Human lung epithelial cells express a functional cold-sensing TRPM8 variant.

Several transient receptor potential (TRP) ion channels sense and respond to changes in ambient temperature. Chemical agonists of TRP channels, including menthol and capsaicin, also elicit sensations of temperature change. TRPM8 is a cold- and menthol-sensing ion channel that converts thermal and chemical stimuli into neuronal signals and sensations of cooling/cold. However, the expression and function of TRPM8 receptors in non-neuronal cells and tissues is a relatively unexplored area. Results presented here document the expression and function of a truncated TRPM8 variant in human bronchial epithelial cells. Expression of the TRPM8 variant was demonstrated by RT-PCR, cloning, and immunohistology. Receptor function was characterized using the prototypical TRPM8 agonist, menthol, and exposure of cells to reduced temperature (18 degrees C). The TRPM8 variant was expressed primarily within endoplasmic reticulum membranes of lung epithelial cells and its activation was attenuated by thapsigargin, the cell-permeable TRPM8 antagonist N-(4-tert-butylphenyl)-4-(3-chloropyridin-2-yl)piperazine-1-carboxamide, and shRNA-induced suppression of TRPM8 expression. Activation of the TRPM8 variant in lung cells was coupled with enhanced expression of the inflammatory cytokines IL-6 and IL-8. Collectively, our results suggest that this novel TRPM8 variant receptor may function as a modulator of respiratory physiology caused by cold air, and may partially explain asthmatic respiratory hypersensitivity to cold air.

Increased transcription of cytokine genes in human lung epithelial cells through activation of a TRPM8 variant by cold temperatures.

Recognition of temperature is a critical element of sensory perception and allows mammals to evaluate both their external environment and internal status. The respiratory epithelium is constantly exposed to the external environment, and prolonged inhalation of cold air is detrimental to human airways. However, the mechanisms responsible for adverse effects elicited by cold air on the human airways are poorly understood. Transient receptor potential melastatin family member 8 (TRPM8) is a well-established cold- and menthol-sensing cation channel. We recently discovered a functional cold- and menthol-sensing variant of the TRPM8 ion channel in human lung epithelial cells. The present study explores the hypothesis that this TRPM8 variant mediates airway cell inflammatory responses elicited by cold air/temperatures. Here, we show that activation of the TRPM8 variant in human lung epithelial cells leads to increased expression of several cytokine and chemokine genes, including IL-1alpha, -1beta, -4, -6, -8, and -13, granulocyte-macrophage colony-stimulating factor (GM-CSF), and TNF-alpha. Our results provide new insights into mechanisms that potentially control airway inflammation due to inhalation of cold air and suggest a possible role for the TRPM8 variant in the pathophysiology of asthma.

Transient receptor potential vanilloid 1 agonists cause endoplasmic reticulum stress and cell death in human lung cells.

Transient receptor potential vanilloid 1 (TRPV1) is a calcium-selective ion channel expressed in human lung cells. We show that activation of the intracellular subpopulation of TRPV1 causes endoplasmic reticulum (ER) stress and cell death in human bronchial epithelial and alveolar cells. TRPV1 agonist (nonivamide) treatment caused calcium release from the ER and altered the transcription of growth arrest- and DNA damage-inducible transcript 3 (GADD153), GADD45alpha, GRP78/BiP, ATF3, CCND1, and CCNG2) in a manner comparable with prototypical ER stress-inducing agents. The TRPV1 antagonist N-(4-tert-butylbenzyl)-N'-(1-[3-fluoro-4-(methylsulfonylamino)-phenyl]ethyl)thiourea (LJO-328) inhibited mRNA responses and cytotoxicity. EGTA and ruthenium red inhibited cell surface TRPV1 activity, but they did not prevent ER stress gene responses or cytotoxicity. Cytotoxicity paralleled eukaryotic translation initiation factor 2, subunit 1 (EIF2alpha) phosphorylation and the induction of GADD153 mRNA and protein. Transient overexpression of GADD153 caused cell death independent of agonist treatment, and cells selected for stable overexpression of a GADD153 dominant-negative mutant exhibited reduced sensitivity. Salubrinal, an inhibitor of ER stress-induced cytotoxicity via the EIF2alphaK3/EIF2alpha pathway, or stable overexpression of the EIF2alpha-S52A dominant-negative mutant also inhibited cell death. Treatment of the TRPV1-null human embryonic kidney 293 cell line with TRPV1 agonists did not initiate ER stress responses. Likewise, n-benzylnonanamide, an inactive analog of nonivamide, failed to cause ER calcium release, an increase in GADD153 expression, and cytotoxicity. We conclude that activation of ER-bound TRPV1 and stimulation of GADD153 expression via the EIF2alphaK3/EIF2alpha pathway represents a common mechanism for cytotoxicity by cell-permeable TRPV1 agonists. These findings are significant within the context of lung inflammatory diseases where elevated concentrations of endogenous TRPV1 agonists are probably produced in sufficient quantities to cause TRPV1 activation and lung cell death.