Human airway trypsin-like protease induces amphiregulin release 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 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 BioMedical 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 proteaseactivated 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 HAT-induced AR release can occur were investigated HATinduced AR gene expression was mediated by extracellular signal-regulated kinase (ERK) pathway, as pretreatment of cells with ERK pathway inhibitor eliminated the effect of HAT on AR mRNA Both HAT and PAR-2 agonist peptide (PAR-2 AP) induced ERK phosphorylation; further, desensitization 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 HATinduced AR release These observations show that HAT induces AR production through the PAR-2 mediated ERK pathway, and then causes AR release by a TACE-dependent mechanism 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 chronic bronchitis and bronchial asthma, based on its protease activity [1] It exists in the sputum as a monomer with a molecular size of 27 kDa as estimated by gel filtration chromatography [1] HAT cDNA has been isolated 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 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 FEBS Journal 272 (2005) 6387–6399 ª 2005 The Authors Journal compilation ª 2005 FEBS 6387 HAT-induced AR release by PAR-2 and TACE M Chokki et al possesses 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 transmembrane 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 tracheal 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 processes 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 guanine 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 epithelium 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 protein in the airway, is known to participate in the pathogenesis 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 airway disease such as bronchial asthma or bronchitis In addition, the effect of HAT was completely negated by 6388 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 NCIH292 stimulation by either HAT or the PAR-2 agonist peptide (PAR-2 AP), neither MUC5AC gene expression nor AR protein release was affected by treatment with PAR-2 AP From these observations, HATinduced MUC5AC gene expression appears to be mediated by the AR-EGFR pathway, and PAR-2 activation 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 observations suggest that the AR-EGFR pathway plays an important role in the induction of mucus overproduction; 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 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 h after treatment (Fig 1A; P < 0.05), suggesting that the effect of HAT on the AR mRNA level occurred earlier than 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 FEBS Journal 272 (2005) 6387–6399 ª 2005 The Authors Journal compilation ª 2005 FEBS M Chokki et al HAT-induced AR release by PAR-2 and TACE HAT induces tyrosine phosphorylation of EGFR mediated by AR Fig HAT regulates AR production at the transcriptional level (A, B) NCI-H292 cells were stimulated with HAT (200 nM) for indicated durations (C) NCI-H292 cells were pretreated with the vehicle alone (Veh), cycloheximide (CHX; lgỈmL)1) or actinomycin D (ActD; 10 lgỈmL)1) for 20 and then stimulated with HAT (200 nM) for 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 (TaqManTM) 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 HATtreated cells in the absence of the inhibitors, Dunnett’s test the requirement of transcriptional regulation of the AR gene on HAT-induced AR release, the effects of actinomycin D and cycloheximide, which are transcription 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 actinomycin D and cycloheximide treatments These observations indicate the HAT-induced AR release is regulated at the transcriptional level To examine whether HAT-induced autocrine AR release activates EGFR, HAT-induced tyrosine phosphorylation 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 heterodimerization following ligand binding (autophosphorylation) 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, phosphorylation of EGFR at Tyr845 and Tyr1068 was not observed until 30 after treatment (Fig 2A), whereas immediate phosphorylation (i.e after the stimulation) of these tyrosine residues occurred during treatment with AR However, 120 after HAT treatment, EGFR phosphorylation at these tyrosine residues had increased and the extent of phosphorylation continued to increase until 480 after the treatment and decreased to the basal level by the last time point measured, 24 h after treatment (Fig 2B) Next, the effect of anti-AR neutralizing antibody on HAT-induced phosphorylation of EGFR was assessed The extent of EGFR phosphorylation at the Tyr1068 residue was used to