Calcium-dependent and independent mechanisms of capsaicin receptor (TRPV1)-mediated cytokine production and cell death in human bronchial epithelial cells

Abstract

Activation of the capsaicin receptor (VR1 or TRPV1) in bronchial epithelial cells by capsaicinoids and other vanilloids promotes pro-inflammatory cytokine production and cell death. The purpose of this study was to investigate the role of TRPV1-mediated calcium flux from extracellular sources as an initiator of these responses and to define additional cellular pathways that control cell death. TRPV1 antagonists and reduction of calcium concentrations in treatment solutions attenuated calcium flux, induction of interleukin-6 and 8 gene expression, and IL-6 secretion by cells treated with capsaicin or resiniferatoxin. Most TRPV1 antagonists also attenuated cell death, but the relative potency and extent of protection did not directly correlate with inhibition of total calcium flux. Treatment solutions with reduced calcium content or chelators had no effect on cytotoxicity. Inhibitors of arachidonic acid metabolism and cyclo-oxygenases also prevented cell death indicating that TRPV1 agonists disrupted basal arachidonic acid metabolism and altered cyclo-oxygenase function via a TRPV1-dependent mechanism in order to produce toxicity. These data confirm previous results demonstrating calcium flux through TRPV1 acts as a trigger for cytokine production by vanilloids, and provides new mechanistic insights on mechanisms of cell death produced by TRPV1 agonists in respiratory epithelial cells.

FIGURE 1

Chemical structures of LJO-328, SC0030, JYL-1433, KMJ-642, 5-iodo-RTX, and capsazepine.

FIGURE 2

(A) Dose-response data for the induction of intracellular calcium flux in TRPV1-overexpressing cells by RTX (squares) and capsaicin (triangles). Data represent the mean fluorescence values for cell populations and standard deviation (n = 4). EC₅₀ values were obtained by nonlinear regression analysis (Prism 4, GraphPad Software, Inc., San Diego, CA) using the one-site binding model. (B) Attenuated capsaicin-induced (20 μM) calcium flux (open bars) in TRPV1-overexpressing cells using reduced calcium solutions (left group), depletion of ER-calcium stores with thapsigargin (1.5 μM, 5 min) (gray bars), and treating with 100 μM EGTA and 10 μM ruthenium red (black bars). Data represent the mean fluorescence values for cell populations and standard deviation (n = 4). *Statistically significant decreases relative to complete media, **significant decreases due to depletion of ER calcium stores, and ***additional decreases afforded by EGTA and ruthenium red (p ≤ 0.05) are identified.

FIGURE 3

(A) Inhibition of cell death (1 μM capsaicin) in TRPV1-overexpressing cells by various TRPV1 selective antagonists. SC0030 (upside-down open triangles, solid line), JYL-1433 (filled diamonds, dashed line), capsazepine (stars, dashed line), and 5-iodo-RTX (open diamonds, solid line). (B) Inhibition of cell death by LJO-328 (stars, dashed line), KMJ-642 (filled diamonds, solid line), antagonist A (upside-down open triangles, solid line), and antagonist B (filled diamonds, dashed line). Data are representative of the mean viability and standard deviation (n = 3). For clarity, statistical significance has not been noted in the figures. (C) The effects of LJO-328 and 5-iodo-RTX on cell death induced by vanilloid treatment. TRPV1-overexpressing cells were treated with 1 μM capsaicin or 10 nM RTX with increasing concentrations of LJO-328 or 5-iodo-RTX for 24 h. Data represent the mean and standard deviation (n = 3). Data are as follows: 10 nM RTX plus 5-iodo-RTX (circles), 10 nM RTX plus LJO-328 (triangles), and 1 μM capsaicin plus LJO-328 (squares). Statistically significant changes in cell viability relative to capsaicin- or RTX-treated controls (p ≤ 0.05) are identified with an asterisk. (D) Dose-response cytotoxicity data for TRPV1-overexpressing cells treated with increasing concentrations of capsaicin in the presence (triangles) and absence of 20 μM LJO-328 (squares). Data represent the mean and standard deviation (n = 4).

FIGURE 4

(A) Induction of IL-6 and IL-8 gene expression in TRPV1-overexpressing cells by capsaicin and inhibition by LJO-328. Cells were treated as shown in the figure for 4 h, harvested, and changes in gene expression assessed by RT-PCR, as described under the materials and methods section. (*) Represents a statistical increase over untreated control cells while (#) represents significant differences from treated and control cells. (B) The effects of multiple TRPV1 antagonists and modulators of TRPV1 function or calcium concentration on the induction of IL-6 (open bars) and IL-8 (shaded bars) genes in TRPV1-overexpressing cells treated with capsaicin (1 μM) for 4 h at 37°C. Concentrations of antagonist were SC0030 (100 nM), capsazepine (15 μM), EGTA (75 μM), and ruthenium red (150 μM). Points at which statistically greater levels of gene expression were observed versus untreated control cells are indicated by an asterisk (*), while lower levels of expression relative to capsaicin-treated cells are represented with an open circle (°). Data represent the mean and standard deviation (n = 5). (C) Inhibition of capsaicin- and RTX-induced IL-6 secretion by TRPV1-overexpressing cells with LJO-328. Cells were treated with increasing concentrations of LJO-328 and capsaicin (1 μM) (squares) or RTX (10 nM) (triangles) for 24 h at 37°C. IL-6 concentration in media was determined by ELISA using pooled samples (n = 3). The concentration of IL-6 in untreated control cells was ~265 pg/mL.

FIGURE 5

(A) Dose-response cytotoxicity data for TRPV1-overexpressing cells treated with increasing concentrations of capsaicin in complete (diamonds) and calcium deficient (squares) cell culture media. Data represent the mean and standard deviation (n = 4). Statistical differences (p ≤ 0.05) were not observed. (B) Inhibition of IL-6 production by cells treated with capsaicin (1 μM) in complete and calcium-deficient cell culture media. The concentration of IL-6 in media collected from untreated cells was 270 ± 50 pg/mL. IL-6 was lower than complete cell culture media (*) and in cells treated with capsaicin (°) (p ≤ 0.05). Data represent the mean and standard deviation (n = 3).

FIGURE 6

Inhibition of cell death by inhibitors of arachidonic acid metabolism and COX activity. Inhibition of cell death induced by capsaicin (1 μM) in TRPV1-overexpressing cells using ETYA (upside down triangles), indomethacin (squares), etodolac (triangles), acetyl-salicylic acid (diamonds), and diclofenac (circles). Error bars less than 5% are not shown. Data represent the mean and standard deviation (n = 3). The lowest concentrations of inhibitor at which statistical significance (p ≤ 0.05) was observed are indicated with an asterisk (*).

FIGURE 7

Schematic representation of the proposed mechanism(s) for extracellular calcium-dependent cytokine production and extracellular calcium-independent cell death in TRPV1-overexpressing cells. TRPV1 is represented as shaded ovals on the plasma membrane and endoplasmic reticulum of cells. TRPV1-selective antagonists and overt modifications to extracellular calcium content selectively inhibit cytokine responses, but not cell death. Only lipophilic TRPV1 antagonists prevent cell death, presumably by inhibiting intracellular, thapsigargin-sensitive TRPV1-mediated activities.