Moreover, PAR-2 AP induced AR gene expression subsequent to protein production in the cellular fraction through the ERK pathway indicating that PAR-2-mediated activation of ERK is essent
Trang 1release through a mechanism involving protease-activated receptor-2-mediated ERK activation and TNF a-converting enzyme activity in airway epithelial cells
Manabu Chokki, Hiroshi Eguchi, Ichiro Hamamura, Hiroaki Mitsuhashi and Takashi Kamimura Pharmaceutical Discovery Research Laboratories, Institute for Bio-Medical Research, Teijin Pharma Limited, Tokyo, Japan
Human airway trypsin-like protease (HAT) is a novel
serine protease that can be purified from the sputum
of patients with chronic airway diseases, such as
chro-nic bronchitis and bronchial asthma, based on its
pro-tease activity [1] It exists in the sputum as a monomer
with a molecular size of 27 kDa as estimated by gel fil-tration chromatography [1] HAT cDNA has been iso-lated from a tracheal tissue cDNA library; analysis of this cDNA suggests that HAT is originally translated
as a precursor with a molecular size of 48 kDa and
Keywords
amphiregulin; extracellular signal-regulated
kinase; human airway trypsin-like protease;
protease-activated receptor-2; tumour
necrosis factor a-converting enzyme
Correspondence
M Chokki, Pharmaceutical Discovery
Research Laboratories, Institute for
Bio-Medical Research, Teijin Pharma Limited,
Tokyo, Japan
Tel: +81 42 586 8134
Fax: +81 42 587 5515
E-mail: m.chiyotsuki@teijin.co.jp
(Received 15 September 2005, revised 20
October 2005, accepted 26 October 2005)
doi:10.1111/j.1742-4658.2005.05035.x
Human airway trypsin-like protease (HAT), a serine protease found in the sputum of patients with chronic airway diseases, is an agonist of protease-activated receptor-2 (PAR-2) Previous results have shown that HAT enhances the release of amphiregulin (AR); further, it causes MUC5AC gene expression through the AR-epidermal growth factor receptor pathway
in the airway epithelial cell line NCI-H292 In this study, the mechanisms
by which induced AR release can occur were investigated HAT-induced AR gene expression was mediated by extracellular signal-regulated kinase (ERK) pathway, as pretreatment of cells with ERK pathway inhib-itor eliminated the effect of HAT on AR mRNA Both HAT and PAR-2 agonist peptide (PAR-2 AP) induced ERK phosphorylation; further, desen-sitization of PAR-2 with a brief exposure of cells to PAR-2 AP resulted in inhibition of HAT-induced ERK phosphorylation, suggesting that HAT activates ERK through PAR-2 Moreover, PAR-2 AP induced AR gene expression subsequent to protein production in the cellular fraction through the ERK pathway indicating that PAR-2-mediated activation of ERK is essential for HAT-induced AR production However, in contrast
to HAT, PAR-2 AP could not cause AR release into extracellular space; it appears that activation of PAR-2 is not sufficient for HAT-induced AR release Finally, HAT-induced AR release was eliminated by blockade of tumour necrosis factor a-converting enzyme (TACE) by the TAPI-1 and RNA interference, suggesting that TACE activity is necessary for HAT-induced AR release These observations show that HAT induces AR pro-duction through the PAR-2 mediated ERK pathway, and then causes AR release by a TACE-dependent mechanism
Abbreviations
AR, amphiregulin; EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase; HAT, human airway trypsin-like protease; HGF, hepatocyte growth factor; MEK, ERK kinase; PAR, protease-activated receptor; MMP, matrix metalloprotease; PAR-2 AP, PAR-2 agonist peptide; siRNA, small interfering RNA; TACE, tumour necrosis factor a-converting enzyme.
