INTRODUCTION

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Keywords: asthma; chloride channel; goblet cells

Widely distributed in nature, chloride channels play diverse roles such as maintaining membrane potential and movement of Cl ions for fluid and electrolyte transport. The importance of Cl channels in human health and disease is clear in myotonia (1) and cystic fibrosis, where voltage-dependent Cl channels (ClC) (2) and the cystic fibrosis transmembrane conductance regulator (CFTR) (3-5), respectively, are defective. Recently, a new family of proteins has been discovered that mediates Ca²⁺-activated Cl conductance. One of the most interesting aspects of this family is its wide distribution in human secretory organs (6). Ca²⁺-activated Cl channel 1 (CaCC1) mRNA is present in parts of the digestive tract, such as the colon, small intestine, stomach, and appendix, whereas CaCC2 is mainly expressed in the colon and trachea, and CaCC3 is mainly expressed in the trachea.

In a previous report, using a murine model of allergic asthma we showed that mouse gob-5, a member of the CaCC family, had a selective expression pattern in asthmatic airway goblet cells (7). Furthermore, we proved that gob-5 plays a critical role in bronchial hyperreactivity and mucus overproduction, which are important features of bronchial asthma, using intratracheal administration of a gob-5-expressing adenovirus vector.

Given the high structural homology and similarity of tissue distribution between the two, it is likely that the human counterpart of gob-5 is CaCC1 (8). Therefore, we hypothesized that the expression of CaCC1 would be increased in the airways of patients with asthma and that it would be particularly associated with mucus overproduction. The aims of this study were to determine the expression levels of CaCC family members (CaCC1, CaCC2, and CaCC3) in bronchial tissues from patients with asthma and normal control subjects. Using real-time quantitative polymerase chain reaction (PCR), we found that the expression of CaCC1 was significantly upregulated in patients with bronchial asthma. Additionally, we examined the localization of CaCC1 in the airway using in situ hybridization, and mucin production using immunohistochemistry. Furthermore, we attempted to assess the role of CaCC1 in the regulation of mucus overproduction from a human airway epithelial cell line (NCI-H292) in vitro.

	METHODS

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Subjects

Twenty-one patients with asthma were diagnosed according to the criteria of the American Thoracic Society (9). All patients had typical clinical symptoms, a documented 20% reversibility in forced expiratory volume in 1 second (FEV₁) and increased airway responsiveness to methacholine. Thirteen nonatopic normal control subjects were recruited from the hospital outpatient department. None of the patients had received inhaled or oral corticosteroids for 3 months before the study, and all patients required intermittent inhaled ₂-agonist alone. All the subjects were nonsmokers and had no respiratory tract infections within the 2 weeks preceding the study. The study was approved by the ethics committee of the Toho University School of Medicine, and all subjects gave written informed consent. Subjects' characteristics are summarized in Table E1 in the online data supplement.

Tissues obtained from fiberoptic bronchoscopy and bronchial biopsy were prepared as previously described (10).

Isolation of Total RNA and Real-Time Quantitative PCR

Total RNA from tissues and cells were obtained using ISOGEN (Wako Pure Chemicals, Osaka, Japan), quantitated by spectrophotometry, and reverse transcribed using the TaqMan Gold RT-PCR Kit (Applied Biosystems, Foster City, CA). cDNA samples were measured by real-time quantitative PCR using a Perkin-Elmer Applied Biosystems prism model 7,700 sequence detection instrument. Matching primers and TaqMan probes were designed according to the Primer Express Program (Applied Biosystems; Table E2).

Histochemical Analysis

Digoxigenin (DIG)-labeled 0.5-kb RNA probes were transcribed in vitro from PCR products amplified from CaCC1 cDNA using a DIG RNA labeling kit (Roche Diagnostics, Mannheim, Germany). In situ hybridization was performed on bronchial sections using the ISHR starting kit (Nippon Gene, Tokyo, Japan) as previously described (7).

