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The Human Cathelicidin LL-37 Induces MUC5AC Mucin Production by Airway Epithelial Cells Via Tace-Tgf
The Human Cathelicidin LL-37 Induces MUC5AC Mucin Production by Airway Epithelial Cells Via Tace-Tgf
ORIGINAL ARTICLE
Yuke Zhang,1 Maoxiang Zhu,2 Zhihua Yang,2 Xiujie Pan,2 Yuanyuan Jiang,1 Congcong Sun,1
Qin Wang,1 and Wei Xiao1
1
INTRODUCTION
Airway mucus overproduction is the hallmark of airway inflammatory diseases, including chronic obstructive pulmonary disease (COPD), asthma, and
cystic fibrosis [1, 2]. Excessive mucus secreted into
the airway lumen contributes to morbidity and mortality 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 major component of airway mucus, and its production
is regulated by a signaling pathway involving epidermal 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 activity and various biological functions involving the
regulation of cellular proliferation, apoptosis, inflammation and immune responses [7]. LL-37 is present
at high concentrations in airway epithelium and airway secretions of subjects with COPD [810]. LL-37
is involved in the development of COPD by causing
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Y. Zhang et al.
Cell Culture
The NCI-H292 cells, a human airway epithelial
cell line, were obtained from American Tissue Culture 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 humidified, 5% CO2 /95% air, water-jacketed incubator. After the cells reached near 80% confluence,
they were serum-starved for 24 hours before sequential treatments, and the in vitro studies were done in
serum-free media unless otherwise indicated.
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
ANOVA and the Bonferroni post-test. A P-value <.05
was considered significant.
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) dosedependently 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 liganddependent EGFR phosphorylation in LL-37-induced
MUC5AC mucin production.
Y. Zhang et al.
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 fluorescence 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.
DISCUSSION
Here, we demonstrate for the first time that LL-37
activates TACE in human airway epithelial cells, lead
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Y. Zhang et al.
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.
This pretreatment effectively prevented EGFR phosphorylation and mucin production induced by LL37, implicating ligand-dependent EGFR activation 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 phosphorylation and MUC5AC mucin induction by LL-37.
These results suggest that TGF- is implicated in LL37-induced mucin production by binding to and activating EGFR. To examine whether a metalloprotease
Experimental Lung Research
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-specific. 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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Y. Zhang et al.
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 activity in these processes. TACE is one member of a
disintegrin and metalloprotease (ADAM) family proteases 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 epithelial 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. Altogether, these findings suggest a crucial role of TACEmediated 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 proHB-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 antibodies against AR and HB-EGF have no effect on LL-
[10]
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[12]
[13]
[14]
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