Trang 2possesses a hydrophobic transmembrane domain near
the N terminus [2] Based on this derived structure,
HAT is thought to be a member of the type-II
trans-membrane serine protease family, which includes corin,
enteropeptidase, MT-SP1 (also known as matriptase)
and hepsin [3] Northern blotting results using RNA
that was collected from 17 human tissues showed that
HAT mRNA is most prominently expressed in
tra-cheal tissue, suggesting that HAT is localized in the
airway [2] Additional evidence supporting this was
obtained from the results of using a HAT-specific
mAb to conduct an immunohistochemical analysis of
the airway tissue isolated from healthy subjects These
results show that HAT is specifically found in ciliated
epithelial cells; however, it is not found in the basal
and goblet cells in the epithelium or in the submucosal
gland cells [4] Therefore, it is thought that HAT might
be responsible for regulating certain biological
proces-ses in airway cells
It has been shown that protease-activated receptor
(PAR)-2 functions as a target protein for HAT in
bronchial epithelial cells [5] PAR-2 is a member of
the PAR protein family; this family includes PAR-1,
PAR-3 and PAR-4 [6] PARs are heterotrimeric
guan-ine nucleotide-binding protein-coupled receptors that
are activated by the cleavage of their N-terminal
domain The proteolytic cleavage of the N-terminal
region of each PAR reveals a new N terminus This
newly revealed terminus acts as a tethered ligand that
binds to the receptor and autoactivates it PAR-2 is
activated by trypsin and mast cell tryptase and is
known to mediate airway inflammation both in vitro
[7,8] and in vivo [9] Furthermore, it has been reported
that the PAR-2 mRNA expression in airway
epithe-lium increases in bronchial asthmatic patients [10]
These observations suggest that HAT might mediate
airway inflammation by PAR-2 activation
In addition to airway inflammation, hypersecretion
of airway mucus is a characteristic sign of chronic
obstructive airway diseases, which include bronchitis,
bronchial asthma and cystic fibrosis [11,12] Such
excessive mucus secretion causes airway obstruction,
which contributes to the morbidity and mortality due
to these diseases [11,12] MUC5AC, a prominent
pro-tein in the airway, is known to participate in the
path-ogenesis of mucus hypersecretion in patients with
chronic airway diseases [13–15] In a previous study
[16], HAT was shown to increase MUC5AC gene
expression, leading to mucus production by the airway
epithelial cell line (NCI-H292) in a range of samples
obtained from the sputum of patients with chronic
air-way disease such as bronchial asthma or bronchitis In
addition, the effect of HAT was completely negated by
treating these cells with a neutralizing antibody of either amphiregulin (AR) or its receptor, i.e epidermal growth factor receptor (EGFR) Further, the treatment
of cells with HAT induced AR gene expression, and subsequently, AR protein release Although the PAR-2 activation in NCI-H292 cells, inferred by intracellular calcium mobilization, is thought to occur after NCI-H292 stimulation by either HAT or the PAR-2 agonist peptide (PAR-2 AP), neither MUC5AC gene expres-sion nor AR protein release was affected by treatment with PAR-2 AP From these observations, HAT-induced MUC5AC gene expression appears to be mediated by the AR-EGFR pathway, and PAR-2 acti-vation alone cannot account for the effect of HAT [16] It has been shown that EGFR plays an important role in the induction of mucin gene expression and mucin production [17–19] Further, the activation of the AR-EGFR pathway has been observed in airway epithelial cells exposed to cigarette smoke extract [20,21] and fine particulate matter [22], which are well known as agents causing airway diseases These obser-vations suggest that the AR-EGFR pathway plays an important role in the induction of mucus overproduc-tion; however, the mechanism by which HAT induces
AR release remains unknown In the present study, HAT-induced AR release and the target molecule of HAT were investigated
Results
HAT regulates AR release at the transcriptional level
Results of a previous study, which focused on the effect of HAT on AR mRNA level 2–24 h after the treatment, indicated that a statistically significant increase in the AR mRNA level began 2 h after the start of HAT treatment (P < 0.01), and that these levels returned to the basal level after 24 h [16] To evaluate the kinetics of HAT-induced AR release, the time course of this release was investigated As shown
in Fig 1A, HAT stimulated a time-dependent AR release A statistically significant effect of HAT was observed as early as 2 h after treatment (Fig 1A;
P < 0.05), suggesting that the effect of HAT on the
AR mRNA level occurred earlier than 2 h To define the onset time of HAT-induced increase in AR mRNA level more accurately, changes in the AR mRNA level
of HAT-treated cells were evaluated at 0.5–2 h after stimulation As shown in Fig 1B, the AR mRNA level appeared to increase almost immediately, i.e 0.5 h after the HAT treatment, with a statistically significant difference (P < 0.01) from the control To evaluate
Trang 3the requirement of transcriptional regulation of the AR
gene on HAT-induced AR release, the effects of
actinomycin D and cycloheximide, which are
transcrip-tion and protein synthesis inhibitors, respectively, on
HAT-induced AR release were determined As shown
in Fig 1C, HAT-induced AR release was significantly
and almost completely inhibited by both
actino-mycin D and cycloheximide treatments These
obser-vations indicate the HAT-induced AR release is
regulated at the transcriptional level
HAT induces tyrosine phosphorylation of EGFR mediated by AR
To examine whether HAT-induced autocrine AR release activates EGFR, HAT-induced tyrosine phos-phorylation of EGFR and the involvement of AR in this signalling cascade were investigated Western blots probed with antiphospho-EGFR antibodies were used
