Experimental Lung Research, Early Online, 110, 2014

Copyright 2014 Informa Healthcare USA, Inc.

ISSN: 0190-2148 print / 1521-0499 online
DOI: 10.3109/01902148.2014.926434
ORIGINAL ARTICLE
The human Cathelicidin LL-37 induces MUC5AC mucin
production by airway epithelial cells via
TACE-TGF--EGFR pathway
Yuke Zhang,
1
Maoxiang Zhu,
2
Zhihua Yang,
2
Xiujie Pan,
2
Yuanyuan Jiang,
1
Congcong Sun,
1
Qin Wang,
1
and Wei Xiao
1
1
Department of Respiratory Medicine, Qilu Hospital, Shandong University, Jinan, China
2
Beijing Institute of Radiation Medicine, Beijing, China
ABSTRACT
Aim: To investigate the mechanism for LL-37 inducing MUC5AC mucin production in airway epithelial cells.
Materials and Methods: The airway epithelial NCI-H292 cells were stimulated with various concentrations of
LL-37 synthetic peptide and scrambled LL-37 (sLL-37) synthetic peptide ranged from 2.5 to 10 g/mL. The ef-
fects of LL-37 and sLL-37 on TNF--converting enzyme (TACE) and EGFR activation and MUC5AC mucin pro-
duction were evaluated by fuorescence resonance energy transfer (FRET) assay, Western blotting and ELISA
respectively. Furthermore, we measured changes of transforming growth factor-alpha (TGF-) in culture su-
pernatants. A serious of inhibitors including TACE inhibitor TAPI-1, EGFR inhibitor AG1478, EGFR-neutralizing
antibody, TGF--neutralizing antibody, amphiregulin (AR)-neutralizing antibody, and heparin binding-epidermal
growth factor (HB-EGF)-neutralizing antibody were used to block the signaling pathway. Human serum and FBS
were also used to investigate the effects of serum on LL-37-induced MUC5AC mucin production. Results: LL-37
induced TACE and EGFR activation, as well as TGF- and MUC5AC mucin production by NCI-H292 cells in a
dose-dependent manner. EGFR-neutralizing antibody and AG1478 inhibited LL-37-induced EGFRactivation and
subsequent MUC5AC mucin production, whereas TGF--neutralizing antibody increased LL-37-induced TGF-
production. TAPI-1 inhibited LL-37-induced TGF- production, EGFR activation and subsequent MUC5AC
mucin production, whereas TGF--neutralizing antibody, but not AR- or HB-EGF-neutralizing antibody, inhib-
ited LL-37-induced EGFR activation and subsequent MUC5AC mucin production in NCI-H292 cells. The sLL-37
had no effect on TACE and EGFR activation and MUC5AC mucin production. Additionally, Human serum, rather
than FBS, inhibited LL-37-induced MUC5AC mucin production. Conclusions: LL-37 induces MUC5AC mucin
production by airway epithelial cells via TACE-TGF--EGFR pathway.
KEYWORDS Cathelicidin, COPD, EGFR, mucins, TACE, TGF-
INTRODUCTION
Airway mucus overproduction is the hallmark of air-
way infammatory diseases, including chronic ob-
structive pulmonary disease (COPD), asthma, and
cystic fbrosis [1, 2]. Excessive mucus secreted into
the airway lumen contributes to morbidity and mor-
tality of COPD by causing airway obstruction and
Received 6 March 2014; accepted 15 May 2014
We thank Professor Qinzhi Xu and Dr. Long Xu for technical assistance.
Address correspondence to Wei Xiao, Department of Respiratory Medicine,
Qilu Hospital, Shandong University, Wen-hua-xi-lu Road 107, Jinan, China.
E-mail: xiaowei433@yahoo.com; xiaowei4226@163.com
recurrent infections [3]. MUC5AC mucin is a ma-
jor component of airway mucus, and its production
is regulated by a signaling pathway involving epider-
mal growth factor receptor (EGFR) [2, 46].
The human cationic antimicrobial peptides of
18KD (hCAP-18) is the only member of human
Cathelicidin family with a 37-residue helical peptide
(LL-37) cleaved to display potential antimicrobial ac-
tivity and various biological functions involving the
regulation of cellular proliferation, apoptosis, infam-
mation and immune responses [7]. LL-37 is present
at high concentrations in airway epithelium and air-
