Cold temperature induces mucin hypersecretion from normal human bronchial epithelial cells in vitro through a transient receptor potential melastatin.

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Cold temperature induces mucin hypersecretion from normal human bronchial epithelial cells in vitro through a transient receptor potential melastatin 8 (TRPM8)–mediated mechanism MinChao Li, PhD, Qi Li, PhD, Gang Yang, PhD, Victor P. Kolosov, MD, PhD, Juliy M. Perelman, MD, PhD, Xiang Dong Zhou, MD, PhD Journal of Allergy and Clinical Immunology Volume 128, Issue 3, Pages 626-634.e5 (September 2011) DOI: 10.1016/j.jaci.2011.04.032 Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 TRPM8 expression in bronchial epithelium from the control (no chronic inflammatory airway disease, n = 9) and COPD (n = 8) groups. A-C, Photomicrographs of representative histologic sections (×400). TRPM8+ cells are indicated by arrows. D, Expression of TRPM8 protein is shown as means ± SDs. ▴P < .05 versus control subjects. Fig 1, C-1, Negative control: nonimmune IgG in place of primary antibody. Fig 1, C-2, Blocking experiment to confirm the specificity of staining. Journal of Allergy and Clinical Immunology 2011 128, 626-634.e5DOI: (10.1016/j.jaci.2011.04.032) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Relative quantitation of TRPM8 mRNA and protein in bronchial epithelium from the control (no chronic inflammatory airway disease, n = 9) and COPD (n = 8) groups. A-C, TRPM8 mRNA and protein levels were determined by means of real-time PCR and Western blotting, respectively. Blocking peptide to confirm specificity of immunodetection. TRPM8 mRNA (Fig 2, B) and TRPM8 protein (Fig 2, C) are shown as means ± SDs. ▴P < .05 versus control subjects. Journal of Allergy and Clinical Immunology 2011 128, 626-634.e5DOI: (10.1016/j.jaci.2011.04.032) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 Characteristic function of TRPM8 channel. A, Menthol or vehicle treated for 4 minutes. *P < .05 versus vehicle; ▴P < .05 versus 1 mmol/L menthol. B-D, Effect of BCTC and TRPM8 shRNA on intracellular Ca2+. Fig 3, B and C: *P < .05 versus vehicle/untreated control. Fig 3, D: *P < .05 versus menthol/cold; #P < .05 versus 15 μmol/L BCTC. Data are presented as means ± SDs (n = 5). Journal of Allergy and Clinical Immunology 2011 128, 626-634.e5DOI: (10.1016/j.jaci.2011.04.032) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 A-C, Relative quantitation of MUC5AC mRNA elicited by menthol/cold and effect of BCTC/TRPM8 shRNA. MUC5AC mRNA expression was evaluated by using real-time RT-PCR with the ΔΔCt method. Data represent the fold increase in expression of MUC5AC relative to GAPDH values as means ± SDs of the 2-ΔΔCt values (n = 3). *P < .05 versus untreated/vehicle control; #P < .05 versus cold/menthol-treated cells. Journal of Allergy and Clinical Immunology 2011 128, 626-634.e5DOI: (10.1016/j.jaci.2011.04.032) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 Cold and menthol increased intracellular and secreted MUC5AC protein expression. A-D, Relative quantization of secreted MUC5AC protein (Fig 5, A and B) and intracellular MUC5AC protein (Fig 5, C and D). E, Effect of BCTC and TRPM8 shRNA on MUC5AC protein. Protein levels were measured by means of ELISA. Data represent fold increase from untreated/vehicle control as means ± SDs (n = 5). *P < .05 versus untreated/vehicle control; #P < .05 versus cold/menthol-treated cells. Journal of Allergy and Clinical Immunology 2011 128, 626-634.e5DOI: (10.1016/j.jaci.2011.04.032) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 6 Cold- and menthol-induced hydrolysis of PIP2. A and B, upper panels, Representative confocal images of localization of GFP fluorescence. Lower panels, The fluorescence intensity changes across the white lines were plotted as line intensity histograms. C, Data represent fluorescence ratios of plasma membrane fluorescence intensity (Fm)/cytosolic fluorescence intensity (Fc) normalized to time 0 as means ± SDs (n = 3). Journal of Allergy and Clinical Immunology 2011 128, 626-634.e5DOI: (10.1016/j.jaci.2011.04.032) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 7 PSD-mutant MARCKS hindered MUC5AC secretion. NHBE cells transfected with wild-type MARCKS, PSD-mutant MARCKS, or vector DNA were exposed to cold and menthol (1 mmol/L) or vehicle medium for 8 and 28 hours, respectively. Mucin secretion was analyzed by means of ELISA. Data are presented as means ± SDs (n = 5). *P < .05 versus untreated/vehicle control; #P < .05 versus cold/menthol-treated cells. Journal of Allergy and Clinical Immunology 2011 128, 626-634.e5DOI: (10.1016/j.jaci.2011.04.032) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E1 A-C, The extent of knockdown and dynamic changes in TRPM8 expression were identified by means of Western blotting in TRPM8 shRNA–transfected NHBE cells. TRPM8 protein values are shown as means ± SDs (n = 3). ▴P < .05 versus untransfected or scramble shRNA–transfected NHBE cells. Journal of Allergy and Clinical Immunology 2011 128, 626-634.e5DOI: (10.1016/j.jaci.2011.04.032) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E2 A and B, Relative quantitation of p-ERK1/2 protein by means of Western blotting in G418-selected cells and control cells (before G418 treatment). p-ERK1/2 protein values are shown as means ± SDs (n = 3). ▴P < .05 versus control subjects. Journal of Allergy and Clinical Immunology 2011 128, 626-634.e5DOI: (10.1016/j.jaci.2011.04.032) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions