC5AC antibody (1:100 dilution) at 4 °C
overnight, and then incubated with tetramethyl rhodamin
isothiocyanate (TRITC)-conjugated goat anti-rabbit
antibody at room temperature for 60 min. The nuclei
were stained for DAPI for 10 min in dark. The
fluorescence-labeled cells were examined using a
Zeiss-LSM780 Confocal laser scanning microscope
To determine the protein level of caveolin-1 and
TRPC1, as well as lipid rafts aggregation, BECs were
cultured on cover slips, after pretreated with 10 mM
MβCD or 100 μM 2-APB for 30 min, cells were treated
with 20 ng/ml IL-4 for 15 min in the presence or
absence of M-βCD or 2-APB, BECs were stained with a
mouse monoclonal antibody specific to caveolin-1 and
rabbit anti-TRPC1 polyclonal antibody. Lipid rafts were
visualized by staining with 2 μg/ml cholera toxin subunit
B- Alexa Fluor 488 for 20 min.
Flotation of lipid rafts by sucrose gradient centrifugation
To isolate lipid raft fractions from the cell membrane,
BECs were lysed in 1.5 ml buffer containing 10 mM
Tris·HCl, 150 mM NaCl, 5 mM EDTA, 1 mMPMSF,
3 mM Na3VO4, protease inhibitors cocktail and 1%
Triton X-100 (pH 7.4). Cell extracts were homogenized
with five passages through a 25-gauge needle.
Homogenates were adjusted with 60% sucrose density gradient
medium to 40% and overlaid with 2 ml 90% sucrose,
4 ml 35% sucrose and 4 ml 5% sucrose Density Gradient
Samples were centrifuged at 39000 rpm and 4°C for
18 h using a SW32.1 rotor. Fractions were collected
from top to bottom, each sample had 11 fractions. For
immunoblot analysis of lipid raft-associated proteins,
these fractions were precipitated by mixing with equal
volume of 100% trichloroacetic acid and 30 min of
incubation on ice, and then samples were centrifuged at
15000 g and 4°C for 15 min, protein sediments were
washed with ice acetone twice, air dried, and then
resuspended in 1 M Tris·HCl (pH 8.0), which was ready
for immunoblot analysis. Western blots were performed
to detect protein levels of caveolin-1 and TRPC1 in each
Results are shown as the mean ± SD for n experiments,
n means cells from n rats. Differences between groups
were analyzed using unpaired t tests or two-way analysis
of variance. A P value less than 0.05 was considered to
be statistically significant.
2-APB prevented IL-4-induced Ca2+ influx in BECs
To study the effect of IL-4 on [Ca2+]i in BECs, we
treated BECs with IL-4 in the presence or absence of
2APB, an inhibitor of Ca2+ release. As shown in Fig. 1a-d,
IL-4 significantly increased the level of [Ca2+]i, but this
was abolished by 2-APB. After careful analysis of Ca2+
influx duration and amplitude, we found both Ca2+
influx duration and amplitude were increased with the
treatment of IL-4 in BECs (Fig. 1e, f ). These data
suggested that IL-4 induced Ca2+ influx could be prevented
by 2-APB in BECs.
Extracellular Ca2+ signal mediated IL-4-induced MUC5AC
protein synthesis in BECs
To investigate the role of Ca2+ influx in MUC5AC
synthesis, we detected MUC5AC mRNA and protein levels
in BECs treated by IL-4. As shown in Fig. 2a and b, IL-4
increased the levels of MUC5AC mRNA and protein
expression, and these were prevented by 2-APB (Fig. 2a,
b). Because 2-ABP is nonspecific and blocks both TRP
channels and intracellular inositol triphosphate receptor
(IP3R), to distinguish between extracellular and
intracellular Ca2+ source, we blocked extracellular Ca2+ using
EGTA. As shown in Fig. 2c and d, IL-4 induced
increases in MUC5AC expression, but this was attenuated
by EGTA at a dose-dependent manner. These data
suggested extracellular Ca2+ influx played a role in IL-4
induced MUC5AC expression.
