Inflammatory Effects of Menthol vs. Non-menthol Cigarette Smoke Extract on Human Lung Epithelial Cells: A Double-Hit on TRPM8 by Reactive Oxygen Species and Menthol

Abstract

Clinical studies suggest that smokers with chronic obstructive pulmonary disease who use menthol cigarettes may display more severe lung inflammation than those who smoke non-menthol cigarette. However, the mechanisms for this difference remain unclear. Menthol is a ligand of transient receptor potential melastatin-8 (TRPM8), a Ca²⁺-permeant channel sensitive to reactive oxygen species (ROS). We previously reported that exposure of human bronchial epithelial cells (HBECs) to non-menthol cigarette smoke extract (Non-M-CSE) triggers a cascade of inflammatory signaling leading to IL-8 induction. In this study, we used this in vitro model to compare the inflammatory effects of menthol cigarette smoke extract (M-CSE) and Non-M-CSE and delineate the mechanisms underlying the differences in their impacts. Compared with Non-M-CSE, M-CSE initially increased a similar level of extracellular ROS, suggesting the equivalent oxidant potency. However, M-CSE subsequently produced more remarkable elevations in intracellular Ca²⁺, activation of the mitogen-activated protein kinases (MAPKs)/nuclear factor-κB (NF-κB) signaling, and IL-8 induction. The extracellular ROS responses to both CSE types were totally inhibited by N-acetyl-cysteine (NAC; a ROS scavenger). The intracellular Ca²⁺ responses to both CSE types were also totally prevented by NAC, AMTB (a TRPM8 antagonist), or EGTA (an extracellular Ca²⁺ chelator). The activation of the MAPK/NF-κB signaling and induction of IL-8 to both CSE types were suppressed to similar levels by NAC, AMTB, or EGTA. These results suggest that, in addition to ROS generated by both CSE types, the menthol in M-CSE may act as another stimulus to further activate TRPM8 and induce the observed responses. We also found that menthol combined with Non-M-CSE induced greater responses of intracellular Ca²⁺ and IL-8 compared with Non-M-CSE alone. Moreover, we confirmed the essential role of TRPM8 in these responses to Non-M-CSE or M-CSE and the difference in these responses between the both CSE types using HBECs with TRPM8 knockdown and TRPM8 knockout, and using HEK293 cells transfected with hTRPM8. Thus, compared with exposure to Non-M-CSE, exposure to M-CSE induced greater TRPM8-mediated inflammatory responses in HBECs. These augmented effects may be due to a double-hit on lung epithelial TRPM8 by ROS generated from CSE and the menthol in M-CSE.

Keywords: TRPM8; calcium; cigarette smoke; lung epithelial cell; lung inflammation; menthol; reactive oxygen species; signaling pathway.

Figure 1

Roles of reactive oxygen species (ROS) and TRPM8 in IL-8 induction by non-menthol cigarette smoke extract (Non-M-CSE) and menthol cigarette smoke extract (M-CSE) in human bronchial epithelial cells (HBECs). (A) Cells were exposed to medium alone, 0.5–2% Non-M-CSE or M-CSE for 24 h. (B,C) Cells were pretreated with 5–20 μM AMTB (a TRPM8 antagonist) for 1 h and then exposed to medium alone, 1% Non-M-CSE or 1% M-CSE for 24 h. (D) Cells were pretreated with N-acetyl-cysteine (NAC, a ROS scavenger; 1 mM), AMTB (20 μM), or EGTA (an extracellular Ca²⁺ chelator; 500 μM) for 1 h and then exposed to medium alone, 1% Non-M-CSE, or M-CSE for 24 h. Protein levels of IL-8 were analyzed by Western blot. Data from each group are means ± SEM from four independent experiments. ^*p < 0.05 vs. the medium group (A–D); ^@p < 0.05 vs. the Non-M-CSE group with the same concentrations (A,D); #p < 0.05 vs. the same type of CSE group without pretreatment (B–D).

Figure 2

Suppression of IL-8 induction by M-CSE or Non-M-CSE in HBECs caused by knockdown or knockout of TRPM8. (A) Cells were incubated with or without TRPM8 siRNA for 24 h. (B) Cells were pretreated with 50 nM TRPM8 siRNA or scramble siRNA for 24 h and then exposed to medium alone, 1% Non-M-CSE or 1% M-CSE for 24 h. (C) Cells were transfected with control plasmid (control) or TRPM8 Double Nickase plasmid (KO). (D) Cells were transfected with control plasmid or TRPM8 KO for 48 h and then exposed to medium alone, 1% Non-M-CSE, or 1% M-CSE for 24 h. Protein levels of TRPM8 or IL-8 were analyzed by Western blot in all panels. mRNA levels of TRPM8 were analyzed by RT-PCR in (C). Data from each group are means ± SEM from four independent experiments. ^*p < 0.05 vs. the medium group (A,B,D); ^@p < 0.05 vs. the Non-M-CSE group without pretreatment (B,D); #p < 0.05 vs. the same type of CSE group without pretreatment (B,D).