evaluate the EGFR activation because phosphorylation 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 neutralizing antibody, from the onset time of HAT-induced EGFR phosphorylation (120 after treatment) to the peak of phosphorylation (480 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 anti-AR neutralizing antibody These results indicate that the HAT-induced EGFR phosphorylation almost completely depends on AR, and HAT-induced MUC5AC production is mediated through the AR-EGFR pathway FEBS Journal 272 (2005) 6387–6399 ª 2005 The Authors Journal compilation ª 2005 FEBS 6389 HAT-induced AR release by PAR-2 and TACE M Chokki et al A B C D Fig HAT induces phosphorylation of EGFR mediated by AR in NCIH292 cells NCI-H292 cells were stimulated with HAT (200 nM) or AR (3 ngỈmL)1) for the indicated durations (A, B) NCI-H292 cells were pretreated with the vehicle alone or anti-AR neutralizing antibody (aAR; 10 lgỈmL)1) for 20 and then either (C) stimulated with HAT (200 nM) for the indicated durations or (D) stimulated with HAT (300 nM) for 24 h in the presence of the antibodies (A, B, C) Immunoblotting, 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 compared with vehicle-treated cells and ##P < 0.01 when compared with HAT-treated cells in the absence of the antibodies, Dunnett’s test Involvement of extracellular-signal regulated kinase pathway in HAT-induced AR gene expression The cellular mechanism responsible for HAT-induced AR gene expression was examined As it has been reported that the extracellular-signal regulated kinase (ERK) pathway involves AR release from airway epithelial cells exposed to fine particulate matter [22], the role of the 6390 Fig Involvement of ERK in HAT-induced AR gene expression (A, B) NCI-H292 cells were pretreated with the vehicle alone (Veh), PD98059 (PD; 10 lM) or U0126 (U; lM) for 20 (A) Cells were then stimulated with HAT (200 nM) for h in the presence of the inhibitor or vehicle, total RNA was extracted and quantitative realtime RT ⁄ PCR (TaqManTM) analysis was used to determine the amounts of AR and b-actin mRNA (B) Cells were stimulated with HAT (200 nM) for 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 ERK signal transduction pathway in HAT-induced AR gene expression was investigated using PD98059 and U0126, which are potent and selective chemical inhibitors of ERK kinase (MEK) As shown in Fig 3A, pretreatment with PD98059 completely eliminated the stimulatory effect of HAT on AR mRNA level Similarly, and consistent with findings showing that HATinduced 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 FEBS Journal 272 (2005) 6387–6399 ª 2005 The Authors Journal compilation ª 2005 FEBS M Chokki et al antiphospho-ERK antibodies was performed Timedependent 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 of the HAT treatment; however, dephosphorylation occurred 30 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 after the HAT stimulation and lasted until 480 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 and 480 was examined independently by the following steps For the 5-min time point, NCI-H292 cells were pretreated with PD98050 20 before the treatment, while for the 480 time point, NCI-H292 A B C Fig HAT induces biphasic activation of ERK (A, B) NCI-H292 cells were stimulated with HAT (200 nM) for the indicated durations (C) NCI-H292 cells were pretreated with the vehicle alone or with PD98059 (PD; 10 lM) for 20 and then stimulated with HAT (200 nM) for During evaluation at a culture period of 480 min, NCI-H292 cells were stimulated with HAT for 30 and then treated with the vehicle alone or with PD98059 (PD; 10 lM) Further, they were cultured up to 480 Immunoblotting, with repeated probing using the antibodies indicated on the left side of the figure, was used to analyse the cell lysates HAT-induced AR release by PAR-2 and TACE cells were treated with PD98059 30 after HAT stimulation; at this time, the first round of phosphorylation is completed (Fig 4A) When assessed in this manner, HAT-induced p90RSK phosphorylation at and 480 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 accompanied by the enhancement of its kinase activity Next, the involvement of AR in the HAT-induced biphasic ERK activation was investigated using an antiAR neutralizing antibody The HAT-induced initial ERK activation (5 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 activation (480 after stimulation with HAT) was inhibited by the PD98059 treatment or the anti-AR neutralizing antibody treatment (Fig 5B) A timecourse 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 after HAT treatment was not affected by the anti-AR neutralizing antibody; however, the activation of ERK was completely inhibited by the anti-AR neutralizing antibody at 120 and 240 after HAT treatment (Fig 5C) In