title: Cold temperature induces mucin hypersecretion from normal human bronchial epithelial cells in vitro through a transient receptor potential melastatin 8 (TRPM8)-mediated mechanism.
abstract: 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.
authors.affiliation: Department of Respiratory Medicine, Second Affiliated Hospital of Chongqing Medical University, Chongqing, China.
authors.forename: MinChao
authors.initials: M
authors.lastname: Li
authors.forename: Qi
authors.initials: Q
authors.lastname: Li
authors.forename: Gang
authors.initials: G
authors.lastname: Yang
authors.forename: Victor P
authors.initials: VP
authors.lastname: Kolosov
authors.forename: Juliy M
authors.initials: JM
authors.lastname: Perelman
authors.forename: Xiang Dong
authors.initials: XD
authors.lastname: Zhou
mesh_terms: D000368:Aged; D000818:Animals; D001980:Bronchi / Q000166:cytology / Q000378:metabolism*; D002460:Cell Line; D003080:Cold Temperature; D004847:Epithelial Cells / Q000378:metabolism*; D005260:Female; D005786:Gene Expression Regulation; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D008297:Male; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008875:Middle Aged; D055271:Mucin 5AC / Q000235:genetics / Q000378:metabolism*; D009077:Mucins / Q000378:metabolism; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D029424:Pulmonary Disease, Chronic Obstructive / Q000378:metabolism; D050053:TRPM Cation Channels / Q000235:genetics / Q000378:metabolism*
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; C000619710:MARCKS protein, human; C524678:MUC5AC protein, human; D008565:Membrane Proteins; D055271:Mucin 5AC; D009077:Mucins; D050053:TRPM Cation Channels; C425966:TRPM8 protein, human; D000076250:Myristoylated Alanine-Rich C Kinase Substrate