to determine HAT-induced tyrosine phosphorylation of EGFR Since EGFR is known to be phosphorylated by its intrinsic receptor kinase through homo- and hetero-dimerization following ligand binding (autophosphory-lation) and by nonreceptor tyrosine kinases such as Src family kinases [23,24], phosphorylation of EGFR at Tyr845 (known to be phosphorylated by Src [24]) and Tyr1068 (known as an autophosphorylation site [23]) were investigated In HAT-treated cells, phosphoryla-tion of EGFR at Tyr845 and Tyr1068 was not observed until 30 min after treatment (Fig 2A), whereas imme-diate phosphorylation (i.e 3 min after the stimulation)
of these tyrosine residues occurred during treatment with AR However, 120 min after HAT treatment, EGFR phosphorylation at these tyrosine residues had increased and the extent of phosphorylation continued
to increase until 480 min after the treatment and decreased to the basal level by the last time point meas-ured, 24 h after treatment (Fig 2B) Next, the effect of anti-AR neutralizing antibody on HAT-induced phos-phorylation of EGFR was assessed The extent of EGFR phosphorylation at the Tyr1068 residue was used to evaluate the EGFR activation because phos-phorylation on this tyrosine residue functions as the direct binding site for the signal-transducing adapter molecule Grb2 [25], leading to ERK activation [26,27] following MUC5AC gene expression in NCI-H292 cells [19] As shown in the time course analysis in Fig 2C, HAT-induced EGFR phosphorylation was almost completely inhibited in the presence of anti-AR neutral-izing antibody, from the onset time of HAT-induced EGFR phosphorylation (120 min after treatment) to the peak of phosphorylation (480 min after treatment) Results of a previous study suggest that AR is involved
in HAT-induced MUC5AC gene expression [16] In this study, the effect of AR on HAT-induced MUC5AC production was also investigated at the protein level
As shown in Fig 2D, the MUC5AC protein content of NCI-H292 cells increased twofold 24 h after the HAT treatment; however, this effect was almost completely negated in the presence of AR neutralizing anti-body These results indicate that the HAT-induced EGFR phosphorylation almost completely depends on
AR, and HAT-induced MUC5AC production is medi-ated through the AR-EGFR pathway
Fig 1 HAT regulates AR production at the transcriptional level (A,
B) NCI-H292 cells were stimulated with HAT (200 n M ) for indicated
durations (C) NCI-H292 cells were pretreated with the vehicle alone
(Veh), cycloheximide (CHX; 5 lgÆmL)1) or actinomycin D (ActD;
10 lgÆmL)1) for 20 min and then stimulated with HAT (200 n M ) for
2 h in the presence of these inhibitors (A, C) ELISA was used to
determine the AR concentrations in the culture supernatant (B)
Total RNA was extracted, and quantitative real-time RT ⁄ PCR
(Taq-Man TM ) was used to determine the AR and b-actin mRNA amounts.
The results are expressed as the mean ± SD (n ¼ 3) *P < 0.05,
**P < 0.01 when compared with vehicle-treated cells at the same
time point and # P < 0.05, ## P < 0.01 when compared with
HAT-treated cells in the absence of the inhibitors, Dunnett’s test.
Trang 4Involvement of extracellular-signal regulated kinase
pathway in HAT-induced AR gene expression
The cellular mechanism responsible for HAT-induced
ARgene expression was examined As it has been
repor-ted that the extracellular-signal regularepor-ted kinase (ERK)
pathway involves AR release from airway epithelial cells
exposed to fine particulate matter [22], the role of the
ERK signal transduction pathway in HAT-induced AR gene expression was investigated using PD98059 and U0126, which are potent and selective chemical inhibi-tors of ERK kinase (MEK) As shown in Fig 3A, pre-treatment with PD98059 completely eliminated the stimulatory effect of HAT on AR mRNA level Simi-larly, and consistent with findings showing that HAT-induced AR release is regulated at the transcriptional level, the HAT-induced AR release was also completely inhibited by treatment with either PD98059 or U0126 (Fig 3B) These results suggest that HAT induces AR gene expression through the MEK-ERK pathway
HAT induces biphasic ERK activation through AR-dependent and -independent pathways
To determine whether HAT activates the MEK-ERK pathway, western blotting using antiphospho-MEK and
A
B
C
D
Fig 2 HAT induces phosphorylation of EGFR mediated by AR in
NCI-H292 cells NCI-NCI-H292 cells were stimulated with HAT (200 n M ) or AR
(3 ngÆmL)1) for the indicated durations (A, B) NCI-H292 cells were
pretreated with the vehicle alone or anti-AR neutralizing antibody
(aAR; 10 lgÆmL)1) for 20 min and then either (C) stimulated with
HAT (200 n M ) for the indicated durations or (D) stimulated with HAT
(300 n M ) for 24 h in the presence of the antibodies (A, B, C)
Immuno-blotting, with repeated probing using the antibodies indicated on the
left side of the figure, was used to analyse the cell lysates (D)
MUC5AC protein level in cell lysates was determined by ELISA The
results are presented as mean ± SD (n ¼ 3) **P < 0.01 when
com-pared with vehicle-treated cells and ## P < 0.01 when compared with
HAT-treated cells in the absence of the antibodies, Dunnett’s test.
Fig 3 Involvement of ERK in HAT-induced AR gene expression (A, B) NCI-H292 cells were pretreated with the vehicle alone (Veh), PD98059 (PD; 10 l M ) or U0126 (U; 5 l M ) for 20 min (A) Cells were then stimulated with HAT (200 n M ) for 1 h in the presence of the inhibitor or vehicle, total RNA was extracted and quantitative real-time RT ⁄ PCR (TaqMan TM ) analysis was used to determine the amounts of AR and b-actin mRNA (B) Cells were stimulated with HAT (200 n M ) for 2 h in the presence of the inhibitor or vehicle and ELISA was used to determine the AR concentrations in the culture supernatant The results are presented as mean ± SD (n ¼ 3).