addition, AR-induced ERK phosphorylation occurred within of the stimulation and sustained up to 480 (Fig 5D); these kinetics were similar to those of the HAT-induced second round of ERK activation (Fig 4A) Considered together, these observations suggest 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 mmL)1, Fig 5E), which is similar to the concentration observed in mucoid sputum from patients with either chronic bronchitis (23.46 ± 18.03 mmL)1) or bronchial asthma (46.96 ± 43.96 mmL)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 FEBS Journal 272 (2005) 6387–6399 ª 2005 The Authors Journal compilation ª 2005 FEBS 6391 HAT-induced AR release by PAR-2 and TACE M Chokki et al A A B B C C D Fig Desensitization of PAR-2 blocks HAT-induced initial ERK activation (A, B) NCI-H292 cells were stimulated with PAR-2 AP (300 lM) or HAT (200 nM) for the indicated durations (C) NCI-H292 cells were pretreated with the vehicle alone or with PAR-2 AP (300 lM) for 20 (C) Cells were then stimulated with HAT (200 nM), PAR-2 AP (300 lM) or HGF (20 ngỈmL)1) for in the presence of inhibitors or the vehicle Immunoblotting, with repeated probing using the antibodies indicated on the left side of the figure, was used to analyse the cell lysates E Fig HAT induces biphasic activation of ERK through AR-dependent and -independent pathways (A, C) NCI-H292 cells were pretreated with the vehicle alone (Veh), PD98059 (PD; 10 lM), anti-AR neutralizing antibody (aAR; 10 lgỈmL)1) or normal mouse IgG1 (IgG; 10 lgỈmL)1) for 20 The cells were then stimulated with HAT (200 nM) for (A) or the indicated durations (C) in the presence of the indicated inhibitors or antibody (B) For the 480 culture period, NCI-H292 cells were stimulated with HAT for 30 and then treated with the vehicle alone or with PD98059 (PD; 10 lM), anti-AR neutralizing antibody (aAR; 10 lgỈmL)1) or normal mouse IgG1 (IgG; 10 lgỈmL)1) and further cultured for 480 (D) Cells were stimulated with AR (3 lgỈmL)1) for the indicated durations (E) Cells were stimulated with increasing concentrations of HAT for Immunoblotting, with repeated probing using the antibodies indicated on the left side of the figure, was used to analyse the cell lysates by PAR-2, experiments using PAR-2 AP, which specifically 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 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 6392 ERK activation was not observed within 480 of treatment (Fig 6A) Consistent with findings that show that a second round of HAT-induced ERK activation is mediated by the AR-EGFR pathway (Fig 5B and C), the activation of EGFR was not observed within 480 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 stimulation, 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 inhibition of PAR-2 AP-induced ERK activation whereas hepatocyte growth factor (HGF)-induced ERK activation was unaffected Further, HAT-induced initial ERK activation was completely inhibited by pretreatment 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 FEBS Journal 272 (2005) 6387–6399 ª 2005 The Authors Journal compilation ª 2005 FEBS M Chokki et al AP on the AR gene expression during the 0.5–2-h period after the treatment was examined In PAR-2 AP-treated NCI-H292 cells, AR mRNA level significantly 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 h after the treatment (Fig 1B), the AR mRNA level returned to the basal level h after treatment in PAR-2 AP-treated cells and did not significantly increase until h after the treatment (Fig 7A) These results demon- HAT-induced AR release by PAR-2 and TACE strate that the activation of PAR-2 causes a rapid transient increase in AR mRNA level, although no statistically significant change in AR protein concentration 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 concentration in the culture supernatant and cellular lysates was evaluated in cell cultures treated with PAR-2 AP Similar to the results of a previous study [16], PAR-2 AP treatment had no effect on the AR protein concentration in the culture supernatant and h after treatment (Fig 7B) However, the AR protein concentration in cell lysates prepared from PAR-2 AP-treated cells showed a statistically significant increase (P < 0.01) between and h after the treatment The effect of PAR-2 AP on AR protein production was mediated by the ERK pathway since the PAR-2 AP-induced increase in AR protein concentration in cellular lysate was completely eliminated by pretreatment with PD98059 (Fig 7C) These observations 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 Fig Activation of PAR-2 causes AR gene expression and AR protein production but does not evoke AR release (A, B) NCI-H292 cells were stimulated with PAR-2 AP (300 lM) for the indicated durations (C) NCI-H292 cells were pretreated with the vehicle alone (Veh) or with PD98059 (PD; 10 lM) for 20 and then stimulated with PAR-2 AP (300 lM) for h (A) Total RNA was extracted, and quantitative real-time RT ⁄ PCR (TaqManTM) 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-treated 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 Results of the present study suggest that the activation of PAR-2 is sufficient to induce AR protein production, 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 broadspectrum metalloprotease inhibitor, and TAPI-1, a relatively 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 metalloprotease (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 whether TACE is required for HAT-induced AR release, endogenous TACE expression was blocked using a small interfering RNA (siRNA) Silencing of TACE FEBS Journal 272 (2005) 6387–6399 ª 2005 The Authors Journal compilation ª 2005 FEBS 6393 HAT-induced AR release by PAR-2 and TACE M Chokki et al was 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 Further, 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 h stimulation and this effect was inhibited by pretreatment with PD98059, suggesting that AR induces AR gene expression through the ERK pathway Further, the HAT-induced increase in AR mRNA level occurring h after the HAT treatment 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 feedback loop A B C D E Discussion Fig TACE is involved in HAT-induced AR release, which prolongs HAT-induced AR gene expression by positive feedback loop (A, D, E) NCI-H292 cells were pretreated with the vehicle alone (Veh), GM6001 (GM; lM), TAPI-1 (TAPI; lM), PD98059 (PD; 10 lM) or anti-AR neutralizing antibody (aAR; 10 lgỈmL)1) for 20 (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 nM) for 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 ngỈmL) for h (D) or HAT (200 nM) for h (E) and the total RNA was extracted, and quantitative real-time RT ⁄ PCR (TaqManTM) 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 6394 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 suggested 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 sputum 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 Therefore, finding a mechanism by which HAT regulates AR-EGFR activation might elucidate the basic mechanisms of airway disease pathogenesis Further, this finding may also provide an additional benefit in terms of leading to the development of new therapeutic strategies to treat diseases marked by airway mucus hypersecretion The results of the present study showed that HAT activates EGFR through a pathway that includes FEBS Journal 272 (2005) 6387–6399 ª 2005 The Authors Journal compilation ª 2005 FEBS M Chokki et al PAR-2 mediating ERK-dependent AR gene expression and TACE-dependent AR protein release In the time course analysis of tyrosine phosphorylation of EGFR, AR-induced EGFR activation could be detected within of stimulation; however, HATinduced EGFR activation was observed h after stimulation 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 activation corresponded to that of HAT-induced AR release; further, the activation of EGFR and the induction of MUC5AC protein production were completely inhibited in the presence of anti-AR neutralizing antibody Considered along with our previous observation that among several EGFR ligands (EGF, HB-EGF, transforming growth factor-a, AR), only anti-AR neutralizing 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 HATinduced 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 agonists have not yet been reported in any cells It has been reported that the EGFR ligand family, which includes AR, is originally translated as precursors 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 TACEdependent release of AR has been shown to function HAT-induced AR release by PAR-2 and TACE 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 NCIH292 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 activation, was eliminated by blocking TACE using siRNA (Fig 8C) These observations indicate that the activation of ERK through PAR-2 results in the production 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-independent mechanism Although the cellular process of TACE activation has not been defined, the mechanisms 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 activation of EGFR occurred 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, lipopolysaccharide 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 MMPdependent 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 clarify the mechanisms that lead to HAT-induced AR release, including TACE activity regulation In conclusion, Fig depicts the mechanism of HAT-induced AR in NCI-H292 cells It schematically reflects the major findings of the present study, which are as follows: (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 FEBS Journal 