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

Chronic bronchitis 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 studies 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. Mucin synthesis in response to various stimuli is regulated by the epidermal growth factor receptor (EGFR) system.

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 upregula-tion of MUC5AC at both messenger RNA (mRNA) and protein levels.

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 asthmatics, and COPD patients. In addition, transfection of the hCLCA1 gene into the human muco-epidermoid cell line NCI-H292 induces mucin production. 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, and was markedly induced with MUC5AC in respiratory syncytial virus-infected, allergically sensitized mice. Furthermore, some polymorphisms and haplotypes within the hCLCA1 gene have been shown to be associated with bronchial asthma and COPD. 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.

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.

Gene ExpressionsRNA 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 antiGob-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-phos-phate-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. 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 described 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 piper-azine-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. Smoke SolutionAt 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 μmo]/L), AG-1478 (10 μmo]/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]-CTCGCTGGCCATTGCTATTAT-GCCC-[MGB]-3′, sense primer 5′-TCCACCATATACCGCCA-CAGA-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. 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 μmo]/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.

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