Chest Email Content Delivery
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Guest Access | Sign In via User Name/Password
doi:10.1378/chest.06-2031
(Chest. 2007; 131:1149-1156)
© 2007 American College of Chest Physicians
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Article Archive
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via ISI Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hegab, A. E.
Right arrow Articles by Sekizawa, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hegab, A. E.
Right arrow Articles by Sekizawa, K.

Niflumic Acid and AG-1478 Reduce Cigarette Smoke-Induced Mucin Synthesis*

The Role of hCLCA1

Ahmed E. Hegab, PhD; Tohru Sakamoto, MD; Akihiro Nomura, MD; Yukio Ishii, MD; Yuko Morishima, MD; Takashi Iizuka, MD; Takumi Kiwamoto, MD; Yosuke Matsuno, MD; Shinsuke Homma, MD and Kiyohisa Sekizawa, MD

* From the Department of Pulmonary Medicine, Institute of Clinical Medicine, University of Tsukuba, Ibaraki, Japan.

Correspondence to: Tohru Sakamoto, MD, Department of Pulmonary Medicine, Institute of Clinical Medicine, University of Tsukuba, 1–1-1 Tennoudai, Tsukuba, Ibaraki 305-8575, Japan; e-mail: t-saka{at}md.tsukuba.ac.jp

Abstract

Background: Cigarette smoke induces bronchial mucus secretion. However, the mechanism of this induction is still unidentified. In this study, we investigated the role of the putative calcium-activated chloride channel 1 (CLCA1) and its blocker, niflumic acid, in cigarette smoke-induced mucin synthesis both in vivo and in vitro.

Methods and results: Sprague-Dawley rats were exposed to cigarette smoke for 4 weeks. The CLCA1, epidermal growth factor receptor (EGFR), and MUC5AC expressions were increased in the trachea and lung tissues. Goblet-cell hyperplasia with marked mucin staining was detected in the tracheal and bronchial epithelium. In the human bronchial epithelial cell line NCI-H292, cigarette smoke solution also induced mucin production as well as the RNA and protein expressions of CLCA1, EGFR, and MUC5AC. Both in vivo and in vitro, the induction of MUC5AC and mucin synthesis were inhibited by niflumic acid, and/or a selective EGFR tyrosine kinase inhibitor, AG-1478. Niflumic acid also blocked the epidermal growth factor-induced MUC5AC and mucin staining in the NCI-H292 cell line.

Conclusion: Both EGFR and niflumic acid-sensitive chloride channels (probably CLCA1) are dependently affecting the mucin production as a part of a single complex signaling pathway. CLCA1 may be a key signaling member that can be targeted with pharmacologic interventions to treat mucus hypersecretion.

Key Words: calcium-activated chloride channel • COPD • epidermal growth factor receptor • MUC5AC

Chronic bronchitis is a clinical syndrome characterized by chronic sputum production and is a major phenotype of COPD. Cigarette smoking is the main risk factor for COPD. Although several studies1 have shown that cigarette smoke can induce mucus production, the mechanism of this induction remains unexplained. MUC5AC is the major respiratory mucin in goblet-cell secretion.2 Mucin synthesis in response to various stimuli is regulated by the epidermal growth factor receptor (EGFR) system.3 Cigarette smoke causes activation of EGFR via phosphorylation of tyrosine residues that leads to extracellular signal-regulated kinase activation that is required for activator protein 1-containing response element to bind JunD and Fra-2 causing upregulation of MUC5AC at both messenger RNA (mRNA) and protein levels.4

Human calcium-activated chloride channel 1 (hCLCA1) has been shown to be critical for airway mucus production. The expression of hCLCA1 is strongly induced in the airway goblet cells in human asthmatics56 and COPD patients.7 In addition, transfection of the hCLCA1 gene into the human mucoepidermoid cell line NCI-H292 induces mucin production.8 Its murine counter part, murine calcium-activated chloride channel 3 (mCLCA3), was the most highly induced gene at all time points in the asbestos-induced mucus production,9 and was markedly induced with MUC5AC in respiratory syncytial virus-infected, allergically sensitized mice.10 Furthermore, some polymorphisms and haplotypes within the hCLCA1 gene have been shown to be associated with bronchial asthma11 and COPD.12 Taken together, these data indicate that calcium-activated chloride channel 1 (CLCA1) is involved in several pulmonary diseases that are characterized by increased mucus production.