272 (2005) 6387–6399 ª 2005 The Authors Journal compilation ª 2005 FEBS 6395 HAT-induced AR release by PAR-2 and TACE M Chokki et al (Minneapolis, MN, USA) The antiphospho-MEK (Ser217 ⁄ 221), antiphospho-ERK (Thr202 ⁄ Tyr204), anti-ERK, antiphospho-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 Fig 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-2mediated initial ERK activation causes the production of AR precursor, and then, biologically active AR is released by PAR-2independent mechanism involving TACE diseases through its effect on the PAR-2 and EGFR signalling pathways Experimental procedures Reagents and antibodies Recombinant HAT (60 mg)1 protein) was prepared as previously described [1,2,5] In brief, HAT was expressed in insect cells infected with a recombinant baculovirus carrying the HAT cDNA [2] Benzamidine affinity chromatography 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 SerLeu-Ile-Gly-Lys-Val-NH2 [6] was from Bachem AG (Bubendorf, 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, Calbiochem (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 6396 NCI-H292 cells were from the American Type Culture Collection (Rockville, MD, USA) and were cultured in RPMI 1640 medium supplemented with 10% (v ⁄ v) fetal bovide serum, 50 mL)1 penicillin and 50 lgỈmL)1 streptomycin (Gibco BRL, Grand Island, NY, USA) in a humidified incubator at 37 °C 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 conditions, quickly placed on ice, and washed twice in ice-cold NaCl ⁄ Pi The cells were then lysed in M-PERÒ Mammalian 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 at room temperature Each lysate was transferred to a separate centrifuge tube, and the lysates were centrifuged at °C for 10 at 15 000 g The cleared supernatants were collected separately, and a BioRad protein assay system (Bio-Rad, Hercules, CA, USA) was used to determine the protein content in each supernatant 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 at 99 °C, 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 proteins 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 h, washed three times for with TBST and treated with one of the following antibody preparations at °C overnight: antiphospho ERK antibody (diluted to : 2000 in 5% nonfat milk TBST), mouse anti-EGFR mAb (diluted to : 2500 in 5% nonfat milk TBST), antiphospho-EGFR (Tyr845) antibody, anti- FEBS Journal 272 (2005) 6387–6399 ª 2005 The Authors Journal compilation ª 2005 FEBS M Chokki et al HAT-induced AR release by PAR-2 and TACE phospho-EGFR (Tyr1068) antibody, anti-ERK antibody, antiphospho-MEK antibody, or antiphospho-p90RSK antibody (diluted to : 1000 in 5% BSA TBST) Following treatment with the antibody preparations, the blots were washed three times for in TBST, and incubated for h with peroxidase-conjugated secondary IgG (Amersham Biosciences; diluted to : 10000 in 5% nonfat milk TBST) The bands were detected using a chemiluminescence detection kit (ECL-plus; Amersham Biosciences) In some cases, blots were separated from the bound antibody using the RestoreTM Western Blot Stripping Buffer (Pierce) in accordance to the manufacturer’s instructions 20) was added to each well, incubated for 15 at room temperature, and after washing, incubated with the peroxidase substrate (TMB substrate; Kirkegaard & Perry Laboratories, Gaithersburg, MD, USA) for 30 at room temperature The reaction was terminated by the addition of molỈL)1 sulphuric acid, and a microplate reader (Thermomax; Molecular Devices, Sunnyvale, CA, USA) was used to determine the optical density at 450 nm The concentration of AR in each sample was determined by interpolation from the standard curve using softmax pro software (Molecular Devices) The limit of assay sensitivity is 4.096 pgỈmL)1 Real-time RT/PCR to measure AR mRNA Determination of the amount of MUC5AC protein A GeneAmp 5700 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) was used to conduct real-time PCR to measure the expression of AR mRNA by a previously described method [16] In brief, an RNeasy Mini Kit (Qiagen, Hilden, Germany), which included a DNase I digestion step (RNase-free DNase set; Qiagen), was used to extract total cellular RNA Approximately 0.5 lg of each total RNA preparation was reverse transcribed with Omniscript RT Kit (Qiagen); random hexamers were used to prime the reactions PCR was performed using a TaqManTM Universal PCR Master Mix Kit (Applied Biosystems) with specific primers and probes for AR and b-actin The method reported by Takeyama et al [17], with some modifications, was used to determine the amount of MUC5AC protein in the cellular lysates of NCI-H292 cells In brief, the cell lysates were prepared as described previously Each lysate was diluted with bicarbonate-carbonate buffer, applied to a separate well of