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 Ca(2+) 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 Ca(2+) 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 Ca(2+)-PLC-PIP2-MARCKS signaling pathway.
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 Ca(2+) imaging and spatiotemporal dynamics of phospholipase C (PLC) δ1-pleckstrin homology-green fluorescent protein, respectively. The expression of MUC5AC mRNA and MUC5ACmucin 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 Ca(2+) 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 Ca(2+)-PLC-PIP2-MARCKS signaling pathway.