Materials and Methods

In Vivo Studies
Animals and Exposure to Cigarette Smoke:
Male Sprague-Dawley rats weighing 200 to 250 g were randomly assigned to the nonsmoking group (n = 4), the smoke-exposed group (n = 20; 4 animals killed at 2, 7, 14, 21, and 28 days), or the smoke-exposed treatment group (n = 12; 4 animals for each treatment protocol). Rats were exposed to the whole smoke from six nonfiltered cigarettes per day, 5 d/wk, for 2 to 28 days by the whole-body exposure method at the rate of one puff per minute. After the exposure of one cigarette smoke, animals were allowed to breathe fresh air for 20 min before the next cigarette smoke was administered.

Treatment With Niflumic Acid and/or AG-1478:
Niflumic acid (Sigma-Aldrich; St. Louis, MO) is a blocker of calcium-activated chloride channels (CLCAs). AG-1478 (LC Laboratories; Woburn, MA) is a selective EGFR tyrosine kinase inhibitor. To examine the effect of inhibiting CLCA1 and/or EGFR on the cigarette smoke-induced mucus production, the animals in the smoke-exposed treatment group were injected intraperitoneally 30 min/d before the smoke exposure with either vehicle, niflumic acid (3 mg/kg), AG-1478 (3 mg/kg), or both niflumic acid and AG-1478.

RNA Isolation and Quantification of Gene Expressions:
Two hours after the last smoke exposure, the animals were killed; RNA was then isolated from the trachea and lungs. RNA expression measurement was done using the real-time TaqMan-polymerase chain reaction technology (ABI Prism 7000 Sequence Detection System; Applied Biosystems; Foster City, CA); the quantification of CLCA1 and EGFR was done using the Assays-on-Demand gene expression assay primer/probe. The quantification of MUC5AC was done using the following sequences: probe 5'-[FAM]-CTGTCCATTCACGTGCC-[MGB]-3', sense primer 5'-TGGAGAAGCCATACCAACAACA-3' and antisense primer 5'-CAAGGCTGGTATACTTGGTTTTCA-3'. The TaqMan 18s recombinant RNA primer/probe mixture (Applied Biosystems) was used as a housekeeping gene to normalize for differences in the amounts of mRNAs. Samples were run simultaneously in triplicate and repeated three times.

Western Blotting:
Protein was separated from lung tissues, and its concentration was measured by standard techniques. The samples were separated on polyacrylamide gel, and then electrotransferred onto Immun-Blot polyvinylidene fluoride membrane (Bio-Rad; Hercules, CA). Membranes were blocked in skimmed milk and then probed with the following primary polyclonal antibodies (dilute 1:200 to approximately 1,000): a rabbit anti-Gob-5 (kindly provided by Takeda Pharmaceutical; Tsukuba, Japan), a rabbit anti-EGFR, a goat anti-phosphospecific (Tyr 1173) EGFR (pEGFR), and a goat anti-glyceraldehyde-3-phosphate-dehydrogenase antibodies (Santa Cruze Biotechnology; Santa Cruz, CA). The membranes were subsequently incubated in horseradish peroxidase-coupled secondary antibodies. Band formation was visualized by simple staining with the DAB substrate. The bands were quantified by densitometric scanning using ImageJ (available at: http://rsb.info.nih.gov/ij/index.html). Data are expressed as percentage of control, and the expression of the different proteins were normalized to glyceraldehyde 3-phosphate dehydrogenase readings.

Morphometric Analysis:
Paraffin-embedded lungs with small portions of the tracheas were sectioned transversely and stained with hematoxylin-eosin or with periodic acid-Schiff (PAS) to evaluate the total epithelial area and the area stained for intracellular mucin, respectively. These areas were measured using a digital camera (FX380; Olympus; Tokyo, Japan). Eight different images, four from the trachea and four from major bronchi, were analyzed for each rat. Data are expressed as the percentage of PAS-stained area to the total epithelial area.

In Vitro Studies
Smoke Solution Preparation:
Smoke solution was prepared as previously described13 with some modifications. In brief, one cigarette and a 50-mL syringe were attached to a three-way stopcock. Then 1, 4, or 10 puffs of smoke were repeatedly withdrawn to the syringe and bubbled into 20 mL of serum-free RPMI 1640 medium containing 25 mmol/L hydroxylethyl piperazine-ethanesulfonic acid buffer. The smoke solution was filtered and used immediately.