Figure 3

Roles of ROS and TRPM8 in the increased intracellular Ca²⁺ level induced by M-CSE and Non-M-CSE in HBECs. Intracellular Ca²⁺ levels were measured by Fluo-8 fluorescent probe assay. (A) Cells were exposed to medium alone (control), 1% Non-M-CSE, or 1% M-CSE for 1, 2, 5, 10, and 30 min. (B) Representative images of fluorescence-positive cells at 5 min after exposure. (C) Cells were pretreated with N-acetyl-cysteine (NAC), AMTB, or ethylene glycol tetraacetic acid (EGTA) for 1 h and then exposed to medium alone, 1% Non-M-CSE, or 1% M-CSE for 5 min. (D) Cells were transfected with control plasmid or TRPM8 Double Nickase plasmid (KO) and then exposed to medium alone, 1% Non-M-CSE, or 1% M-CSE for 5 min. Data from each group are means ± SEM from four independent experiments. ^*p < 0.05 vs. the medium group (A,C,D); ^@p < 0.05 vs. the Non-M-CSE group without pretreatment (A,C,D); #p < 0.05 vs. the same type of CSE group without pretreatment (C,D).

Figure 4

Role of TRPM8 in the increased extracellular and intracellular ROS levels induced by M-CSE and Non-M-CSE in HBECs. Cells were pretreated with NAC, AMTB, or EGTA for 1 h and then exposed to medium alone, 1% Non-M-CSE, or 1% M-CSE for 5 (A) and 30 min (B). The ROS levels were assessed by the membrane-permeable probe hydroethidine, which was converted to red fluorescent ethidium (ETH) by ROS. The medium and cells were separately collected to measure the extracellular and intracellular levels of ROS, respectively. Data from each group are means ± SEM from four independent experiments. ^*p < 0.05 vs. the medium group; #p < 0.05 vs. the same type of CSE group without pretreatment.

Figure 5

Role of ROS and TRPM8 in the activation of the associated signaling pathway by M-CSE and Non-M-CSE in HBECs. Cells were pretreated with NAC, AMTB, or EGTA for 1 h and then exposed to medium alone, 1% Non-M-CSE, or 1% M-CSE for 6 (A,B) and 12 h (C), respectively. The activation of the signaling pathway is reflected by increases in phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) in cell lysates and upregulation in the expression of p65 (a subunit of NF-κB) in nuclear extracts. Protein levels were analyzed by Western blot. p-, t-, and H-1 represent phospho-, total-, and histone H1 proteins, respectively. Data from each group are means ± SEM from four independent experiments. ^*p < 0.05 vs. the medium group; ^@p < 0.05 vs. the Non-M-CSE group without pretreatment; #p < 0.05 vs. the same type of CSE group without pretreatment.

Figure 6

Responses of intracellular Ca²⁺ and IL-8 to menthol alone or in combination with Non-M-CSE in HBECs. Intracellular Ca²⁺ levels were measured by Fluo-8 fluorescent probe assay at 1, 2, 5, 10, and 30 min after exposure. IL-8 levels were analyzed by Western blot at 24 h after exposure. Pretreatment with AMTB was performed 1 h prior to exposure. Cells were exposed to medium alone (control) or menthol (0.5–2.5 mM) (A), medium alone, 1% Non-M-CSE, or a combination of Non-M-CSE and menthol (2.5 mM) (B), or medium alone, menthol (2.5 mM), 1% Non-M-CSE, or a combination of Non-M-CSE and menthol (C). Data in each group are means ± SEM from four independent experiments. ^*p < 0.05 vs. the medium group; ^@p < 0.05 vs. the Non-M-CSE group without pretreatment; #p < 0.05 vs. the combination group without pretreatment.

Figure 7

Responses of intracellular Ca²⁺ and IL-8 to M-CSE and Non-M-CSE in HEK293 cells transfected with human TRPM8 (hTRPM8). (A) Cells were transfected with control (pCMV6) or Flag-tagged hTRPM8 vector for 24 h. Anti-Flag and anti-TRPM8 antibodies were used to perform Western blot to confirm successful transfection. (B) Control cells or hTRPM8-expressing cells were exposed to 1% Non-M-CSE or M-CSE for 1, 2, 5, 10, and 30 min. Intracellular Ca²⁺ levels were measured by Fluo-8 fluorescent probe assay. The hTRPM8-expressing cells were exposed to medium alone, 1% Non-M-CSE, 1% M-CSE, or menthol for 2 min (C) and 24 h (D) with or without pretreatment with AMTB. IL-8 levels were analyzed by ELISA. Data from each group are means ± SEM from four independent experiments. ^*p < 0.05 vs. the control vector group (B) or medium group (C,D); ^@p < 0.05 vs. the Non-M-CSE group without pretreatment (B–D); #p < 0.05 vs. the same type of CSE group without pretreatment (C,D).

Figure 8

Oxidative stress-related events induced by M-CSE and Non-M-CSE in HBECs. (A) Cells were pretreated with apocynin (APO, a NADPH oxidase inhibitor; 150 μM) for 1 h and then exposed to medium alone, 1% Non-M-CSE, or M-CSE for 24 h. (B,C) Cells were pretreated with N-acetyl-cysteine (NAC) for 1 h and then exposed to medium alone, 1% Non-M-CSE, or M-CSE for 2 h (B) or for 24 h (C). Levels of IL-8 and 4-HNE were measured using cell lysates, and the level of Nrf2 was measured using nuclear extracts. Protein levels were analyzed by Western blot. Data from each group are means ± SEM from four independent experiments. ^*p < 0.05 vs. the medium group (A–C); ^@p < 0.05 vs. the Non-M-CSE group without pretreatment (A,B); #p < 0.05 vs. the same type of CSE group without pretreatment (A–C).