way secretions of subjects with COPD [810]. LL-37
is involved in the development of COPD by causing
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airway infammation and alveolar apoptosis, as well
as airway mucus overproduction [11, 12].
Several studies in airway epithelial cells showed
that TNF--converting enzyme (TACE) is responsi-
ble for the transactivation of EGFR and subsequent
MUC5AC mucin production [4, 5, 13]. TACE plays
an essential role in the ectodomain shedding of mul-
tiple cell surface proteins, resulting in the generation
of soluble mature transforming growth factor-alpha
(TGF-), amphiregulin (AR) and heparin-binding
epidermal growth factor (HB-EGF) [13]. It has been
reported that cigarette smoke extract (CSE) and hu-
man neutrophil elastase (HNE) induce MUC5AC
mucin production by causing activation of TACE,
which cleaves pro-TGF- into soluble TGF-, lead-
ing to phosphorylation of EGFR and subsequent
mucin production [4, 5]. In our recent study re-
garding COPD, we have shown that LL-37 induces
MUC5AC mucin production in airway epithelial
NCI-H292 cells through a pathway involving EGFR
[12]. However, the precise mechanism for LL-37-
induced activation of EGFR is yet to be determined.
Here, we hypothesize that TACE mediated TGF-
release is responsible for the transactivation of EGFR
and subsequent MUC5AC mucin induction by
LL-37 in airway epithelial cells. In the present study,
we examined the effects of LL-37 on TACE activity
and soluble TGF- release, and then investigated
the role of TACE-mediated TGF- release in LL-
37-induced EGFR phosphorylation and subsequent
MUC5AC mucin production in airway epithelial
NCI-H292 cells. On the other hand, as TACE is also
responsible for the generation of AR and HB-EGF
which are implicated in the transactivation of EGFR
induced by CSE or LL-37 in multiple cell types
[1416], we investigated the involvement of AR and
HB-EGF in LL-37-induced EGFR phosphorylation
and MUC5AC mucin production in airway epithelial
cells.
MATERIALS AND METHODS
Materials
LL-37 (amino acid sequence: LLGDF-
FRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES)
and scrambled LL-37 (sLL-37: RSLEGTDRF-
PFVRLKNSRKLEFKDIKGIKREQFVKIL) were
synthesized by GL Biochem, Shanghai, China. Hu-
man MUC5AC ELISA kit was from USCN, Hous-
ton, USA. AG1478, TAPI-1, EGFR-neutralizing
monoclonal antibody (Ab-3) were from Calbiochem,
San Diego, USA. EGFR (1005) polyclonal anti-
body and p-Tyr (PY99) monoclonal antibody were
from Santa Cruz Biotechnology, Santa Cruz, USA.
SensoLyte 520 TACE activity assay kit was from
AnaSpec, San Jose, USA. Human TGF- ELISA
kit, TGF--neutralizing monoclonal antibody,
amphiregulin-neutralizing monoclonal antibody and
HB-EGF-neutralizing monoclonal antibody were
from R&D systems, Minneapolis, USA. Human
serum (type AB) and fetal bovine serum (FBS) were
from Sigma, St. Louis, USA. The M-PER mam-
malian protein extraction reagent, Halt phosphatase
inhibitor cocktail and BCAprotein assay kit were
from Thermo Fisher Scientifc, Rockford, USA.
Preparation for LL-37 and sLL-37 Solutions
The LL-37 concentrations used in the present study
ranged from 2.5 to 10 g/mL, which was determined
by MTT method in our previous study [12]. Under
this condition, the cell viability was not less than
80%. The LL-37 synthetic peptide was dissolved in
serum-free RPMI 1640 medium to prepare the stock
solution (1 mg/mL), which was further diluted to
working concentrations indicated above in following
experiments. As a control peptide, equivalent con-
centrations of sLL-37 were prepared and used in
selected experiments.
Cell Culture
The NCI-H292 cells, a human airway epithelial
cell line, were obtained from American Tissue Cul-
ture Collection. Cells were cultured in RPMI 1640
medium supplemented with FBS (10%), penicillin
(100 U/ml) and streptomycin (100 g/mL) at 37