Cell Culture and Exposure to Smoke Solution:
The human bronchial mucoepidermoid carcinoma cell line NCI-H292 (American Type Culture Collection; Rockville, MD) were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 µg/mL) at 37°C in a humidified atmosphere of 5% CO2. The cells were cultured in 100-mm culture dishes for RNA studies and in Lab-Tek eight-chamber slides (Nalge Nunc International; Naperville, IL) for PAS staining studies. At confluence, the medium was changed into serum-free RPMI 1640 and the cells were cultured for 24 h more. Next, the cells were incubated in the smoke solution for 1 h. The cells were then washed and cultured with serum-free medium for 1, 18, 24, or 36 h. In treatment studies, the culture medium was supplemented with niflumic acid (300 µmol/L), AG-1478 (10 µmol/L), or both from 1 h before changing the medium into the smoke solution to the end of the culture. As controls, cells were incubated with serum-free medium alone or supplemented with the same drugs for the same duration. Since dimethyl sulfoxide (DMSO) was used as a solvent for niflumic acid and AG-1478, we also exposed the cells to the same concentration of DMSO alone to examine its effect on the gene expressions. All samples were managed simultaneously in triplicate, and the experiments were repeated at least three times.

RNA Isolation and Quantification of Gene Expressions:
The quantification of hCLCA1 and EGFR was done using the Assays-on-Demand gene expression assay (Applied Biosystems). The quantification of MUC5AC was done using the following sequences: probe 5'-[FAM]-CTCGCTGGCCATTGCTATTATGCCC-[MGB]-3', sense primer 5'-TCCACCATATACCGCCACAGA-3' and antisense primer 5'-TGGACCGACAGTCAC TGTCAAC-3'.

Western Blotting:
The cells were harvested and resuspended in 1 mL of water, sonicated for 30 s on ice, and then cell debris was removed by centrifugation. The rest of the procedures were the same as mentioned in the in vivo studies except that the antibody used for detection of human CLCA1 was polyclonal rabbit anti-CLCA1 antibody (kindly provided by Takeda).

Determination of Mucous Glycoconjugate Production in NCI-H292 Cells
Confluent cells cultured in eight-chamber slides were serum starved for 24 h and then exposed to smoke solution with and without the treatments with the same concentrations and durations as those used for culture dishes mentioned above. The cells were then fixed with formalin, and mucous glycoconjugates were visualized by PAS staining.

Is hCLCA1 Part of the EGFR-MUC5AC Pathway?
Several ligands have been identified to bind to EGFR causing its activation. Binding of epidermal growth factor (EGF) to EGFR stimulates MUC5AC expression and mucin staining in NCI-H292 cells.14 To determine whether this pathway is dependant on the hCLCA1, serum-starved confluent NCI-H292 cells were incubated with 25 ng/mL EGF (Sigma) alone or combined with 300 µmol/L niflumic acid or 10 µmol/L AG-1478 for 18 h. Then the hCLCA1, EGFR, and MUC5AC mRNA expressions were quantified as described above. Cells grown in eight-chamber slides were treated similarly and stained by PAS stain. Furthermore, CLCA1, EGFR, and pEGFR protein expression was examined by Western blot.

Statistical Analysis
Data are presented as mean ± SD of at least three separate experiments. One-way analysis of variance was used to determine statistically significant differences between groups; p < 0.05 was considered to be statistically significant.

Results

In Vivo Studies
Cigarette Smoke Up-regulates CLCA1 and EGFR mRNA and Protein Expressions and MUC5AC mRNA Expression in Rats:
In the nonsmoking group, CLCA1, EGFR, and MUC5AC mRNA expressions were constitutively expressed in rat lung and trachea. The smoke-exposed group showed significant upregulation of all the three mRNA expressions from day 21 onwards (p < 0.001) [Fig 1 ]. This effect was stronger in the trachea than in the lung. Similarly, the protein expressions of CLCA1 and both resting EGFR and activated pEGFR were significantly increased on day 28 (p < 0.001) [Fig 2 ].


Figure 1
View larger version (12K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 1. Effect of cigarette smoke inhalation on the mRNA expressions of CLCA1, EGFR, and MUC5AC in rats. Cigarette smoke caused significant upregulation of CLCA1 (circles), EGFR (squares), and MUC5AC (triangles) genes starting from 21 days onwards. *p < 0.001 compared to the control.

 

Figure 2
View larger version (16K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 2. Effect of cigarette smoke inhalation with or without a blocker of CLCAs—niflumic acid (NFA), tyrosine kinase inhibitor (AG-1478), or both—on CLCA1, EGFR, and pEGFR protein expressions at 28 days in rats. Cigarette smoke caused significant upregulation of CLCA1 (triangles), EGFR (squares), and pEGFR (diamonds) genes. This upregulation was not affected by niflumic acid, while AG-1478 partially inhibited and abolished the EGFR and pEGFR upregulations, respectively. #p < 0.001 compared to the control, *p < 0.001 compared to the smoke only, and ¥ p < 0.05 compared to the smoke only.