*P < 0.05, **P < 0.01 when compared with vehicle-treated cells and #P < 0.05,##P < 0.01 when compared with HAT-treated cells
in the absence of inhibitors, Dunnett’s test.
Trang 5antiphospho-ERK antibodies was performed
Time-dependent effects of HAT on phosphorylation of MEK
and ERK were examined until 24 h after the treatment
Phosphorylation of MEK and ERK was observed
within 5 min of the HAT treatment; however,
de-phosphorylation occurred 30 min after treatment After
completion of the rapid transient phosphorylation of
MEK and ERK, a second, less extensive round of
MEK and ERK phosphorylation was observed, which
began 120 min after the HAT stimulation and lasted
until 480 min after treatment (Fig 4A) In order to
assess the effect of HAT on the kinase activity of ERK,
phosphorylation of the downstream kinase p90RSK at
Ser359 and Thr363 (residues known to be directly
phosphorylated by ERK [28]) was examined Similar
to HAT-induced MEK-ERK phosphorylation, biphasic
phosphorylation of p90RSK was induced by HAT
treatment (Fig 4B) To confirm this result, the effect of
PD98059 on HAT-induced p90RSK phosphorylation at
5 min and 480 min was examined independently by the
following steps For the 5-min time point, NCI-H292
cells were pretreated with PD98050 20 min before the
treatment, while for the 480 min time point, NCI-H292
cells were treated with PD98059 30 min after HAT sti-mulation; at this time, the first round of phosphoryla-tion is completed (Fig 4A) When assessed in this manner, HAT-induced p90RSK phosphorylation at
5 min and 480 min was inhibited in the presence
of PD98059 (Fig 4C), suggesting that p90RSK was directly phosphorylated by activated ERK Thus, the HAT-induced biphasic ERK phosphorylation is accom-panied by the enhancement of its kinase activity Next, the involvement of AR in the HAT-induced biphasic ERK activation was investigated using an
anti-AR neutralizing antibody The HAT-induced initial ERK activation (5 min after stimulation with HAT) was inhibited by a PD98059 treatment but not by the anti-AR neutralizing antibody treatment (Fig 5A), while the HAT-induced second round of ERK activa-tion (480 min after stimulaactiva-tion with HAT) was inhib-ited by the PD98059 treatment or the anti-AR neutralizing antibody treatment (Fig 5B) A time-course study of HAT-induced ERK activation in the presence or absence of anti-AR neutralizing antibody was also conducted in order to confirm the involvement
of AR ERK activation at 5 min after HAT treatment was not affected by the anti-AR neutralizing antibody; however, the activation of ERK was completely inhib-ited by the anti-AR neutralizing antibody at 120 min and 240 min after HAT treatment (Fig 5C) In addi-tion, AR-induced ERK phosphorylation occurred within 5 min of the stimulation and sustained up to
480 min (Fig 5D); these kinetics were similar to those
of the HAT-induced second round of ERK activation (Fig 4A) Considered together, these observations sug-gest that the HAT increases AR gene expression through initial ERK activation and that a second round
of ERK activation is induced through EGFR that is activated by autocrine AR stimulation In addition, these events appear to occur in the airway of patients with chronic airway diseases since the HAT-induced initial ERK activation was observed at a low HAT concentration of 6.6 nm (equivalent to 10.8 mUÆmL)1, Fig 5E), which is similar to the concentration observed
in mucoid sputum from patients with either chronic bronchitis (23.46 ± 18.03 mUÆmL)1) or bronchial asthma (46.96 ± 43.96 mUÆmL)1[29])
Desensitization of PAR-2 blocks HAT-induced ERK phosphorylation
HAT and PAR-2 AP-induced activation of PAR-2 has been observed in NCI-H292 cells [16] In addition, it has been reported that the activation of PAR-2 causes ERK phosphorylation [8,30–34] To clarify whether the HAT-induced initial ERK activation is mediated
A
B
C
Fig 4 HAT induces biphasic activation of ERK (A, B) NCI-H292
cells were stimulated with HAT (200 n M ) for the indicated
dura-tions (C) NCI-H292 cells were pretreated with the vehicle alone or
with PD98059 (PD; 10 l M ) for 20 min and then stimulated with
HAT (200 n M ) for 5 min During evaluation at a culture period of
480 min, NCI-H292 cells were stimulated with HAT for 30 min and
then treated with the vehicle alone or with PD98059 (PD; 10 l M ).
Further, they were cultured up to 480 min Immunoblotting, with
repeated probing using the antibodies indicated on the left side of
the figure, was used to analyse the cell lysates.