C
in a humidifed, 5% CO
2
/95% air, water-jacketed in-
cubator. After the cells reached near 80%confuence,
they were serum-starved for 24 hours before sequen-
tial treatments, and the in vitro studies were done in
serum-free media unless otherwise indicated.
Western Blot Analysis for EGFR
Phosphorylation
After treatments, cells were lysed in M-PER mam-
malian protein extraction reagent, and protein
concentrations were determined using BCA protein
assay. Samples containing an equal amount of total
protein (30 g) were subjected to SDS-PAGE on the
8% glycine-based gel, and dissolved proteins were
transferred to a polyvinylidene difuoride membrane
(300 mA, 4 hours). After the nonspecifc binding
sites were blocked with 5% BSA, the membranes
were incubated with primary Abs against phospho-
rylated EGFR (diluted 1:200 with TBST buffer)
overnight at 4

C and then incubated with secondary

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LL-37 Induces Mucin Production via TACE-TGF--EGFR Pathway 3
HRP-conjugated antibodies (1:5000 dilution with
TBST buffer) for 1 hours after washing three times
with TBST buffer. The ECL western blotting de-
tection system was used to reveal immunoreactivity.
The membrane was then stripped and reprobed
with primary antibodies against EGFR (diluted
1:200 with TBST buffer) and secondary HRP-
conjugated antibodies (1:5000 dilution with TBST
buffer) following the same steps specifed above.
The representative bands presented are from three
independent repeated experiments.
Assay of TACE Activity
The TACE activity of cell lysates were measured
by fuorescence resonance energy transfer (FRET)
method using Sensolyte 520 TACE activity assay
kit according to the manufacturers instruction. Af-
ter treatments, NCI-H292 cells were lysed in Assay
buffer (Component C) containing 0.1% (v/v) Triton-
X 100, and cell lysates were incubated at 4

C for
10 minutes before cell supernatants were collected for
use. The TACE substrate solution was freshly pre-
pared with TACE substrate (Component A) diluted
in Assay buffer by the proportion of 1:100. Equal
amounts of cell supernatants were incubated with
50 L of TACE substrate solution for 30 minutes at
37

C, and 50 Lof stop solution (Component E) was

then added to end the reaction. The fuorescence in-
tensity was monitored by the fuorescence microplate
reader with 490-nm excitation and 520-nm emission
settings, and was expressed as relative fuorescence
units (rfu).
ELISA for MUC5AC Mucin and Soluble TGF-
The MUC5AC mucin production were determined
as previously described [5]. After treatments, the cell
culture media were collected and the cells were lysed
with the M-PER Mammalian Protein Extraction
Reagent. The cell culture media and cell lysates were
centrifuged at 12,000 g for 5 minutes at 4