NF-κB rather than STAT6 activation mediated IL-4
induced MUC5AC protein synthesis
To study downstream signals of IL-4 in BECs, we
investigate STAT6 and NF-κB pathways. As shown in Fig. 3a
and b, IL-4 induced time-dependent phosphorylation of
STAT6 and P65 which suggested both signals were
activated in IL-4 treated BECs. To further distinguish the
role of STAT6 and NF-kB pathway in IL-4-induced
MUC5AC expression, we used specific STAT6 inhibitor
AS1517499 and a selective IκB kinase (IKK) inhibitor
IKK 16 to treat cells. As shown in Fig. 3c, IKK 16
induced significant down-regulation of MUC5AC protein,
but AS1517499 had no effect on IL-4 induced
upregulation of MUC5AC protein. These data suggested
that NF-κB rather than STAT6 signal pathway mediated
MUC5AC up-regulation induced by IL-4. To further
dissect the role of NF-κB as well as the relationship with
Ca2+ influx, we detected NF-κB activity. As shown in
Fig. 3d, IL-4 enhanced NF-κB activity, and this was
blocked by 2-APB. These data suggested extracellular
Ca2+ influx played a role in IL-4 induced NF-κB
activation and further MUC5AC expression.
Caveolin-1 containing lipid rafts aggregation was involved in TRPC1 activation by IL-4 in BECs
To explore the potential mechanism underlying
extracellular Ca2+ influx induced by IL-4, we next focused on
the lipid rafts clustering in BECs. We detected the
colocalization and clustering of caveolin-1 and ganglioside
GM1 enriched lipid rafts. Cells were first incubated with
an anti-calveolin-1 antibody, followed by staining with
cholera toxin subunit B-Alexa Fluor 488, which is a
specific clustering agent for GM1 enriched lipid rafts. As
shown in Fig. 4a and b, IL-4 induced clustering and
colocalization of GM1 enriched lipid rafts and
caveolin1. Moreover, when the cells were treated with the
inducer of lipid rafts disruption (M-βCD), IL-4 failed to
induce clustering of lipid rafts. By using sucrose gradient
ultracentrifugation and immunoblotting, lysates were
separated into lipid raft and non-lipid raft fractions. As
shown in Fig. 4c, caveolin-1 was mainly found in
nonlipid raft fractions in the control. However, the treatment
of IL-4 led to significantly increased caveolin-1 in
lipid raft fractions, and this was prevented by M-βCD
(Fig. 4c, d). These data suggested that IL-4 induced
caveolin-1 containing lipid rafts aggregation.
TRPC activation is necessary in extracellular Ca2+
influx when IP3 binds with IP3R, so to further uncover
the relationship between extracellular Ca2+ influx and
caveolin-1-containing lipid rafts aggregation, we studied
the colocalization of TRPC1 and caveolin-1. As shown
in Fig. 4e and f, IL-4 induced clustering and
colocalization of caveolin-1 and TRPC1. After sucrose gradient
ultracentrifugation, the immunoblotting images revealed
that TRPC1 was found in non-lipid raft fractions in
normal control, but IL-4 caused significant increase of
TRPC1 in lipid raft fractions (Fig. 4g, h). These data
suggested that IL-4 induced colocalization of TRPC1 in
caveolin-1-containing lipid rafts aggregation.
Blocking lipid rafts aggregation prevented extracellular
Ca2+ influx induced by IL-4
To further confirm the colocalization of TRPC1 in
caveolin-1-containing lipid rafts aggregation, we
detected [Ca2+]i levels. BECs were exposed to IL-4 in the
presence or absence of M-βCD. As shown in Fig. 5, IL-4
triggered increases in [Ca2+]i including amplitude and
duration, but after disruption of lipid raft clustering with
M-βCD, IL-4 did not induce any change in [Ca2+]i
signal. These data suggested that IL-4 induced
intracellular calcium response is mediated by lipid rafts
Caveolin-1-containing lipid rafts aggregation induced by
IL-4 contributed to MUC5AC synthesis
To study the role of IL-4-induced caveolin-1-containing
lipid rafts aggregation in MUC5AC synthesis in BECs,
we used M-βCD to disrupt lipid rafts aggregation, and
then detected changes in NF-κB activity and MUC5AC
levels. As shown in Fig. 6a, IL-4 induced increases in
NF-κB activity, and this was blocked by M-βCD. IL-4
significantly increased the intracellular MUC5AC mRNA
and protein levels (Fig. 6b, c), disruption of lipid raft by
M-βCD attenuated IL-4-induced increases of MUC5AC.
These data suggest that IL-4 induced
caveolin-1containing lipid rafts aggregation which contributed to
In this study, we provided substantial evidence linking
asthmatic cytokine IL-4 and airway epithelial cells
MUC5AC overproduction. Our additional data revealed
that MUC5AC proteins were over-expressed in asthmatic
rat airway epithelial cells in vivo (Additional file 1). We
found that IL-4 induced caveolin-1-containing lipid
rafts aggregation, and co-localization with TRPC1 in
the lipid rafts domain. Disruption of lipid rafts or
blocking of TRPC1 attenuated IL-4 induced calcium
signals and NF-κB activation, consequently prevented
MUC5AC over-expression. Thus, these observations
indicated that IL-4 induced MUC5AC overproduction
via cell membrane lipid rafts aggregation and
activation of TRPC1 (Fig. 7).