a 96-well plate (NUNC Maxisorp) and incubated at 40 °C until it was dry The plates were washed three times with NaCl ⁄ Pi and blocked with NaCl ⁄ Pi containing 1% (w ⁄ v) BSA for h at room temperature The plates were again washed for five times with PBST and mouse anti-MUC5AC mAb [0.5 lgỈmL)1 in NaCl ⁄ Pi containing 1% BSA and 0.05% (v ⁄ v) Tween 20] was added to the wells The plates were incubated for h at room temperature The plates were then washed again, and biotinylated antimouse antibody (Dako, Glostrup, Denmark; diluted to : 10000 in NaCl ⁄ Pi containing 1% BSA and 0.05% Tween 20) was added to each well The plates were then incubated for h at room temperature After washing, streptavidin-conjugated HRP was added to each well and the plates were incubated for 15 at room temperature After washing, addition of peroxidase substrate, termination of reaction and the measuring the optical density were done as described previously Data are expressed as ratio of the observed value to the mean value of the control group Determination of the amount of AR protein The amount of AR in the culture supernatant of NCIH292 cells and in the cellular lysates of NCI-H292 cells treated with the vehicle HAT or PAR-2 AP was measured as follows Anti-AR mAb (clone 12111.333, Genzyme) was used as a capture antibody, and a biotinylated antihuman AR polyclonal antibody (Genzyme) was used as a detection antibody for ELISA Sulpho-NHS-LC-Biotin (Pierce) was used to biotinylate the antibody Cellular lysates of treated NCI-H292 cells were prepared as described previously The capture antibody (2 lgỈmL)1 in NaCl ⁄ Pi) was used to coat the bottom of the wells of 96-well plates (NUNC Maxisorp; Fisher Scientific, Santa Clara, CA, USA) by incubating the plates containing the capture antibody overnight at °C The wells were washed with PBST [NaCl ⁄ Pi, 0.05% (v ⁄ v) Tween 20], blocked by the addition of NaCl ⁄ Pi containing 1% (w ⁄ v) BSA for h at 37 °C and washed again The culture medium, cellular lysates or standards (human recombinant AR in NaCl ⁄ Pi containing 1% BSA) were then added to the wells The plates were incubated for h at room temperature, washed and incubated with biotinylated detection antibody (150 ngỈmL)1 in NaCl ⁄ Pi containing 1% BSA and 0.05% Tween 20) at room temperature for h After washing, peroxidase-labelled streptavidin (diluted : 6000 in NaCl ⁄ Pi containing 1% BSA and 0.05% Tween Transfection of siRNA Predesigned human TACE siRNA (siRNA ID no 113003) was purchased from Ambion (Austin, TX, USA) The sequences of the 21-nucleotide siRNAs were sense: GCAG CAUUCGGUAAGAAAAtt and antisense: UUUUCU UACCGAAUGCUGCtg Silencer Negative Control no siRNA (Ambion) was used as a control siRNA NCI-H292 cells in 6-well plates were transfected for 72 h prior to the HAT treatment with 100 pmol TACE or control siRNA by using the Lipofectamine 2000 Reagent (Invitrogen) according to the manufacturer’s instructions Specific silencing of TACE was confirmed by flow cytometry as described below FEBS Journal 272 (2005) 6387–6399 ª 2005 The Authors Journal compilation ª 2005 FEBS 6397 HAT-induced AR release by PAR-2 and TACE M Chokki et al Flow cytometry To detect cell-surface TACE, flow cytometry was performed NCI-H292 cells were nonenzymatically collected by incubation in NaCl ⁄ Pi containing mm EDTA for at 37 °C After washing in NaCl ⁄ Pi containing 1% FBS, the cells were stained with anti-TACE mAb or control IgG at °C for 30 After washing with NaCl ⁄ Pi containing 1% FBS, cells were incubated with fluorescein isothiocyanate-conjugated goat antimouse IgG (DAKO) for 30 at °C and washed again with NaCl ⁄ Pi containing 1% FBS The cells were analysed on a FACS Calibur cytometer (BD Bioscience, Mountain View, CA, USA) Statistical analysis Data are presented as the mean ± SD of at least three separate experiments For statistical analysis, Dunnett’s two-tailed test was used to test the statistically significant differences The commercial statistical software Super anova ver 1.1 (Abacus Concepts Inc., Berkeley, CA, USA) was used to perform the tests Tests that returned P-values < 0.05 were considered to represent significant differences 10 Acknowledgements The authors would like to thank Dr S Yasuoka (Department of Nutrition, University of Tokushima School of Medicine) for his valuable advice and support 11 12 References Yasuoka S, Onishi T, Kawano S, Tsuchihashi S, Ogawara M, Masuda K, Yamaoka K, Takahashi M & Sano T (1997) Purification, 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HAT-induced initial ERK activation (5 after stimulation with HAT) was inhibited by a. .. epithelium increases in bronchial asthmatic patients [10] These observations suggest that HAT might mediate airway in? ??ammation by PAR-2 activation In addition to airway in? ??ammation, hypersecretion of airway