For immunohistochemical analysis, the N-terminal region of CaCC1 was produced in Escherichia coli AD494 (DE3) as a hybrid protein fused to thioredoxin using the pET Trx Fusion System 32 (Novagen, Madison, WI). The CaCC1-fusion protein was purified with a His Bind Purification Kit (Novagen). An anti-CaCC1 antibody was produced in rabbits by injection of purified fusion protein mixed with Freund's adjuvant (Sigma, St. Louis, MO). Frozen sections were incubated with anti-CaCC1 antibody or anti-MUC5AC antibody clone 45M1 (Lab Vision Corporation, Fremont, CA). Biotinylated goat anti-rabbit IgG or biotinylated horse anti-mouse IgG, followed by streptavidin-peroxidase complex (Elite ABC kit; Vector Laboratories, Burlingame, CA) treatment was used to visualize antigen-antibody complexes.

Periodic acid Schiff (PAS) staining was performed to detect mucins and to identify goblet cells. Sections were counterstained with hematoxylin.

Determination of Mucus Production in NCI-H292 Cells

NCI-H292 cells (ATCC CRL-1848) were cultured in 24-well plates and transfected with 3 µg pcDNA-CaCC1 (7) or vector alone using FuGENE6 (Roche Diagnostics) as a carrier. After 4 days in culture, cells were fixed with formalin and stained with PAS or immunostained with anti-CaCC1 or anti-MUC5AC antibody. PAS-stained areas were measured using the computerized image analysis system Mac SCOPE (Mitani Corp., Fukui, Japan). MUC5AC gene detection was determined by real-time quantitative PCR.

Statistical Analysis

All data are expressed as the mean ± SEM. Statistical analyses were performed using SAS software (SAS Institute Inc., Cary, NC). Student's t test was used, and p less than 0.05 was considered significant.

	RESULTS

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Quantitative Detection of CaCC Family Members in the Bronchial Tissues

To examine the expression levels of CaCC family members, a sensitive real-time quantitative PCR assay was used. After initial optimization of the PCR protocols, all primer pairs were confirmed to amplify single products of the expected sequence by agarose gel electrophoresis and sequencing. No products were detected in control subjects without reverse transcriptase, demonstrating that the assays were specific for the transcribed products (data not shown). To obtain absolute quantification, standard curves were generated for each run using the DNA fragments containing the genes of interest (dilutions ranging from 1 to 1,000,000 copies) and demonstrated the linearity of the assays with no change in the amplification efficiency (correlation coefficient exceeded 0.98). Next, we examined the copy numbers of CaCC family (CaCC1, CaCC2, and CaCC3) transcripts in the bronchial tissues of 21 patients with asthma and 13 control subjects. Expression levels of CaCC1 transcripts were significantly higher in patients with asthma than in control subjects (3.44 ± 0.98 copies versus 0.88 ± 0.14 copies, p < 0.05). Expression levels of other CaCC family transcripts were also slightly higher in asthmatics (CaCC2, 2.86 ± 0.54 copies versus 1.94 ± 0.37 copies; CaCC3, 1.98 ± 0.64 copies versus 0.64 ± 0.30 copies) (Figure 1).

View larger version (13K): [in this window] [in a new window]   Figure 1.   Gene expression of CaCC family members in the bronchial tissues of patients with asthma (n = 21) and normal control (n = 13) subjects. Expression levels are displayed as copy number per nanogram of total RNA. Each circle represents the copy number from an individual, with the mean per group indicated by a horizontal line. *p < 0.05 (compared with control). These data are representative of two independent experiments.

CaCC1 Expression in the Airway Mucus-Producing Cells of Patients with Asthma

To identify the cell specific expression of CaCC1, in situ hybridization was performed in the bronchial tissues of patients with asthma. Strong expression was detected throughout the bronchial tissues from patients with asthma but not in tissue from control subjects (Figures 2A and 2C). The CaCC1-expressing areas coincided completely with the surface areas positively stained by the PAS reaction (Figure 2D), which indicated that CaCC1 expression was strictly localized to the airway epithelium, and especially concentrated in mucus-producing cells. Hybridization with the sense probe revealed no staining, confirming the specificity of the assay (Figure 2B).

View larger version (123K): [in this window] [in a new window]   Figure 2.   CaCC1 expression in the mucus-producing cells of asthmatic bronchial tissues. Bronchial sections of asthmatic (A and B) and normal control (C  ) subjects were in situ hybridized with DIG-labeled, single-stranded antisense (A and C  ) and sense (B) RNA probes (0.5 kb). The same sections from patients with asthma were stained with PAS and counterstained with hematoxylin (D). Arrows indicate areas of CaCC1 expression (A) and mucus-producing cells (D). L = lumen; E = epithelium. Original magnification: ×400. Bar = 50 µm.

