Original Contribution
Induction of apoptosis by cigarette smoke via ROS-dependent endoplasmic reticulum stress and CCAAT/enhancer-binding protein-homologous protein (CHOP)

https://doi.org/10.1016/j.freeradbiomed.2008.03.003Get rights and content

Abstract

In this report, we investigated a role of endoplasmic reticulum (ER) stress in cigarette smoke (CS)-induced apoptosis of human bronchial epithelial cells (hBEC). Exposure of hBEC to CS or CS extract (CSE) caused expression of endogenous ER stress markers GRP78 and CHOP and induction of apoptosis evidenced by nuclear condensation, membrane blebbing, and activation of caspase-3 and caspase-4. In vivo exposure of mice to CS also caused induction of GRP78 and CHOP in the lung. Attenuation of ER stress by overexpression of ER chaperone GRP78 or ORP150 significantly attenuated CSE-triggered apoptosis. Exposure of hBEC to CSE caused generation of reactive oxygen species, and treatment with antioxidants inhibited CSE-induced apoptosis. Interestingly, antioxidants including a scavenger of O2radical dot- blunted induction of CHOP by CSE without affecting the level of GRP78, and dominant-negative inhibition of CHOP abolished CSE-induced apoptosis. Furthermore, a generator of O2radical dot- selectively induced CHOP and apoptosis in hBEC. Our results revealed that: (1) CS induces ER stress in vitro and in vivo, (2) ER stress mediates CS-triggered apoptosis downstream of oxidative stress, (3) CS-initiated apoptosis is caused through oxidative stress-dependent induction of CHOP, (4) O2radical dot- may play a dominant role in this process, and (5) oxidative stress-independent induction of GRP78 counterbalances the proapoptotic action of CHOP.

Introduction

Chronic obstructive pulmonary disease (COPD) is one of the most popular causes of death in the world, with its mortality rate still increasing [1]. Cigarette smoke, a complex mixture of oxidants, nicotine, heavy metals, aromatic hydrocarbons, aldehyde, and phenolics, has been regarded as the main risk factor that triggers development of COPD [2]. Cigarette smoking causes several pathological events underlying the development of COPD, e.g., inflammation, proteolysis, and oxidative stress, all of which may lead to apoptosis of pulmonary cells [3]. In general, there are two major intracellular organelles that operate apoptosis, i.e., mitochondria and the endoplasmic reticulum (ER). Accumulating evidence suggests that, in addition to mitochondria, the ER also serves as an important apoptotic control point, and a number of classic death signals may utilize both gateways [4]. The mitochondria-mediated pathway initiates signaling via increases in mitochondrial membrane permeability, which promotes release of cytochrome c and activation of caspases [5]. On the other hand, the ER-mediated pathway is triggered by ER stress, which leads to proapoptotic unfolded protein response including induction of CCAAT/enhancer-binding protein-homologous protein (CHOP), activation of the apoptosis signal-regulating kinase 1 (ASK1)–c-Jun-N-terminal kinase (JNK) pathway and cleavage of ER resident caspases including caspase-12 (in rodent) and caspase-4 (in human) [6], [7].

Previous reports suggested that oxidative stress plays a critical role in the pathogenesis of COPD [8]. In addition to reactive oxygen species (ROS) in cigarette smoke per se, cellular ROS may arise in mitochondria under pathologic conditions. In patients with COPD, elevated levels of oxidative stress markers have been observed in the airspace, sputum, breath, lung tissues, and blood. ROS may cause oxidative inactivation of antiproteases and surfactants, mucus hypersecretion, membrane lipid peroxidation, alveolar epithelial injury, remodeling of extracellular matrix, and apoptosis, all of which contribute to the development and exacerbation of COPD [8]. Mossman et al. recently reported that oxidants in cigarette smoke activated mitogen-activated protein kinases in lung epithelial cells in vitro and in vivo. They also showed that consequent activation of activator protein 1 may contribute to injury of lung epithelial cells and development of COPD [9].

Although accumulative evidence suggests roles of ROS and the mitochondrial pathway in the cytotoxic effects of cigarette smoke on the lung, involvement of ER stress has never been reported. Previous studies indicated that some components in cigarette smoke including ROS, nicotine, heavy metals, and aldehyde may have the potential to induce unfolded protein response in nonpulmonary cells [10], [11], [12], [13]. It is reasonable to speculate that cigarette smoke causes ER stress and thereby contributes to apoptosis of lung epithelial cells. The present investigation was initiated to examine this possibility.

Section snippets

Reagents and cigarette smoke extract (CSE)

Hoechst33258, dichlorofluorescein-diacetate (DCFH-DA), N-acetylcysteine (NAC), menadione, and tunicamycin were purchased from Sigma-Aldrich Japan (Tokyo, Japan), and manganese(III)-5,10,15,20-tetrakis(N-methylpyridinium-2yl)porphyrin pentachloride (MnTM-2-PyP) was purchased from Calbiochem (San Diego, CA). To prepare CSE, mainstream smoke was made from 10 mg-tar cigarettes using constant vacuum flow (10.5 L/min) and collected in a glass bottle, as described previously [14]. The smoke from 20

Induction of ER stress by cigarette smoke

To examine whether cigarette smoke has the potential to induce ER stress in human bronchial epithelial cells, we tested effects of CSE on the expression of endogenous markers for ER stress. BEAS-2B cells were treated with 5% CSE for 4 h, and expression of GRP78 and CHOP was examined by Northern blot analysis. As shown in Fig. 1A, exposure of the cells to CSE resulted in induction of the ER stress markers. Subsequent experiments revealed that the effect of CSE was dose dependent and observed at

Discussion

Cigarette smoke causes injury of bronchial epithelial cells via multiple mechanisms including oxidative stress. However, molecular mechanisms downstream of oxidative stress have not been fully elucidated. In particular, a role of ER stress, a major mechanism that mediates apoptotic cell death, has not been reported yet. In the present investigation, we examined a role of ER stress in cigarette smoke-induced apoptosis of bronchial epithelial cells. Our results elucidated that cigarette smoke

Summary

Cigarette smoke has been regarded as the crucial factor that triggers development of COPD. Previous reports suggested that the mitochondrial pathway and oxidative stress play a role in the injury of the lung during the development of COPD. However, little has been known about the involvement of ER stress in cigarette smoke-triggered pulmonary injury. In the present report, we elucidated for the first time that; (1) cigarette smoke induces ER stress in bronchial epithelial cells in vitro and in

Acknowledgments

We appreciate Dr. Hidetoshi Hayashi (Nagoya City University, Nagoya, Japan), Dr. Richard C. Austin (Henderson Research Center, Ontario, Canada), Dr. Kazutoshi Mori (Kyoto University, Kyoto, Japan), Dr. Sampath Parthasarathy (Emory University School of Medicine, Atlanta, GA), Dr. David Ron (New York University School of Medicine, NY), and Dr. Kazunori Imaizumi (University of Miyazaki, Miyazaki, Japan) for kindly providing us with plasmids. This work was supported by a grant from the Smoking

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