C, and the
supernatants were stored at 80

C until assay. The

levels of MUC5ACmucin in the culture supernatants
and cell lysates were measured with a specifc ELISA
kit and summed for each sample. Simultaneously, the
total protein levels in cell lysates were determined
using the BCA Protein Assay Kit. The summed to-
tal MUC5AC mucin in each sample was normal-
ized to total protein in cell lysates and was expressed
as g/mg protein. Soluble TGF- in the cell cul-
ture supernatants of each sample was measured using
TGF- specifc ELISA kit following the manufac-
turers instructions and was expressed as pg/10
6
cells.
Statistical Analysis
Data were expressed as mean SEM. Statistical
analyses were performed using SPSS for Windows
(version 13.0, Chicago, USA). Comparisons between
multiple treatment groups were performed using
ANOVAand the Bonferroni post-test. AP-value <.05
was considered signifcant.
RESULTS
Ligand-dependent EGFR Phosphorylation is
Required for LL-37-induced MUC5AC Mucin
Production
LL-37 induced EGFR phosphorylation (Figure 1A)
and MUC5AC mucin production (Figure 1B) dose-
dependently in NCI-H292 cells. These effects were
blocked by pretreatment of the cells with AG1478 (a
selective inhibitor of EGFR phosphorylation, Figure
1C and D), implicating EGFR phosphorylation
in LL-37-induced MUC5AC mucin production.
These effects were also prevented by preincubation
of the cells with an EGFR-neutralizing antibody
(Ab-3), which was used to prevent ligand binding
and internalization of the receptor-bound ligand
(Figure 1C and D). These results implicate ligand-
dependent EGFRphosphorylation in LL-37-induced
MUC5AC mucin production.
LL-37 Induces Shedding of Soluble TGF- via
Activation of TACE
Pro-TGF- is constitutively expressed on the mem-
brane surface of NCI-H292 cells and can be cleaved
by TACE to release soluble TGF- [17]. LL-37 in-
creased TACE activity time- and dose-dependently
in NCI-H292 cells (Figure 2A and B). LL-37 also
increased TGF- release by NCI-H292 cells time-
and dose-dependently (Figure 2C and D). In addi-
tion, TAPI-1 (a relatively selective TACE inhibitor)
inhibited LL-37-induced TACE activation (Figure
2E) and TGF- release (Figure 2F). These results
suggest an essential role of TACE activation in LL-
37-induced TGF- shedding.
TACE-mediated TGF- Release Plays an
Essential role in LL-37-induced EGFR
Phosphorylation and MUC5AC Mucin
Production
Preincubation of the cells with a TGF--neutralizing
antibody prevented LL-37-induced EGFR phospho-
rylation (Figure 3A) and MUC5AC mucin produc-
tion (Figure 3B), and preincubation of the cells with
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FIGURE 1. LL-37 induces ligand-dependent EGFR phosphorylation and MUC5AC mucin
production. (A and B) NCI-H292 cells were treated with LL-37 at various concentrations for 15
minutes and analyzed for EGFR phosphorylation by Western blotting analysis (A), or stimulated for
24 hours and analyzed for MUC5AC mucin production by ELISA (B). (C and D) NCI-H292 cells were
pretreated with EGFR-neutralizing antibody (Ab-3, 4 g/mL) and the selective inhibitor of EGFR
phosphorylation AG1478 (5 M) for 30 minutes, and then stimulated with LL-37 (7.5 g/mL) for
15 min and analyzed for EGFR phosphorylation by Western blotting analysis (C), or stimulated for
24 hours and analyzed for MUC5AC mucin production by ELISA (D). Data are expressed as mean
SEM of three independent experiments.

P < .01 versus untreated control, P < .01 versus LL-37 alone.
an EGFR-neutralizing antibody (Ab-3) increased the
accumulation of soluble TGF- (Figure 3C), sug-
gesting the involvement of EGFR ligand TGF-
in LL-37-induced responses. Additionally, pretreat-
ment of cells with TACE inhibitor TAPI-1 prevented
LL-37-induced EGFR phosphorylation (Figure 3A)
and mucin production (Figure 3B), as well as pre-
vented LL-37-induced TGF- accumulation even in
the presence of EGFR-neutralizing antibody (Ab-3)
(Figure 3C), suggesting the involvement of TACE
in LL-37-induced responses. All these results indi-
cate that TACE-mediated TGF- plays an essential
role in LL-37-induced EGFR phosphorylation and
MUC5AC mucin production.
AR and HB-EGF are not Involved
in LL-37-induced EGFR Activation
and MUC5AC Mucin Production
AR and HB-EGF are potential EGFR ligands that
can be generated by airway epithelial cells through
the ectodomain shedding by TACE [13, 14, 16].
We examined the involvement of AR and HB-
EGF in LL-37-induced EGFR phosphorylation and
MUC5AC mucin production. Pretreatment of the
cells with either AR-neutralizing antibody or HB-
EGF-neutralizing antibody did not affect LL-37-
induced EGFR phosphorylation (Figure 4A) and
MUC5AC mucin production (Figure 4B). These re-
sults suggest that AR and HB-EGF are not involved
in LL-37-induced responses.
The Effects of LL-37 on NCI-H292 Cells
are Peptide-specic
To exclude a nonspecifc effect of LL-37, different
concentraions of a scrambled version of LL-37 (sLL-
37) were used to treat cells. The sLL-37 control
peptide had no effect on MUC5AC mucin produc-
tion (Figure 5A), EGFR activation (Figure 5B), and
TACE activation (Figure 5C). These results suggest
that LL-37 activates TACE-TGF--EGFR pathway
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LL-37 Induces Mucin Production via TACE-TGF--EGFR Pathway 5
FIGURE 2. LL-37 induces TGF- release involving TACE activity. (A) NCI-H292 cells were treated with LL-37 (7.5 g/mL) for
various time periods and analyzed for TACE activity by fuorescence resonance energy transfer (FRET) assay. (B) NCI-H292 cells were
treated with LL-37 at different concentrations for 3 hours and analyzed for TACE activity by FRET assay. (C) NCI-H292 cells were
treated with LL-37 (7.5 g/mL) for various time periods and analyzed for soluble TGF- release by ELISA. (D) NCI-H292 cells were
treated with LL-37 at different concentrations for 6 h and analyzed for soluble TGF- release by ELISA. (E and F) After pretreatment
with a selective TACE inhibitor TAPI-1 (10 M) for 30 minutes, NCI-H292 cells were stimulated with LL-37 (7.5 g/mL) for 3 hours
and analyzed for TACE activity by FRET assay (E), or stimulated for 6 hours and analyzed for soluble TGF- release by ELISA (F).
Data are expressed as mean SEM of three independent experiments.