 
Niflumic Acid, AG-1478, or Both Inhibit the Cigarette Smoke-Induced MUC5AC Gene Expression:
Niflumic acid is a blocker of the CLCAs, and AG-1478 is an inhibitor of EGFR tyrosine kinase. In the smoke-exposed treatment group, niflumic acid, AG-1478, or both significantly inhibited the upreglation of MUC5AC mRNA expression (p < 0.001) without affecting the upregulation of CLCA1 and EGFR mRNAs. Combining both drugs did not show a significant synergistic effect on MUC5AC mRNA expression (Fig 3 ). On the protein level, neither niflumic acid, AG-1478, nor both could inhibit the upregulation of CLCA1 protein by cigarette smoke. However, EGFR upregulation was partially inhibited by AG-1478, while pEGFR expression was abolished by it (p < 0.001). Niflumic acid had no effect on the protein expressions of EGFR and pEGFR (Fig 2).


Figure 3
View larger version (16K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 3. Effect of a blocker of CACLs—niflumic acid, tyrosine kinase inhibitor (AG-1478), or both—on cigarette smoke-induced gene expressions at 28 days in rats. Both drugs inhibited the cigarette smoke-induced MUC5AC (triangles) gene expression without affecting the CLCA1 (circles) and EGFR (squares). Combining the drugs is not synergistic. #p < 0.001 compared to the control; *p < 0.001 compared to the smoke only. See Figure 2 footnote for expansion of abbreviation.

 
Cigarette Smoke Increases Mucin Staining and Number of Goblet Cells in Rats, an Effect That Is Inhibited by Treatment With Niflumic Acid, AG-1478, or Both:
The smoke-exposed group showed marked increase in the intensity of PAS staining as well as in the percentage area of goblet cells in the tracheal epithelium from day 21. The area of the mucin glycoconjugate in the epithelium was 1.7 ± 1.3% in the nonsmoking group while it was 24.0 ± 6.9% in the smoke-exposed group on the 28th day (p < 0.001) The areas were 2.6 ± 1.5%, 5.1 ± 2.9%, and 4.0 ± 1.1% in the smoke-exposed niflumic treated, AG-1478 treated, and both treated groups, respectively (p < 0.001) [Fig 4 ]. In a post hoc analysis of animal sample size and power calculation, as the change in the primary outcome variable (mucin production) was > 85% and the within-sample variation was < 10%, the number of animals used gives a 95% power to detect a significance level of 0.05.


Figure 4
View larger version (80K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 4. Effect of cigarette smoke on PAS staining of rat airway epithelium. Left, A: Control rat showed minimal staining. Center, B: Cigarette smoke exposure significantly increased the mucin staining. Right, C: Cigarette smoke-induced increase in mucin staining was significantly inhibited by treatment with a blocker of CLCAs: niflumic acid, tyrosine kinase inhibitor (AG-1478), or both. Representative slides of several independent experiments (original x 40; bar = 100 µm).

 
In Vitro Studies
Smoke Solution Up-regulates CLCA1, EGFR mRNA and Protein Expressions, and MUC5AC mRNA Expressions in NCI-H292 Cells:
In the control condition, EGFR and MUC5AC mRNAs were constitutively expressed in the NCI-H292 cells. By contrast, hCLCA1 mRNA expression was undetectable. However, when the cells were exposed to the smoke solution, all the three genes were significantly up-regulated (p < 0.001) [Fig 5 ]. This up-regulation was dose and time dependent (data not shown). As for the protein expressions, CLCA1 and pEGFR protein expressions were significantly elevated by smoke solution (p < 0.001), while no significant elevation in the EGFR protein expression was detected (Fig 6 ).


Figure 5
View larger version (15K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 5. Effect of a blocker of CLCAs—niflumic acid, tyrosine kinase inhibitor (AG-1478), or both—on mRNA expressions of hCLCA1, EGFR, and MUC5AC in smoke-exposed NCI-H292 cells. Smoke solution (10 puffs for 18 h) caused significant increase in the expression of hCLCA1 (circles), EGFR (squares), and MUC5AC (triangles) genes. NFA, AG-1478, or both significantly inhibited smoke-induced MUC5AC expression without affecting the induction of hCLCA1 and EGFR. DMSO-only partly inhibited the smoke-induced MUC5AC expression. *p < 0.001 and ¥ p < 0.05 compared to the control; # p < 0.001 compared to the smoke only. See Figure 2 footnote for expansion of abbreviation.