Trang 6by PAR-2, experiments using PAR-2 AP, which
specif-ically activates PAR-2 [6], were conducted First, the
effect of PAR-2 AP on the extent and pattern of ERK
activation was examined In PAR-2 AP-treated cells,
the ERK activation was observed within 5 min of
treatment (Fig 6A); however, unlike the HAT-induced
biphasic ERK activation (Fig 4A), PAR-2 AP-induced
ERK activation was transient, and a second round of
ERK activation was not observed within 480 min of treatment (Fig 6A) Consistent with findings that show that a second round of HAT-induced ERK acti-vation is mediated by the AR-EGFR pathway (Fig 5B and C), the activation of EGFR was not observed within 480 min of PAR-2 AP treatment (Fig 6B) Next, the effect of a brief exposure to PAR-2 AP (to desensitize PAR-2 [30,31]); prior to the HAT stimula-tion, on the HAT-induced initial ERK activation was examined As shown in Fig 6C, brief exposure of NCI-H292 cells to PAR-2 AP resulted in specific inhi-bition of PAR-2 AP-induced ERK activation whereas hepatocyte growth factor (HGF)-induced ERK activa-tion was unaffected Further, HAT-induced initial ERK activation was completely inhibited by pretreat-ment with PAR-2 AP (Fig 6C) These observations suggest that at least a part of the HAT-induced initial ERK activation was mediated by PAR-2
ERK activation through PAR-2 induces AR protein production but not protein release into the culture supernatant
Since HAT-induced initial ERK activation results in induction of AR gene expression, the effect of PAR-2
A
B
C
D
E
Fig 5 HAT induces biphasic activation of ERK through
AR-depend-ent and -independAR-depend-ent pathways (A, C) NCI-H292 cells were
pre-treated with the vehicle alone (Veh), PD98059 (PD; 10 l M ), anti-AR
neutralizing antibody (aAR; 10 lgÆmL)1) or normal mouse IgG1
(IgG; 10 lgÆmL)1) for 20 min The cells were then stimulated with
HAT (200 n M ) for 5 min (A) or the indicated durations (C) in the
presence of the indicated inhibitors or antibody (B) For the
480 min culture period, NCI-H292 cells were stimulated with HAT
for 30 min and then treated with the vehicle alone or with
PD98059 (PD; 10 l M ), anti-AR neutralizing antibody (aAR;
10 lgÆmL)1) or normal mouse IgG1 (IgG; 10 lgÆmL)1) and further
cultured for 480 min (D) Cells were stimulated with AR (3 lgÆmL)1)
for the indicated durations (E) Cells were stimulated with
increas-ing concentrations of HAT for 5 min Immunoblottincreas-ing, with
repea-ted probing using the antibodies indicarepea-ted on the left side of the
figure, was used to analyse the cell lysates.
A
B
C
Fig 6 Desensitization of PAR-2 blocks HAT-induced initial ERK activation (A, B) NCI-H292 cells were stimulated with PAR-2 AP (300 l M ) or HAT (200 n M ) for the indicated durations (C) NCI-H292 cells were pretreated with the vehicle alone or with PAR-2 AP (300 l M ) for 20 min (C) Cells were then stimulated with HAT (200 n M ), PAR-2 AP (300 l M ) or HGF (20 ngÆmL)1) for 5 min in the presence of inhibitors or the vehicle Immunoblotting, with repea-ted probing using the antibodies indicarepea-ted on the left side of the figure, was used to analyse the cell lysates.
Trang 7AP on the AR gene expression during the 0.5–2-h
per-iod after the treatment was examined In PAR-2
AP-treated NCI-H292 cells, AR mRNA level
signifi-cantly increased at 0.5 h after the treatment (Fig 7A)
In contrast to the continuous increase in AR mRNA
level observed in HAT-treated cells until 4 h after the
treatment (Fig 1B), the AR mRNA level returned to
the basal level 1 h after treatment in PAR-2
AP-trea-ted cells and did not significantly increase until 2 h
after the treatment (Fig 7A) These results
demon-strate that the activation of PAR-2 causes a rapid transient increase in AR mRNA level, although no sta-tistically significant change in AR protein concentra-tion has been observed in the culture supernatant of PAR-2 AP-treated NCI-H292 cells in a previous study [16] To investigate whether PAR-2 AP has any effect
on AR protein production, the AR protein concentra-tion in the culture supernatant and cellular lysates was evaluated in cell cultures treated with PAR-2 AP Sim-ilar to the results of a previous study [16], PAR-2 AP treatment had no effect on the AR protein concen-tration in the culture supernatant 2 and 4 h after treatment (Fig 7B) However, the AR protein con-centration in cell lysates prepared from PAR-2 AP-treated cells showed a statistically significant increase (P < 0.01) between 2 and 4 h after the treat-ment The effect of PAR-2 AP on AR protein produc-tion was mediated by the ERK pathway since the PAR-2 AP-induced increase in AR protein concentra-tion in cellular lysate was completely eliminated by pretreatment with PD98059 (Fig 7C) These observa-tions suggest that activation of PAR-2 mediated ERK pathway results in the induction of AR gene expression subsequent to the production of AR protein that is bound to, or otherwise associated with, the cells, for example, bound to the cell surface
Tumour necrosis factor a-converting enzyme activity is required for HAT-induced AR release that prolongs HAT-induced AR gene expression
by a positive feedback loop Results of the present study suggest that the activation