P < .01 versus untreated control, P< .01 versus LL-37 alone.
specifcally to induce MUC5ACmucin production in
NCI-H292 cells.
Human Serum Inhibits LL-37-induced
MUC5AC Mucin Production
LL-37 has been reported to be able to interact with
some components in serum [18, 19], we investigated
whether serum affects LL-37-induced responses in
cultured airway epithelial NCI-H292 cells. LL-37-
induced MUC5AC mucin production was inhibited
by human serum whereas FBS enhanced MUC5AC
mucin production induced by LL-37 (Figure 6).
These results suggest that some components from
human serum, rather than FBS, may prevent LL-
37 from exerting downstream effects on NCI-H292
cells.
DISCUSSION
Here, we demonstrate for the frst time that LL-37
activates TACEin human airway epithelial cells, lead-
ing to cleavage of pro-TGF- into mature soluble
TGF-, resulting in EGFRactivation and subsequent
mucin production. We also show that EGFR ligands
AR and HB-EGF are not involved in these LL-37-
induced responses.
Airway epithelial cells are exposed to LL-37 in
higher concentrations in pathological conditions such
as cystic fbrosis and COPD [8, 9, 11]. In our pre-
vious study regarding COPD, we have implicated
EGFR-ERK1/2 signaling pathway in LL-37-induced
MUC5AC mucin production by blocking EGFR-
ERK1/2 pathway with corresponding pharmacolog-
ical inhibitors [12]. However, the precise mechanism
for LL-37-induced EGFRactivation is unclear. Stim-
uli relevant to COPD such as cigarette smoke and
HNE have been shown to induce mucin overpro-
duction in airway epithelial cells with TACE-TGF-
-EGFR axis established [4, 5]. In the present study,
we investigated whether TACE-mediated TGF- re-
lease is responsible for LL-37-induced EGFR activa-
tion and subsequent MUC5AC mucin production.
EGFR activation plays an essential role in mucin
induction by various stimuli [2, 4, 5, 20]. We
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FIGURE 3. TACE-mediated TGF- release plays an essential role in LL-37-induced EGFR
phosphorylation and MUC5AC mucin production. (A and B) NCI-H292 cells were pretreated with
TGF--neutralizing antibody (1 g/mL) and TAPI-1 (10 M) for 30 minutes, and then stimulated with
LL-37 (7.5 g/mL) for 15 minutes and analyzed for EGFR phosphorylation by Western blotting analysis
(A), or stimulated for 24 hours and analyzed for MUC5AC mucin production by ELISA (B). (C) Cells
were pretreated with EGFR-neutralizing antibody (Ab-3) alone (4 g/mL) or EGFR-neutralizing
antibody (4 g/mL) plus TAPI-1 (10 M), and then stimulated with LL-37 (7.5 g/mL) for 6 hours
and analyzed for soluble TGF- release by ELISA. Data are expressed as mean SEM of three
independent experiments.