 

Figure 6
View larger version (13K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 6. Effect of smoke solution with or without a blocker of CLCAs—niflumic acid, tyrosine kinase inhibitor (AG-1478), or both—on CLCA1, EGFR, and pEGFR protein expressions in NCI-H292 cells. Smoke solution (10 puffs for 18 h) caused significant upregulation of hCLCA1 (triangles) and pEGFR (diamonds) genes with no significant effect on EGFR (squares). This upregulation was not affected by niflumic acid, while AG-1478 abolished the pEGFR upregulation. #p < 0.001 compared to the control; *p < 0.001 compared to the smoke only. See Figure 2 footnote for expansion of abbreviation.

 
Niflumic Acid, AG-1478, or Both Inhibited the Baseline and Smoke-Induced MUC5AC Gene Expression in NCI-H292 Cells:
Treating the cells exposed to smoke solution with niflumic acid, AG-1478, or both significantly inhibited the upreglation of the MUC5AC gene expression to the control level (p < 0.001) without affecting the upregulation of hCLCA1 and EGFR mRNAs. Combining both drugs did not show a significant synergistic effect (Fig 5). Pretreating the smoke-exposed cells with DMSO also could inhibit, although partially, the MUC5AC mRNA expression without affecting the upregulation of hCLCA1 and EGFR mRNAs (Fig 5). On the protein levels, neither niflumic acid, AG-1478, nor both could inhibit the upregulation of CLCA1 by cigarette smoke. However, pEGFR upregulation was abolished by AG-1478 (p < 0.001). Niflumic acid had no effect on EGFR or pEGFR (Fig 6).

Interestingly, treating naive cells (unexposed to smoke solution) with niflumic acid, AG-1478, or both significantly down regulated MUC5AC mRNA expression below its baseline (p < 0.001) without affecting the CLCA1 and EGFR expressions (Fig 7 ). However, treating the native cells with DMSO did not significantly affect the baseline expression of MUC5AC mRNA, hCLCA1, or EGFR gene expressions (Fig 7).


Figure 7
View larger version (13K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 7. Effect of niflumic acid, AG-1478, or both on mRNA expressions of hCLCA1, EGFR, and MUC5AC in naive NCI-H292 cells. NFA, AG-1478, or both significantly decreased MUC5AC expression level several folds below the baseline gene expression of naïve cells, while DMSO-only had no effect. The expressions of CLCA1 and EGFR were not affected by any of them. See Figure 2 footnote for expansion of abbreviation.

 
Smoke Solution Increases Mucin Staining in NCI-H292 Cells, an Effect That Was Inhibited by Niflumic Acid, AG-1478, or Both:
In the control condition, few cells were positive by PAS staining. However, the number of PAS-positive cells and the intensity of PAS staining in each cell significantly increased after the treatment with the smoke solution (Fig 8 , left, A, and center, B). Treatment of the cells exposed to the smoke solution with niflumic acid, AG-1478, or both significantly inhibited the increase of the PAS-stained area (Fig 8, right, C).


Figure 8
View larger version (94K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 8. Effect of smoke solution without and with a blocker of CLCAs—niflumic acid, tyrosine kinase inhibitor AG-1478, or both—on PAS staining in NCI-H292 cells. Left, A: Control cells showed minimal staining. Center, B: Cells treated with smoke solution showed significant increase in the number of PAS-positive cells and the intensity of PAS staining in each cell. p < 0.001 compared to the control. Right, C: Pretreating the smoke-exposed cells with niflumic acid, AG-1478, or both significantly inhibited the increase in PAS-stained area. p < 0.001 compared to the smoke only (bar = 100 µm). The figure shows representative results from three different experiments.

 
CLCAs Are Part of the EGFR-MUC5AC Pathway:
EGF caused significant upregulation of MUC5AC expression (p < 0.001) without affecting CLCA1 and EGFR expression (Fig 9 ). Pretreatment of these cells with AG-1478 led to complete inhibition of the EGF-induced MUC5AC expression, confirming that the EGF-mediated upregulation of MUC5AC in NCI-H292 cells goes through the activation of EGFR tyrosine kinase. Pretreatment of the cells stimulated by EGF with niflumic acid also led to complete inhibition of the EGF-induced MUC5AC expression. These results suggest that the CLCAs are part of the EGFR-MUC5AC pathway, probably downstream to EGFR, and that sequential activation of EGFR and the chloride channels are essential for the MUC5AC gene upregulation (Fig 9). This was further confirmed by the finding that EGF caused significant increase in PAS staining, which was inhibited by the pretreatment with niflumic acid or AG-1478 (data not shown). On the protein level, treatments by EGF alone or by EGF combined with niflumic acid or AG-1478 had no effect on CLCA1 expression, while EGF significantly upregulated the pEGFR expression, an effect that was partially inhibited by niflumic acid but markedly abolished by AG-1478 (data not shown).


