Transient Receptor Potential Melastatin 8 (TRPM8)-Based Mechanisms Underlie Both the Cold Temperature-Induced Inflammatory Reactions and the Synergistic Effect of Cigarette Smoke in Human Bronchial Epithelial (16HBE) Cells

Abstract

Transient receptor potential melastatin 8 (TRPM8) is a major receptor of cold environment. Recently, we found that cigarette smoke extract (CSE) upregulated TRPM8 mRNA and protein expression in bronchial tissues that made them more sensitive to cold stimuli. In our present study, we found that cold temperature (18°C)-induced activation of TRPM8 in 16HBE (human bronchial epithelial) cells facilitated Ca²⁺ influx and subsequently led to the increased expression of interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF)-α via the upregulation of p-extracellular signal-regulated kinase (ERK) and the activation of NF-κB. In addition, 16HBE cells that co-stimulated with 18°C and CSE were used to explore the synergistic effect of CSE on cold temperature-induced inflammatory cytokine production as well as the possible involved signaling pathway. RT-PCR and western blot analysis revealed that CSE upregulated TRPM8 mRNA and protein level in 16HBE cells. Ca²⁺ imaging, western blot, and luciferase assay showed more robust increase in intracellular Ca²⁺ and promoted phosphorylated ERK, P38, and NF-κB activity, respectively, in 16HBE cells co-stimulated with CSE and cold temperature, and such alteration was attenuated by TRPM8 short hairpin RNA (shRNA) transfection and BCTC pretreatment. Furthermore, enhanced levels of IL-6, IL-8, and TNF-α showed by enzyme-linked immunosorbent assay (ELISA) were reduced by specific inhibitors of ERK and NF-κB. Collectively, our results suggest that mitogen-activated protein kinase (MAPK)/NF-κB signaling is involved in TRPM8-mediated cold temperature-induced inflammatory cytokine expression. In addition, CSE synergistically amplifies cold temperature-induced inflammatory factors release via upregulating TRPM8 expression and enhancing MAPK/NF-κB signaling pathway.

Keywords: airway inflammation; chronic obstructive pulmonary disease; cigarette smoke extract; cold temperature; transient receptor potential melastatin 8.

FIGURE 1

Cell viability unaltered by cold temperature (18°C) and/or CSE (3%) at 8, 16, 24, and 36 h that determined by MTT assay. Data are expressed as mean ± SD (n = 4), one-way ANOVA.

FIGURE 2

Effects of cold temperature or/and CSE on TRPM8 expression in 16HBE cells relative quantification of transient receptor potential melastatin 8 (TRPM8) mRNA and protein in 16HBE cells exposed to18°C or/and CSE. (A) The levels of the TRPM8 protein were determined by western blot analysis. (B) The levels of TRPM8 mRNA were determined by real-time RT-PCR, and a comparative Ct method (2^−ΔΔCt) was used for the relative mRNA quantification. (C) Densitometry quantification of the bands in A was performed using Quantity One software, and the results are expressed as the ratio of the expression of TRPM8 to β-actin. The values in B and C are shown as the means ± SD; n = 4. ^∗p < 0.05 vs. control.

FIGURE 3

Roles of cold temperature or/and CSE in TRPM8 mediated increase in intracellular Ca²⁺ level in 16HBE cells. Intracellular Ca²⁺ levels were measured by Fluo3-AM fluorescent probe assay. (A) Cells were exposed to 18°C or/and CSE for 30 min. (B) Representative images of fluorescence-positive cells were exposed to18°C for 6 min, to CSE for 3 min, to both cold temperature and CSE for 6 min and with or without transfection of TRPM8 shRNA or scramble shRNA. (C) Fluorescence intensity for intracellular calcium concentration was analyzed with the quantification tools. Data in each group are mean ± SD; n = 4. ^∗p < 0.05 vs. control, ^#p < 0.05 vs. 18°C alone, ^Δp < 0.05 vs. CSE alone.

FIGURE 4

Effects of CSE on cold temperature-induced production of inflammatory cytokines. Quantification of IL-6, IL-8, and TNF-α mRNA and protein in 16HBE cells exposed to 18°C or/and CSE. mRNA and protein expression were evaluated by using real-time PCR with the ΔΔCt method and ELISA assay. Data in each group are mean ± SD; n = 4. ^∗p < 0.05 vs. control, ^#p < 0.05 vs. cold alone, ^Δp < 0.05 vs. CSE alone.

FIGURE 5

Roles of MAPKs/NF-κB signaling pathway in the cold temperature-induced production of inflammatory cytokines in 16HBE cells. (A) Protein expression of ERK, P38, JNK, and IκBα was analyzed by western blotting in 16HBE cells exposed to medium, 18°C, and 18°C with TRPM8 shRNA transfection or scramble shRNA transefection, respectively. (B) NF-KB activity was measured with luciferase assay in 16HBE cells exposed to medium, 18°C, and 18°C with TRPM8 shRNA transfection or scramble shRNA transefection, respectively. (C) mRNA and protein expression of IL-6, IL-8, and TNF-α were separately measured with RT-RCR and ELISA in 16HBE cells exposed to 18°C with pretreatment with an ERK inhibitor (U0126), a P38 inhibitor (SB203580), or a NF-κB inhibitor (BAY 11-7085; BAY), p- and t- represent phospho- and total-, respectively. Data in each group are mean ± SD n = 3. ^∗p < 0.05 vs. control.

FIGURE 6

Roles of MAPKs/NF-κB signaling pathway in the synergistic effect of CSE on the cold temperature-induced production of inflammatory cytokines in 16HBE cells. (A) Protein expression of ERK, P38, JNK, and IκBα was analyzed by western blotting in 16HBE cells exposed to medium, CSE, both CSE and 18°C with or without transfection of TRPM8 shRNA or scramble shRNA, respectively. (B) NF-KB activity was measured with luciferase assay in 16HBE cells exposed to medium, CSE, both CSE and 18°C with or without transfection of TRPM8 shRNA or scramble shRNA, respectively. (C) mRNA and protein expression of IL-6, IL-8, and TNF-α were separately measured with RT-RCR and ELISA in 16HBE cells exposed to both CSE and 18°C with pretreatment with an ERK inhibitor (U0126), a P38 inhibitor (SB203580), or a NF-κB inhibitor (BAY 11-7085). p- and t- represent phospho- and total-, respectively. Data in each group are mean ± SD; n = 3. ^∗p < 0.05 vs. control, ^#p < 0.05 vs. cold alone, ^Δp < 0.05 vs. CSE alone.