P <.01 versus untreated control, P < .01 versus LL-37 alone.
found that LL-37 induces EGFR phosphorylation
and MUC5AC mucin production, and the in-
duction can be effectively inhibited by selective
EGFR tyrosine kinase inhibitor AG1478, suggest-
ing the involvement of EGFR in LL-37-induced
mucin production. It has been reported that two
different processes are involved in EGFR activa-
tion: ligand dependent EGFR phosphorylation and
ligand-independent EGFR phosphorylation. A grow-
ing body of evidence indicates that EGFR can be
transactivated by metalloprotease-dependent shed-
ding of EGFR ligands. These results suggest that
some of the EGFR activation previously considered
ligand-independent are essentially ligand-dependent.
To investigate whether ligand binding is required for
EGFR phosphorylation by LL-37, we preincubated
cells with a neutralizing anti-EGFR antibody to block
the EGFR ligand binding sites on the cell surface.
This pretreatment effectively prevented EGFR phos-
phorylation and mucin production induced by LL-
37, implicating ligand-dependent EGFRactivation in
mucin induction by LL-37.
EGFR ligand TGF- is constitutively expressed in
airway epithelial cells [17]. Pro-TGF- (2022 kDa)
is synthesized as a transmembrane precursor of TGF-
. The newly synthesized pro-TGF- is cleaved by
metalloproteases, resulting in the release of mature
soluble TGF- (6 kDa) [21, 22]. Our data showed
that LL-37 promoted the release of soluble TGF-
by NCI-H292 cells, and that blocking EGFR ligand
binding sites caused a accumulation of soluble TGF-
while blocking TGF- prevented EGFR phospho-
rylation and MUC5AC mucin induction by LL-37.
These results suggest that TGF- is implicated in LL-
37-induced mucin production by binding to and acti-
vating EGFR. To examine whether a metalloprotease
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LL-37 Induces Mucin Production via TACE-TGF--EGFR Pathway 7
FIGURE 4. AR and HB-EGF are not involved in LL-37-induced EGFR activation and MUC5AC
mucin production. (A) NCI-H292 cells were pretreated with AR-neutralizing antibody (20 g/mL) and
HB-EGF-neutralizing antibody (4 g/mL) for 30 minutes, and then stimulated with LL-37
(7.5 g/mL) for 15 minutes and analyzed for EGFR phosphorylation by Western blotting analysis. (B)
NCI-H292 cells were pretreated with AR-neutralizing antibody (20 g/mL) and HB-EGF-neutralizing
antibody (4 g/mL) for 30 minutes, and then stimulated with LL-37 (7.5 g/mL) for 24 hours and
analyzed for MUC5AC mucin production by ELISA. Data are expressed as mean SEM of three
independent experiments.

P < .01 versus untreated control.
FIGURE 5. LL-37-induced responses in NCI-H292 cells are peptide-specifc. NCI-H292 cells were
treated with LL-37 (5 g/mL) or sLL-37 (5 g/mL and 10 g/mL) for different time periods. (A) After
stimulated for 24 hours, the MUC5AC mucin production was analyzed by ELISA. (B) After stimulated
for 15 minutes, the EGFR phosphorylation was analyzed by Western blotting. (C) After stimulated for
6 hours, the TACE activity was analyzed by FRET assay. Data are expressed as mean SEM of three
independent experiments.

P < .01 versus untreated control.
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8 Y. Zhang et al.
FIGURE 6. Human serum inhibits LL-37-induced MUC5AC
mucin production. NCI-H292 cells were treated with LL-37 for
24 hours in the absence of serum or in the presence of human
serum (HS, 5%, 10%) and fetal bovine serum (FBS, 5%, 10%),
the MUC5AC mucin were analyzed by ELISA. Data are
expressed as mean SEM of three independent experiments.