Figure 9
View larger version (11K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 9. Effect of EGF without and with a blocker of CLCAs—niflumic acid or tyrosine kinase inhibitor (AG-1478)—on the expressions of hCLCA1, EGFR, and MUC5AC in NCI-H292 cells. EGF caused significant increase in the expression of only MUC5AC (triangles) with no effect on the hCLCA1 (circles) or EGFR (squares). The EGF-induced MUC5AC expression was significantly inhibited by niflumic acid or AG-1478. *p < 0.001 compared to the control; #p < 0.001 compared to the EGF only. See Figure 2 footnote for expansion of abbreviation.

 
Discussion

This study demonstrated that cigarette smoke exposure increases MUC5AC mRNA and mucin synthesis via a signaling pathway that involves upregulation of EGFR and CLCA1 both in vivo and in vitro. It has been shown that cigarette smoke upregulates EGFR expression leading to mucin production in the airway epithelium.13 This is the first study to show a critical role for CLCA1 in cigarette smoke-induced mucin production, which is one of the major features of COPD.

In this study, we showed that either a blocker of CLCAs, niflumic acid, or a selective EGFR tyrosine kinase inhibitor, AG-1478, are completely able to inhibit cigarette smoke-induced mucin synthesis as well as the upregulation of MUC5AC mRNA expression, and that the effect of combining both drugs is not synergistic. This indicates that both CLCA1 and EGFR are dependently affecting mucin production as a part of a single complex signaling pathway, but it does not exclude the participation of CLCAs other than CLCA1. It seems that CLCA1 is involved in the pathologic induction of mucin, while a different CLCAs are related to the baseline physiologic mucin secretion. Although niflumic acid is a known blocker of the CLCAs and has been shown to block the function of hCLCA1,1516 it is nonspecific to CLCA1. Therefore the contribution of its blocking of any other CLCAs cannot be accurately evaluated. In fact, despite the increasing number of discovered CLCAs both in animals and human and their seemingly important functions, understanding these channels has been limited by the absence of specific blockers and the fact that their molecular identities remain in question.17

Our results indicate that CLCAs are part of the EGFR-MUC5AC pathway because niflumic acid can inhibit the EGF-induced MUC5AC expression and mucin staining in NCI-H292 cells, which was the same level of inhibition caused by AG-1478. Several previous reports1819 have shown that activation of several EGFR-related tyrosine kinases open different types of outwardly rectifying chloride channels, which might mean that CLCA1 is probably downstream to EGFR. Furthermore, Leverkoehne and Gruber20 demonstrated that the mCLCA3 (mouse counterpart of hCLCA1) protein is exclusively associated with mucin granule membranes inside the goblet cells. They suggested that mCLCA3 is involved in the synthesis, condensation, or secretion of mucins. Collectively, we suggest that cigarette smoke up-regulates the EGFR and CLCA1 mRNA and protein expressions in goblet cells and it also leads to activation of the EGFR, which then activates the CLCA1 in the mucin granule membranes causing upregulation of the MUC5AC mRNA and induction of mucin synthesis.

Several reports have pointed to a link between CLCA1621 or EGFR22 and the T-helper type 2 (Th2) cytokines: interleukin (IL)-4, IL-9, and IL-13. However, Th2 cytokines were also shown to play a role in smoke-induced COPD.23242526 Taken together, these findings support our results that suggest that the mechanism of cigarette smoke-induced mucin synthesis involves the EGFR and CLCA1 stimulation induced by these Th2 cytokines.

Tumor necrosis factor (TNF)-{alpha} is another mediator that has been shown to induce hCLCA1 leading to upregulation of MUC5AC mRNA and mucus.27 Since TNF-{alpha} increases EGFR expression in airway epithelium,22 it is possible that the induction of hCLCA1 and mucin production by TNF-{alpha} in the airway is mediated via the EGFR signaling pathway. Cigarette smoke can induce TNF-{alpha} both in vivo and in vitro.28 Therefore, we speculated that the upregulation of the EGFR and hCLCA1 genes in the present study may be partly mediated by TNF-{alpha}.

We demonstrated in this study that DMSO partially inhibited the smoke-induced MUC5AC gene expression in NCI-H292 cells. DMSO is a known potent antioxidant agent. Cigarette smoke contains free radicals and other oxidants in abundance. It has been shown that oxidative stress causes mucin synthesis via ligand-independent EGFR activation.29 Taken together, it is suggested that at least some part of the mucin upregulation induced by cigarette smoke is mediated through the oxidants.