In the current study, firstly we found IL-4 induced Ca2
+ influx and MUC5AC overproduction in BECs which
prevented by 2-APB. Because 2-ABP is nonspecific and
blocks both TRP channels and IP3R, to distinguish
between extracellular and intracellular Ca2+ source, we
depleted extracellular Ca2+ using EGTA and found
extracellular Ca2+ influx played a role in IL-4 induced
MUC5AC expression. We next found NF-κB rather than
STAT6 activation mediated IL-4-induced MUC5AC
For the first time, we found that IL-4 induced
caveolin-1-containing lipid rafts aggregation in BECs.
Membrane lipid rafts are highly ordered membrane
domains that are enriched in cholesterol, sphingolipids and
gangliosides, and function as platforms for the
recruitment of signaling proteins to facilitate protein-protein
interaction and signal transduction. An increasing
number of proteins involved in signal transduction have been
found to locate in these ordered membrane domains. It
is well known that lipid rafts organize receptors, ion
channels and their downstream acting molecules to
regulate intracellular signaling pathways [
Recent studies have demonstrated that lipids rafts also
contribute to the organization and function of Ca2+
signaling microdomains. TRPC1 channelosome responsible for
SOCE is located in caveolar lipid raft domains [
Destabilization of the caveolar lipid raft domains by
MβCD treatment or deletion of caveolin-1 prevents SOCE
]. Caveolin-1 gene knockout disrupts
TRPC1-STIM1-Orai1 complex [
]. In the current study,
we demonstrated that IL-4 induced TRPC1 co-localization
with caveolin-1-containing lipid rafts aggregation, which
mediated Ca2+ influx in BECs.
Calcium signal was observed to be related with NF-κB
activation. Sequence analyses of MUC5AC promoter
revealed the presence of NF-κB response elements within
]. NF-κB activity is crucial for mucus production
] . Zhu et al. have reported that Ca2+ oscillation
frequency regulated NF-κB transcriptional activity via
increased cumulated Ca2+ spike duration [
data showed here that IL-4 induced single calcium peak,
blocking TRPC1 with 2-APB prevented calcium signal
and suppressed NF-κB activity. These data suggest that
IL-4 induced calcium signaling through
caveolin-1containing-lipid-rafts/TRPC1 pathway that resulted in
NF-κB activation and MUC5AC over-production.
In the current study, we found IL-4 caused aggregation
of caveolin-1-containing lipid rafts which activated
TPRC1, subsequently activated calcium signal and
NFκB, and finally increased MUC5AC synthesis in BECs.
This study elucidates a new mechanism underlying
asthmatic cytokines induce mucus overproduction, which
may be beneficial in developing novel strategies for the
treatment of asthma.
Additional file 1: Online supplementary data. (PDF 316 kb)
[Ca2+]i: intracellular Ca2+; 2-APB: 2-Aminoethyl diphenylborinate;
BECs: Bronchial epithelial cells; EGTA: Ethylene glycol tetraacetic acid;
IL4: Interleukin-4; IP3R: Inositol triphosphate receptor; M-βCD:
Methyl-βcyclodextrin; SOCE: Store-operated Ca2+ entry; Th2: T helper type 2 cells;
TRPC1: Transient receptor potential canonical 1
We thank all members from Key Laboratory of Respiratory Diseases, Ministry
of Health of China (Wuhan Hubei, China) for their invaluable help. Present
affiliation of Shan-Shan Rao: Department of Pathology, the Central Hospital
This work was supported in part by grants from the National Natural Science
Foundation of China (No. 81570087, 30770943 to HY; No. 81600071 to LX;
No. 81573485, 81370186 to WLM; No. 81200020 to PCC).
Availability of data and materials
Essential datasets supporting the conclusion are included in this published
YX, PCC and HY designed the study; YX, PCC, FY, LX and SSR performed
experiments; XLH, FC and XPY analyzed the data; WLM and HY wrote the
manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate
In the current study, bronchial epithelial cells were isolated from large
bronchial airway of rat. The experiments were performed in accordance with
the Guide for the Care and Use of Laboratory Animals and approved by the
Institutional Animal Care and Use Committee (IACUC) of the Tongji Medical
College, Huazhong University of Science and Technology.
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in
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