P < .01 versus untreated control, P < .01 versus LL-37 alone.
is involved in the ectodomain shedding of pro-TGF-
induced by LL-37 in NCI-H292 cells, we utilized
TAPI-1, an inhibitor of metalloprotease. We found
that TAPI-1 inhibited soluble TGF- release, EGFR
phosphorylation and MUC5AC mucin production
induced by LL-37, implicating metalloprotease ac-
tivity in these processes. TACE is one member of a
disintegrin and metalloprotease (ADAM) family pro-
teases and is constitutively expressed in NCI-H292
cells [13, 23]. It has been reported to be responsible
for the ectodomain shedding of TGF- in various ep-
ithelial tissues [13, 24]. In this study, we demonstrate
that LL-37 possesses the ability to increase TACE
activity in NCI-H292 cells and that LL-37-induced
TACE activity could be inhibited by TAPI-1. Alto-
gether, these fndings suggest a crucial role of TACE-
mediated TGF- release in LL-37-induced EGFR
activation and mucin production with TACE-TGF-
-EGFR axis established.
Previous studies have reported that TACE is also
responsible for the generation of AR and HB-EGF by
ectodomain shedding of pro-amphiregulin and pro-
HB-EGF in membrane surface of epithelial cells [13,
14, 16]. AR and HB-EGF are considered potential
EGFR ligands which bind to and activate EGFR.
In the present study, we show that pretreatment of
the cells with corresponding neutralizing antibod-
ies against AR and HB-EGF have no effect on LL-
37-induced EGFR phosphorylation and MUC5AC
mucin production. These results suggest that AR
and HB-EGF are not mediators between TACE
and EGFR activation in LL-37-induced mucin
production.
Importantly, in this study, we examined the speci-
fcity of LL-37-induced responses in NCI-H292 cells.
We demonstrate that a scrambled version of LL-37
(sLL-37) had no effect on TACE and EGFR activa-
tion, as well as MUC5AC mucin production, which
can be induced by LL-37. These results suggest
that LL-37-induced responses in NCI-H292 cells are
peptide-specifc. In addition, as most in vitro exper-
iments were preformed in the absence of serum, we
investigated whether the effects of LL-37 were infu-
enced by serum. We demonstrate that human serum
inhibits LL-37-induced MUC5AC mucin produc-
tion. It has been reported that some special compo-
nents existed in human serum, such as apolipopro-
tein A-1 and high-density lipoprotein (HDL), could
bind to LL-37 and thus suppress its cytotoxicity [18,
19, 25]. Such components may also prevent LL-37
from interacting with NCI-H292 cells by binding to
LL-37 in the presence of human serum. However, de-
spite the potential effect of serum on LL-37-induced
responses, epithelial cells in human airways, which
generally reside in serum-free environments, are not
infuenced by serum and thus may be sensitive to
high concentrations of LL-37 encountered during in-
fammation such as observed in airway surface from
COPD patients.
Some limitations should be noticed in this study.
First, the results presented here were from studies
performed in a cancer cell line (NCI-H292 cells),
which often behave quite differently from primary
airway epitheial cells. Under some circumstances,
the effect of LL-37 may only be observed in well-
differentiated primary cultures [22, 26]. Although
a variety of studies involving mucin induction em-
ployed NCI-H292 cells, studies performed in pri-
mary airway epithelial cells are needed to provide a
more accurate description for the effects of LL-37 on
airway mucin induction. Second, the concentrations
of LL-37 used in this study (2.510 g/mL) might be
supraphysiologic. It is diffcult to determine the ex-
act concentration of LL-37 in the microenvironment
surrounding airway epithelial cells. Several studies
have examined LL-37 levels in human airway, and
the results are variable in magnitude. LL-37 can be
detected at airway mucosal surfaces in healthy indi-
viduals at concentrations of around 25 g/mL, and
is upregulated to approximately 20 g/mL in bron-
choalveolar lavage fuid from children with lung in-
fections [27]. Nevertheless, a recent study reported
Experimental Lung Research
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LL-37 Induces Mucin Production via TACE-TGF--EGFR Pathway 9
elevated LL-37 concentrations in the epithelial lining
fuid (ELF) from COPD patients in the ng/mL range
[10]. Considering the variability in magnitude, a wide
range of LL-37 concentrations should be employed
to further investigate the in vitro effects of LL-37 on
airway mucin production.
In summary, we show that stimulation with LL-37
specifcally induced MUC5AC mucin production via
TACE-TGF--EGFR pathway in airway epithelial
cells. EGFR ligands AR and HB-EGF are not
involved in these LL-37-induced responses. These
fndings are rather important because LL-37 is
closely associated with COPD. The discovery may
help researchers further understand the mechanism
for airway mucus overproduction in COPD.
Declaration of interest: The authors report no
conficts of interest. The authors alone are responsi-
ble for the content and writing of the paper.
This work was supported by Grant 81170041 from
the National Natural Science Foundation of China.
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