Although our results were largely similar between the study of the human cell line and that of the Sprague-Dawley rats, they cannot automatically correspond to humans without further confirmation. Interspecies or even interstrain differences in the response to cigarette smoke are well identified. We have exposed the C57BL/6 and Balb/c mice to cigarette smoke for up to 8 weeks without detecting any effect on the mCLCA3, EGFR, and MUC5AC gene expressions (A.E. Hegab, PhD; unpublished data; March 2005), while these changes were significantly detected in rats within 3 weeks. The difference is even more manifest when it comes to human development of chronic bronchitis, which requires many years of smoking and affects only a subpopulation of smokers.

In conclusion, we are showing for the first time that a blocker of the CLCAs probably acting through inhibition of hCLCA1 prevents cigarette smoke-induced MUC5AC synthesis in vivo and in vitro. More studies are needed to further understand the mechanism of actions and interactions of hCLCA1 that might reveal a key signaling pathway that can be targeted with pharmacologic interventions to treat mucus hypersecretion.

Footnotes

Abbreviations: CLCA = calcium-activated chloride channel; CLCA1 = calcium-activated chloride channel 1; DMSO = dimethyl sulfoxide; EGF = epidermal growth factor; EGFR = epidermal growth factor receptor; hCLCA1 = human calcium-activated chloride channel 1; IL = interleukin; mCLCA3 = murine calcium-activated chloride channel 3; mRNA = messenger RNA; PAS = periodic acid-Schiff; pEGFR = anti-phosphospecific epidermal growth factor receptor; Th2 = T-helper type 2; TNF = tumor necrosis factor

The authors have no conflicts of interest to disclose.

Received for publication September 16, 2006. Accepted for publication November 20, 2006.