of PAR-2 is sufficient to induce AR protein produc-tion, but cannot account for AR release Thus, the mechanism of HAT-induced AR release from a cell was also investigated The effect of GM6001, a broad-spectrum metalloprotease inhibitor, and TAPI-1, a rel-atively selective metalloprotease inhibitor for tumour necrosis factor a-converting enzyme (TACE), on HAT-induced AR release was determined as it is well known that metalloproteases, such as matrix metallo-protease (MMP) and TACE, cause AR release by proteolytic cleavage of the transmembrane precursor [20,35–37] As shown in Fig 8A, HAT-induced AR protein release was significantly inhibited (P < 0.01)
by pretreatment of cells with GM6001 and TAPI-1 (Fig 8A) In addition, these inhibitors did not affect the protease activity of HAT at the concentration used
in this study (data not shown) Next, to evaluate whe-ther TACE is required for HAT-induced AR release, endogenous TACE expression was blocked using a small interfering RNA (siRNA) Silencing of TACE
Fig 7 Activation of PAR-2 causes AR gene expression and AR
pro-tein production but does not evoke AR release (A, B) NCI-H292
cells were stimulated with PAR-2 AP (300 l M ) for the indicated
durations (C) NCI-H292 cells were pretreated with the vehicle
alone (Veh) or with PD98059 (PD; 10 l M ) for 20 min and then
sti-mulated with PAR-2 AP (300 l M ) for 2 h (A) Total RNA was
extrac-ted, and quantitative real-time RT ⁄ PCR (TaqMan TM ) analysis was
used to determine the amounts of AR and b-actin mRNA (B, C)
ELISA was used to determine the AR concentrations in the culture
supernatants and cellular lysates The results are presented as
mean ± SD (n ¼ 3) **P < 0.01 when compared with
vehicle-trea-ted cells at the same time point,##P < 0.01 when compared with
PAR-2 AP-treated cells in the absence of inhibitors, Dunnett’s test.
Trang 8was confirmed by flow cytometry using a mouse mAb that recognizes an ectodomain of TACE TACE was detected on the surface of NCI-H292 cells but was almost reduced to the background level by transfecting with TACE siRNA (Fig 8B) Inhibition of TACE expression significantly suppressed HAT-induced AR release (Fig 8C) These data suggest that HAT causes
AR protein release mediated by TACE activity Fur-ther, it has been reported that the activation of EGFR
by EGF induces an autocrine EGFR ligand expression
in bronchial epithelial cells [21]; therefore, it is possible that the prolonged effect of HAT on AR mRNA expression is mediated by autocrine stimulation of AR
To test this hypothesis, the involvement of AR with HAT-induced AR mRNA expression was assessed using the anti-AR neutralizing antibody As shown in Fig 8D, treatment of exogenous AR causes AR mRNA expression after 1 h stimulation and this effect was inhibited by pretreatment with PD98059, suggest-ing that AR induces AR gene expression through the ERK pathway Further, the HAT-induced increase in
AR mRNA level occurring 2 h after the HAT treat-ment was significantly and almost completely negated when NCI-H292 cells were treated with the anti-AR neutralizing antibody (Fig 8E) Considered together, these results suggest that HAT induces AR release through TACE activity and the released AR prolongs HAT-induced AR gene expression by a positive feed-back loop
Discussion
Recently, the EGFR signalling pathway has been shown to function as a common pathway through which many stimuli induce MUC5AC production
in vitro [17–19] Further, in the airway epithelium of asthmatic patients, the activation of EGFR was sug-gested to be involved in mucus hypersecretion [38], which can have profound effects on health [11,12] In another study, HAT was originally found in the spu-tum of patients with diseases causing airway mucus hypersecretion [1] Subsequent investigations revealed that EGFR and its ligand AR are involved in the HAT-induced MUC5AC gene expression As a result, HAT appeared to prefer EGFR as an activator There-fore, finding a mechanism by which HAT regulates AR-EGFR activation might elucidate the basic mecha-nisms of airway disease pathogenesis Further, this finding may also provide an additional benefit in terms
of leading to the development of new therapeutic strat-egies to treat diseases marked by airway mucus hyper-secretion The results of the present study showed that HAT activates EGFR through a pathway that includes
A
B
C
D
E
Fig 8 TACE is involved in HAT-induced AR release, which
pro-longs HAT-induced AR gene expression by positive feedback loop.
(A, D, E) NCI-H292 cells were pretreated with the vehicle alone
(Veh), GM6001 (GM; 3 l M ), TAPI-1 (TAPI; 3 l M ), PD98059 (PD;
10 l M ) or anti-AR neutralizing antibody (aAR; 10 lgÆmL)1) for
20 min (B, C) NCI-H292 cells were transfected with siRNA for
TACE or control siRNA (cont) and cultured for 72 h (A, C) Cells
were then stimulated with HAT (200 n M ) for 2 h and ELISA was
used to determine the AR concentration in culture supernatant.
(B) Cells were then collected and stained with anti-TACE antibody
or normal mouse IgG1 (background) and analysed for cell surface
TACE density by flow cytometry (D, E) Cells were then stimulated
with AR (3 ngÆmL) for 1 h (D) or HAT (200 n M ) for 2 h (E) and
the total RNA was extracted, and quantitative real-time RT ⁄ PCR
(TaqMan TM ) analysis was used to determine the amounts of AR
and b-actin mRNA The results are presented as the mean ± SD
(n ¼ 3) *P < 0.05, **P < 0.01 when compared with vehicle-treated
cells and ## P < 0.01 when compared with HAT (A, C) or AR
(B)-treated cells in the absence of inhibitors, Dunnett’s test.