Furthermore, we compared CaCC1 expression around the bronchial epithelium of 10 randomly chosen tissue samples taken from patients with asthma with that of 8 tissue samples from control subjects using a semi-quantitative scoring system. Half of the samples from asthmatics showed strong staining (Score: 3; Table E3), whereas most of the samples from control subjects were not stained (Score: 0-1) by DIG-labeled antisense CaCC1 probe.

CaCC1 Production in the Airway Goblet Cells of Patients with Asthma

Next, to examine CaCC1 expression at the protein level, we prepared an antibody directed against the N-terminal region of CaCC1 corresponding to residues 22-124. Immunoblots of intestinal lysates with the antibody revealed proteins of 90 kD (data not shown), which corresponds precisely with the size of a major processed form of CaCC1 (8). The antibody was then used for immunohistochemical detection of CaCC1. CaCC1 protein was detected on the surface of epithelium from patients with asthma but barely detectable in control subjects (Figures 3A and 3D, Table E3), and these areas completely coincided areas stained with PAS (Figure 3B). MUC5AC protein, a marker of goblet cell metaplasia (11), was also detected in the same area by immunohistochemical staining with an anti-MUC5AC antibody (Figure 3C). Staining was almost undetectable on the surface of epithelium from control subjects (Figures 3E and 3F). Expression levels of the CaCC1 protein were well correlated with the levels of MUC5AC protein and PAS-staining from asthmatics (Table E3). These results demonstrate that CaCC1 is induced in mucus-producing airway goblet cells at the protein level.

View larger version (142K): [in this window] [in a new window]   Figure 3.   CaCC1 production in the airway goblet cells of asthmatic bronchial tissues. Bronchial sections of patients with asthma (A, B, and C  ) and normal control (D, E, and F  ) subjects were immunohistochemically stained with an anti-CaCC1 antibody (A and D). CaCC1 immunoreactivity in asthmatic airways was observed in the epithelium (arrows), whereas normal control subjects barely showed CaCC1 protein reactivity. The same sections were stained with PAS (B and E  ), or immunohistochemically stained with an anti-MUC5AC antibody (C and F  ). Cells staining positively for both PAS and MUC5AC increased markedly in asthmatic epithelium samples compared with control epithelium samples. L = lumen; E = epithelium. Original magnification: ×200. Bar = 100 µm. (Upper panels; original magnification: ×1,000. Bar = 20 µm.)

CaCC1 Induces Mucus Production In Vitro

To examine whether CaCC1 induces MUC5AC expression, and therefore produces mucus in vitro, we transiently introduced a CaCC1 expression vector (pcDNA-CaCC1) into the human pulmonary mucoepidermoid cell line NCI-H292. After culture for 4 days, CaCC1 expression was confirmed by immunochemical staining using the anti-CaCC1 antibody (Figures 4A and 4B). Then, MUC5AC production was detected by immunochemical analysis. The number and intensity of MUC5AC-positive cells were increased in CaCC1-transfected NCI-H292 cells (Figure 4D), whereas no changes were observed in mock-transfected cells (Figure 4C). The expression level of the MUC5AC gene in CaCC1-transfected cells increased 2.5-fold compared with that in mock-transfected cells as determined by a quantitative PCR assay (n = 6, p < 0.01; Figure 4E). Next, we examined mucus production by PAS staining. PAS-stained areas were also significantly increased in CaCC1-transfected NCI-H292 cells (Figure 4F).

View larger version (49K): [in this window] [in a new window]   Figure 4.   Mucus production in CaCC1-transfected NCI-H292 cells. Immunochemical staining with an anti-CaCC1 antibody (A and B) and an anti-MUC5AC antibody (C and D) were performed on the mucoepidermoid cell line NCI-H292 after transfection with control vector (pcDNA; A and C) or CaCC1 expression vector (pcDNA-CaCC1; B and D) for 4 days. Real-time quantitative PCR was performed on cDNAs from NCI-H292 cells after transfection with pcDNA, or pcDNA-CaCC1. Expression levels are displayed as copy number per ng of total RNA (E). The same samples were stained with PAS. PAS-stained areas were measured by a computerized image analysis system as described in METHODS (F). The figures are representative of three separate experiments (n = 6-10) **p < 0.01 (compared with control subjects). Bars are the mean ± SEM.

