Abstract
Endoplasmic reticulum (ER) stress causes cell survival or death, which is dependent on the type of cell and stimulus. Capsaicin (8-methyl-N-vanillyl-6-nonenamide) and its analog, dihydrocapsaicin (DHC), induced caspase-3-independent/-dependent signaling pathways in WI38 lung epithelial fibroblast cells. Here, we describe the molecular mechanisms induced by both chemicals. Exposure to capsaicin or DHC caused induction of p53, p21, and G0/G1 arrest. DHC induced massive cellular vacuolization by dilation of the ER and mitochondria. Classic ER stress inducers elicited the unfolded protein response (UPR) and up-regulation of microtubule-associated protein 1 light chain-3 (LC3) II. DHC induced ER stress by the action of heavy chain-binding protein, IRE1, Chop, eukaryotic initiation factor 2α, and caspase-4 and, to a lesser level, by capsaicin treatment. DHC treatment induced autophagy that was blocked by 3-methyladenine (3MA) and accumulated by bafilomycin A1. Blocking of DHC-induced autophagy by 3MA enhanced apoptotic cell death that was completely inhibited by treatment of cells with benzyl-oxcarbonyl-Val-Ala-Asp-fluoromethyl ketone. Knockdown of Ire1 down-regulated the DHC-induced Chop and LC3II and enhanced caspase-3 activation. DHC induced rapid and high-sustained c-Jun NH2-terminal kinase (JNK)/extracellular signal-regulated kinase (ERK) activation, but capsaicin induced transient activation of JNK/ERK. The JNK inhibitor SP600125 down-regulated the expression of IRE1, Chop, and LC3II induced by DHC, thapsigargin, and MG132 [N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal]. Pharmacological blockade or knockdown of ERK down-regulated LC3II. Capsaicin and DHC induced Akt phosphorylation, and the phosphatidylinositol 3-kinase inhibitors, wortmannin and LY294002 [2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride], induced autophagy via ERK activation. Our results indicate that the differential responses of capsaicin and DHC for cell protection are caused by the extent of the UPR and autophagy that are both regulated by the level of JNK and ERK activation.
Footnotes
-
This work was supported by the Korea Science and Engineering Foundation funded by the Korea government [Grant R13-2003-009].
-
doi:10.1124/jpet.108.144113.
-
ABBREVIATIONS: MAPK, mitogen-activated protein kinase; JNK, c-Jun NH2-terminal kinase; DHC, dihydrocapsaicin; ER, endoplasmic reticulum; UPR, unfolded protein response; ATF, activating transcription factor; eIF2α, eukaryotic initiation factor 2α; ERAD, ER-associated degradation; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium; zVAD, benzyl-oxcarbonyl-Val-Ala-Asp-fluoromethyl ketone; DMSO, dimethyl sulfoxide; PBS, phosphate-buffered saline; PI, propidium iodide; GFP, green fluorescent protein; LC3, microtubule-associated protein 1 light chain-3; siRNA, small interfering RNA; ERK, extracellular signal-regulated kinase; rER, rough endoplasmic reticulum; TG, thapsigargin; MG132, N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal; 3MA, 3-methyladenine; PD98059, 2′-amino-3′-methoxyflavone; PI3K, phosphatidylinositol 3-kinase; LY294002, 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride; BaF1, bafilomycin A1; SP600125, anthra(1,9-cd)pyrazol-6(2H)-one 1,9-pyrazoloanthrone; Bip, heavy chain-binding protein; IRE1, mositol requiring 1; PERK, RNA-dependent protein kinase-like endoplasmic reticulum eIF2α kinase.
-
↵
The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material. - Received July 29, 2008.
- Accepted January 9, 2009.
- The American Society for Pharmacology and Experimental Therapeutics
JPET articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|
