Mechanisms of allergy and clinical immunology
Cold temperature induces mucin hypersecretion from normal human bronchial epithelial cells in vitro through a transient receptor potential melastatin 8 (TRPM8)–mediated mechanism

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Background

Cold air stimulus is a major environmental factor that exacerbates chronic inflammatory airway diseases, such as chronic obstructive pulmonary disease (COPD) and asthma. At the molecular level, cold is detected by transient receptor potential melastatin 8 (TRPM8). To date, TRPM8 expression has not been characterized in the airway epithelium of patients with COPD. The role of TRPM8 channels in a series of airway responses induced by cold stimuli and the molecular and biochemical pathways of TRPM8 in regulating cold-induced responses are largely unknown.

Objective

We sought to explore the role of TRPM8 in cold air–provoked mucus hypersecretion and the potential signaling pathway involved in this process.

Methods

The expression of TRPM8 in the bronchial epithelium was examined by means of immunohistochemistry, RT-PCR, and Western blotting. TRPM8 receptor function and hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) were characterized by means of Ca2+ imaging and spatiotemporal dynamics of phospholipase C (PLC) δ1–pleckstrin homology–green fluorescent protein, respectively. The expression of MUC5AC mRNA and MUC5AC mucin protein was measured by using real-time PCR and ELISA, respectively. Four serine residues in the myristoylated alanine-rich C kinase substrate (MARCKS)–phosphorylation site domain were mutated to identify the function of MARCKS in TRPM8-mediated airway mucus hypersecretion.

Results

TRPM8 protein and mRNA expression were significantly increased in patients with COPD compared with expression seen in healthy subjects. Cold produced robust increases in intracellular Ca2+ levels and promoted translocation of PLCδ1–pleckstrin homology–green fluorescent protein. Cold increased expression of MUC5AC mRNA and intracellular and secreted MUC5AC protein in a nonsustained way. Phosphorylation site domain–mutant MARCKS cDNA hindered MUC5AC secretion induced by cold.

Conclusions

These results indicate that the TRPM8 receptor is involved in cold-induced mucus hypersecretion through the Ca2+-PLC-PIP2-MARCKS signaling pathway.

Section snippets

Reagents

PLCδ1–pleckstrin homology (PH)–green fluorescent protein (GFP) plasmids were kind gifts from Tamas Balla (Public Health Service, National Institute of Health, Bethesda, Md). BCTC, exon 18–specific TRPM8 small hairpin RNA (shRNA), and scramble shRNA were kind gifts from Christopher A. Reilly (University of Utah, Salt Lake City, Utah). L-menthol (abbreviated as menthol) was purchased from Crystal Pure Reagent Co Ltd (Shanghai, China) and dissolved in 75% ethanol to the final concentration

TRPM8 mRNA and protein expression were increased in bronchial epithelial cells of patients with COPD

Immunohistochemistry experiments showed that the majority of TRPM8 immunoreactivity was localized to the membranes of basal or small granule cells. A minor amount of TRPM8 immunoreactivity was localized to the membranes and cytoplasm of columnar epithelial or goblet cells (Fig 1, A and B). TRPM8 protein expressed as integrated optical density/area in the bronchial epithelium was increased in lung tissue of patients with COPD (mean, 0.24; SD, 0.049) compared with that of subjects who had no

Discussion

There has been considerable interest in the causes and mechanisms of exacerbation of COPD because such exacerbations are an important cause of the considerable morbidity and mortality found in patients with COPD. Cold-induced COPD exacerbation is a well-known phenomenon and might contribute to the high level of cold-related morbidity from COPD.1, 2 Low ambient temperatures are associated with increased frequency of exacerbation in patients with COPD.2

An intriguing issue is why patients with

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    Supported by the National Nature Science Foundation of China (no. NSF81070031) and the China-Russia Cooperation Research Foundation (no. NSF81011120108; RFBR10-04-91160).

    Disclosure of potential conflict of interest: The authors have declared that they have no conflict of interest.

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