	DISCUSSION

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The CaCC family is a novel family of Cl channels (12-14). Members of the CaCC family offer potential as therapeutic targets of cystic fibrosis because they are involved in the regulation of epithelial fluid and electrolyte secretion (15, 16). We investigated the possibility that they are also associated with other respiratory diseases such as bronchial asthma. Examining the expression levels of CaCC family members, we showed for the first time that CaCC1 gene expression was significantly higher in patients with asthma than in normal control subjects, which indicates CaCC1 expression was upregulated in the development of bronchial asthma. The expression of CaCC2 and CaCC3 also have a tendency to be upregulated in patients with asthma and both may also play a role in the development of asthma through their Ca²⁺-activated Cl channel activity. In this study, a group of atopic, non-asthmatic control subjects was not included because of constraints imposed by the ethics committee, who believed that bronchial biopsy would have a bad influence on individuals with other atopic diseases. Further case study including atopic normal subjects with larger populations will be needed to elucidate the relationship of the CaCC family to bronchial asthma.

Mucus plugging has long been recognized as a major factor contributing to the mortality associated with acute severe asthma (17), and goblet cell metaplasia contributes to this overproduction (18). Because airways in normal healthy subjects contain few goblet cells, abnormal proliferation of goblet cells is necessary for the symptoms of overproduction to occur. A combination of in situ hybridization and PAS staining revealed that CaCC1 expression was localized to the airway epithelium in patients with asthma and correlated with mucus-producing cells. This indicates that CaCC1 transcripts are localized in goblet cells, whose function is to secrete mucins. CaCC1 transcripts and mucus production were barely detected in normal control subjects. CaCC1 is translated as a 125-kD precursor, and processed to yield two cell surface-associated subunits, a 90-kD protein and a group of 37-41-kD proteins (8). We produced an anti-CaCC1 antibody by immunizing with the N-terminal region of the protein and the antibody detects the 90-kD major processing product of CaCC1. Immunohistochemical analysis with the anti-CaCC1 antibody revealed that the CaCC1 protein can also be clearly detected on goblet cells in patients with asthma but scarcely in normal control subjects. These findings suggest that the expression of CaCC1 in the airway epithelium may result in an increased number of PAS-positive mucus-producing cells in bronchial asthma.

It is generally accepted that an increase in mucus production requires increased expression of mucin genes (18). To date, 13 mucin genes have been identified (19) and MUC5AC gene expression is reported to be a major component of respiratory secretions (20, 21). Moreover, cytokine-mediated induction of MUC5AC gene expression correlates well with mucus overproduction (22). Additionally, the MUC5 mucin glycoprotein has been biochemically isolated from lung mucus in a patient with asthma (23, 24) and immunohistochemically identified in lung mucus from patients with asthma and in pooled secretions from healthy subjects (25). Consequently, we examined MUC5AC expression using immunohistochemical staining with an anti-MUC5AC antibody. The stained cells completely correlated with CaCC1-expressing and PAS-positive cells. This result demonstrates that MUC5AC is primarily expressed in airway epithelial goblet cells, confirming previous reports.

To examine whether CaCC1 directly induces mucus production by airway epithelial cells, we transiently transfected a CaCC1-expression vector into NCI-H292 cells and evaluated mucus production and mucin gene expression in vitro. CaCC1-transfected NCI-H292 cells demonstrated increased PAS-staining areas and MUC5AC production. These results support the hypothesis that CaCC1 expression is one of the major factors to induce mucus overproduction in bronchial asthma.

In conclusion, the data presented here support the growing evidence that CaCC1 plays an important role in mucus production in the asthmatic airway. However, additional studies will be needed to determine how CaCC1 works on airway epithelial cells to produce mucins. Therapeutic strategies to target CaCC1 and/or its signaling pathway may prove to be an effective approach for the treatment of bronchial asthma.