Trang 9PAR-2 mediating ERK-dependent AR gene expression
and TACE-dependent AR protein release
In the time course analysis of tyrosine
phosphoryla-tion of EGFR, AR-induced EGFR activaphosphoryla-tion could be
detected within 3 min of stimulation; however,
HAT-induced EGFR activation was observed 2 h after
sti-mulation This observation was in good agreement
with the results of our previous study showing that
HAT-mediated increase in MUC5AC mRNA level
occurs after the EGF-mediated increase in MUC5AC
mRNA level, although both cause MUC5AC gene
expression through EGFR activation In the present
study, the time course of HAT-induced EGFR
activa-tion corresponded to that of HAT-induced AR release;
further, the activation of EGFR and the induction of
MUC5AC protein production were completely
inhib-ited in the presence of anti-AR neutralizing antibody
Considered along with our previous observation that
among several EGFR ligands (EGF, HB-EGF,
trans-forming growth factor-a, AR), only anti-AR
neutral-izing antibody completely inhibited HAT-induced
MUC5AC gene expression, the results of the present
study strongly suggest that AR may be the initial
EGFR ligand responsible for HAT-induced EGFR
activation in NCI-H292 cells
Different steps of the pathway leading to
HAT-induced AR production have been analysed using
pharmacological enzyme inhibitors The following
results suggest the involvement of PAR-2-mediated
activation of ERK: (a) the MEK inhibitor PD98059
and U0126 inhibited HAT-induced AR release; (b)
activation of PAR-2 by PAR-2 AP induced ERK
phosphorylation and desensitization of PAR-2 resulted
in inhibition of HAT-induced initial ERK activation;
and (c) PD98059 inhibited PAR-2 AP-induced AR
protein production Although the mechanisms by
which PAR-2 activates ERK were unknown, our study
aimed to show that PAR-2 mediated ERK activation
is an essential step in HAT-induced EGFR activation
since the induction of gene expression and subsequent
protein production of EGFR ligands by PAR-2
agon-ists have not yet been reported in any cells
It has been reported that the EGFR ligand family,
which includes AR, is originally translated as
precur-sors with transmembrane domains, and these proteins
are located on the exterior surface of the cytoplasmic
membrane In response to an appropriate stimulation,
these precursors are proteolytically cleaved to obtain
their mature forms by metalloproteases such as MMP
and TACE and are released into the extracellular space
[20,35–37] Although membrane-bound EGFR ligands
can engage in juxtacrine signalling [39,40], the
TACE-dependent release of AR has been shown to function
as a key step in transactivating EGFR in tobacco smoke-stimulated bronchial epithelial cells [20] In the present study, HAT and PAR-2 AP stimulate AR gene expression and subsequent AR protein production; however, an increase in AR protein release in NCI-H292 cells was only observed by HAT stimulation Further, the increase in the AR content in PAR-2 AP-treated cells did not result in the induction of EGFR activation (Fig 6B) Moreover, HAT-induced
AR protein release, which could induce EGFR activa-tion, was eliminated by blocking TACE using siRNA (Fig 8C) These observations indicate that the activa-tion of ERK through PAR-2 results in the producactiva-tion
of the AR precursor; however, this is not responsible for the release of active AR HAT stimulates AR release by TACE activity mediated by a PAR-2-inde-pendent mechanism Although the cellular process of TACE activation has not been defined, the mecha-nisms that cause immediate activation of TACE are probably not responsible for HAT-induced activation
of TACE, because time-course analysis results of this study show that the HAT-induced AR-dependent acti-vation of EGFR occurred 2 h after treating cells with HAT (Fig 2B) One possible mechanism is that HAT may increase TACE expression Recently, it has been reported that in alveolar macrophage, lipopolysaccha-ride increases TACE expression which correlates with the catalytic activity of this enzyme [41] In contrast to the results from our study, it is reported that in colon cancer cells, PAR-2 activation induces an MMP-dependent release of transforming growth factor-a, thus suggesting that PAR-2 activation caused the MMP activation [34] These observations suggest that mechanisms that provoke the release of EGFR ligands appear to be heterogeneous and depend upon specific components of signalling molecules expressed within a cell type Thus, further investigation is needed to clar-ify the mechanisms that lead to HAT-induced AR release, including TACE activity regulation In conclu-sion, Fig 9 depicts the mechanism of HAT-induced
AR in NCI-H292 cells It schematically reflects the major findings of the present study, which are as fol-lows: (a) HAT induces AR gene expression subsequent