References

  1. O’Donnell, RA, Richter, A, Ward, J, et al (2004) Expression of ErbB receptors and mucins in the airways of long term current smokers. Thorax 59,1032-1040[Abstract/Free Full Text]
  2. Hovenberg, HW, Davies, JR, Carlstedt, I Different mucins are produced by the surface epithelium and the submucosa in human trachea: identification of MUC5AC as a major mucin from the goblet cells. Biochem J 1996;318,319-324[ISI][Medline]
  3. Burgel, PR, Nadel, JA Roles of epidermal growth factor receptor activation in epithelial cell repair and mucin production in airway epithelium. Thorax 2004;59,992-996[Abstract/Free Full Text]
  4. Gensch, E, Gallup, M, Sucher, A, et al Tobacco smoke control of mucin production in lung cells requires oxygen radicals AP-1 and JNK. J Biol Chem 2004;279,39085-39093[Abstract/Free Full Text]
  5. Hoshino, M, Morita, S, Iwashita, H, et al Increased expression of the human Ca2+-activated Cl- channel 1 (CaCC1) gene in the asthmatic airway. Am J Respir Crit Care Med 2002;165,1132-1136[Abstract/Free Full Text]
  6. Toda, M, Tulic, MK, Levitt, RC, et al A calcium-activated chloride channel (HCLCA1) is strongly related to IL-9 expression and mucus production in bronchial epithelium of patients with asthma. J Allergy Clin Immunol 2002;109,246-250[CrossRef][ISI][Medline]
  7. Hauber, HP, Bergeron, C, Tsicopoulos, A, et al Increased expression of the calcium-activated chloride channel hCLCA1 in airways of patients with obstructive chronic bronchitis. Can Respir J 2005;12,143-146[Medline]
  8. Nakanishi, A, Morita, S, Iwashita, H, et al Role of gob-5 in mucus overproduction and airway hyperresponsiveness in asthma. Proc Natl Acad Sci U S A 2001;98,5175-5180[Abstract/Free Full Text]
  9. Sabo-Attwood, T, Ramos-Nino, M, Bond, J, et al Gene expression profiles reveal increased mClca3 (Gob5) expression and mucin production in a murine model of asbestos-induced fibrogenesis. Am J Pathol 2005;167,1243-1256[Abstract/Free Full Text]
  10. Hashimoto, K, Graham, BS, Ho, SB, et al Respiratory syncytial virus in allergic lung inflammation increases Muc5ac and gob-5. Am J Respir Crit Care Med 2004;170,306-312[Abstract/Free Full Text]
  11. Kamada, F, Suzuki, Y, Shao, C, et al Association of the hCLCA1 gene with childhood and adult asthma. Genes Immun 2004;5,540-547[CrossRef][ISI][Medline]
  12. Hegab, AE, Sakamoto, T, Uchida, Y, et al CLCA1 gene polymorphisms in chronic obstructive pulmonary disease. J Med Genet 2004;41,e27[Free Full Text]
  13. Takeyama, K, Jung, B, Shim, JJ, et al Activation of epidermal growth factor receptors is responsible for mucin synthesis induced by cigarette smoke. Am J Physiol Lung Cell Mol Physiol 2001;280,L165-L172[Abstract/Free Full Text]
  14. Perrais, M, Pigny, P, Copin, MC, et al Induction of MUC2 and MUC5AC mucins by factors of the epidermal growth factor (EGF) family is mediated by EGF receptor/Ras/Raf/extracellular signal-regulated kinase cascade and Sp1. J Biol Chem 2002;277,32258-63227[Abstract/Free Full Text]
  15. Gruber, AD, Elble, RC, Ji, HL, et al Genomic cloning, molecular characterization, and functional analysis of human CLCA1, the first human member of the family of Ca2+-activated Cl- channel proteins, Genomics 1998;54,200-214[CrossRef][ISI][Medline]
  16. Zhou, Y, Shapiro, M, Dong, Q, et al A calcium-activated chloride channel blocker inhibits goblet cell metaplasia and mucus overproduction. Novartis Found Symp 2002;248,150-165[ISI][Medline]
  17. Hartzell, C, Putzier, I, Arreola, J Calcium-activated chloride channels. Annu Rev Physiol 2005;67,719-758[CrossRef][ISI][Medline]
  18. Lepple-Wienhues, A, Szabo, I, Wieland, U, et al Tyrosine kinases open lymphocyte chloride channels. Cell Physiol Biochem 2000;10,307-312[ISI][Medline]
  19. Bali, M, Lipecka, J, Edelman, A, et al Regulation of ClC-2 chloride channels in T84 cells by TGF-{alpha}. Am J Physiol Cell Physiol 2001;280,C1588-C1598[Abstract/Free Full Text]
  20. Leverkoehne, I, Gruber, AD The murine mCLCA3 (alias gob-5) protein is located in the mucin granule membranes of intestinal, respiratory, and uterine goblet cells. J Histochem Cytochem 2002;50,829-838[Abstract/Free Full Text]
  21. Zhou, Y, Dong, Q, Louahed, J, et al Characterization of a calcium-activated chloride channel as a shared target of Th2 cytokine pathways and its potential involvement in asthma. Am J Respir Cell Mol Biol 2001;25,486-491[Abstract/Free Full Text]
  22. Takeyama, K, Dabbagh, K, Lee, HM, et al Epidermal growth factor system regulates mucin production in airways. Proc Natl Acad Sci U S A 1999;96,3081-3086[Abstract/Free Full Text]
  23. Miotto, D, Ruggieri, MP, Boschetto, P, et al Interleukin-13 and -4 expression in the central airways of smokers with chronic bronchitis. Eur Respir J 2003;22,602-608[Abstract/Free Full Text]
  24. Byron, KA, Varigos, GA, Wootton, AM IL-4 production is increased in cigarette smokers. Clin Exp Immunol 1994;95,333-336[ISI][Medline]
  25. Cozen, W, Diaz-Sanchez, D, Gauderman, W James, et al Th1 and Th2 cytokines and IgE levels in identical twins with varying levels of cigarette consumption. J Clin Immunol 2004;24,617-622[CrossRef][ISI][Medline]
  26. Shim, JJ, Dabbagh, K, Ueki, IF, et al IL-13 induces mucin production by stimulating epidermal growth factor receptors and by activating neutrophils. Am J Physiol Lung Cell Mol Physiol 2001;280,L134-L140[Abstract/Free Full Text]
  27. Hauber, HP, Daigneault, P, Frenkiel, S, et al Niflumic acid and MSI-2216 reduce TNF-{alpha}-induced mucin expression in human airway mucosa. J Allergy Clin Immunol 2005;115,266-271[CrossRef][ISI][Medline]
  28. Churg, A, Wang, RD, Tai, H, et al Macrophage metalloelastase mediates acute cigarette smoke-induced inflammation via tumor necrosis factor-{alpha} release. Am J Respir Crit Care Med 2003;167,1083-1089[Abstract/Free Full Text]
  29. Takeyama, K, Dabbagh, K, Jeong Shim, J, et al Oxidative stress causes mucin synthesis via transactivation of epidermal growth factor receptor: role of neutrophils. J Immunol 2000;164,1546-1552[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Article Archive
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via ISI Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hegab, A. E.
Right arrow Articles by Sekizawa, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hegab, A. E.
Right arrow Articles by Sekizawa, K.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS