Abstract
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), GADD45α, 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 (EIF2α) 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 EIF2αK3/EIF2α pathway, or stable overexpression of the EIF2α-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 EIF2αK3/EIF2α 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.
Footnotes
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This study was supported by National Heart, Lung, and Blood Institute Grant HL069813.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.107.119412.
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ABBREVIATIONS: TRPV1, transient receptor potential vanilloid 1 (a.k.a. VR1 or the capsaicin receptor); ER, endoplasmic reticulum; LJO-328, N-(4-tert-butylbenzyl)-N′-(1-[3-fluoro-4-(methylsulfonylamino)phenyl]ethyl)thiourea; 5-iodo-RTX, 5-iodo-resiniferatoxin; ATF, activating transcription factor; EIF2α, eukaryotic translation initiation factor 2, subunit 1 (α, 35kDa); GADD153, growth arrest- and DNA damage-inducible transcript 3 (a.k.a. DDIT3 and CHOP); GADD45α, growth arrest and DNA-damage-inducible, α (a.k.a. DDIT1); BiP/GRP78, glucose-regulated protein, 70 kDa (a.k.a. HSPA5); CCND1, cyclin D1; CCNG2, cyclin G2; EIF2α-P, phosphorylated eukaryotic translation initiation factor 2, subunit 1; DTT, dithiothreitol; PCR, polymerase chain reaction; NHBE, normal human bronchial epithelial; HEK, human embryonic kidney; RT, reverse transcription; ANOVA, analysis of variance; GFP, green fluorescent protein; EtBr, ethidium bromide; EGFP, enhanced green fluorescent protein; SC0030, N-(4-tert-butylbentyl)-N′-[3-fluoro-4-(methylsulfonylamino)benzyl]thiourea.
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↵ The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.
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↵1 Current affiliation: Jacobs Dugway Team, Life Sciences Testing Facility, Dugway, Utah.
- Received January 3, 2007.
- Accepted February 28, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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