to AR protein release through ERK activation; (b) at least a part of HAT-induced initial ERK activation is mediated through PAR-2; (c) only HAT, and not PAR-2 AP, causes AR protein release through TACE activity; and (d) prolonged effect of HAT on AR mRNA is mediated through a positive feedback loop stimulated by autocrine AR The results of the present study shed light on a complex mechanisms of AR release, further suggest that excess HAT activity directly leads to the pathogenesis of chronic airway
Trang 10diseases through its effect on the PAR-2 and EGFR
signalling pathways
Experimental procedures
Reagents and antibodies
Recombinant HAT (60 UÆmg)1 protein) was prepared as
previously described [1,2,5] In brief, HAT was expressed in
insect cells infected with a recombinant baculovirus
carry-ing the HAT cDNA [2] Benzamidine affinity
chromatogra-phy was used to purify recombinant HAT from the cell
lysate [5], and the specific activity of the purified protein
was measured with Boc-Phe-Ser-Arg-MCA as a substrate,
as previously described [1] PAR-2 AP consisting of
Ser-Leu-Ile-Gly-Lys-Val-NH2 [6] was from Bachem AG
(Bub-endorf, Switzerland) Cycloheximide was from Wako Pure
Chemicals (Osaka, Japan); PD98059 and actinomycin D,
Biomol (Plymouth Meeting, PA, USA); U0126, Promega
(Madison, WI, USA); and GM6001 and TAPI-1,
Calbio-chem (San Diego, CA, USA) Human recombinant AR,
anti-TACE mAb (clone 111633) and nonimmune mouse
IgG1, used as negative controls, were from R&D Systems
Inc (Minneapolis, MN, USA) Anti-p90RSK antibody was
from Upstate Biotechnologies (Lake Placid, NY, USA) and
neutralizing mouse mAb against AR (clone 31221.111) and
goat polyclonal antibody against AR were from Genzyme
(Minneapolis, MN, USA) The antiphospho-MEK (Ser217⁄ 221), antiphospho-ERK (Thr202⁄ Tyr204), ERK, anti-phospho-p90RSK (Thr359⁄ Ser363), antiphospho-EGFR (Tyr845) and antiphospho-EGFR (Tyr1068) antibodies were from Cell Signaling (Beverly, MA, USA) The mouse anti-EGFR mAb was from Transduction Laboratories (Lexington, KY, USA) and the mouse anti-MUC5AC mAb (Clone 45M1) were from LAB VISION (Fremont, CA, USA)
Cell culture NCI-H292 cells were from the American Type Culture Col-lection (Rockville, MD, USA) and were cultured in RPMI
1640 medium supplemented with 10% (v⁄ v) fetal bovide serum, 50 UÆmL)1 penicillin and 50 lgÆmL)1 streptomycin (Gibco BRL, Grand Island, NY, USA) in a humidified incubator at 37C in an atmosphere of 5% CO2 Prior to the experiments, confluent NCI-H292 cells were cultured in
a serum-free medium composed of RPMI 1640 medium containing only 0.1% (w⁄ v) BSA (Sigma, St Louis, MO, USA) for 24 h, unless otherwise indicated
Immunoblotting NCI-H292 cells were incubated with the appropriate condi-tions, quickly placed on ice, and washed twice in ice-cold NaCl⁄ Pi The cells were then lysed in M-PERMammalian Protein Extraction Reagent (Pierce, Rockford, IL, USA) containing 1% (v⁄ v) each of protease inhibitor cocktail and phosphatase inhibitor cocktail (Sigma), while gently stirring the cells for 5 min at room temperature Each lysate was transferred to a separate centrifuge tube, and the lysates were centrifuged at 4C for 10 min at 15 000 g The
cleared supernatants were collected separately, and a Bio-Rad protein assay system (Bio-Bio-Rad, Hercules, CA, USA) was used to determine the protein content in each superna-tant by using the Bradford technique Separate samples containing approximately equal amounts of cellular protein were mixed with SDS⁄ PAGE sample buffer containing dithiothreitol, heated for 5 min at 99C, and loaded on 10–20% or 3–10% gradient SDS⁄ polyacrylamide gels Electrophoresis was performed at a constant current (25 mA⁄ 0.75-mm thick gel) After electrophoresis, the pro-teins were electroblotted (100 mA constant current per
100 cm2 gel) onto a polyvinylidene difluoride membrane (Hybond-P; Amersham Biosciences, Piscataway, NJ, USA) The membrane was blocked with 5% (v⁄ v) nonfat milk TBST [10 mm Tris⁄ HCl pH 7.4, 150 mm NaCl and 0.1% (v⁄ v) Tween 20] solution for 1 h, washed three times for
5 min with TBST and treated with one of the following antibody preparations at 4C overnight: antiphospho ERK antibody (diluted to 1 : 2000 in 5% nonfat milk TBST), mouse anti-EGFR mAb (diluted to 1 : 2500 in 5% nonfat milk TBST), antiphospho-EGFR (Tyr845) antibody,
anti-Fig 9 Proposed mechanisms of HAT-induced activation of
AR-EGFR pathway in airway epithelial cell lines According to this
model, HAT-induced activation of AR production is mediated by
PAR-2 dependent or PAR-2 independent mechanisms A
PAR-2-mediated initial ERK activation causes the production of AR
precursor, and then, biologically active AR is released by
PAR-2-independent mechanism involving TACE.