Respiratory epithelial cell responses to cigarette smoke: The unfolded protein response
ABSTRACT Cigarette smoking exposes the respiratory epithelium to highly toxic, reactive oxygen nitrogen species which damage lung proteins in the endoplasmic reticulum (ER), the cell organelle in which all secreted and membrane proteins are processed. Accumulation of damaged or misfolded proteins in the ER, a condition termed ER stress, activates a complex cellular process termed the unfolded protein responses (UPR). The UPR acts to restore cellular protein homeostasis by regulating all aspects of protein metabolism including: protein translation and syntheses; protein folding; and protein degradation. However, activation of the UPR may also induce signaling pathways which induce inflammation and cell apoptosis. This review discusses the role of UPR in the respiratory epithelial cell response to cigarette smoke and the pathogenesis of lung diseases like COPD.
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ABSTRACT: Formaldehyde (FA) is toxic to the respiratory system, and nitric oxide (NO) dysfunction stimulates the onset of respiratory diseases. The involvement of dimethylarginine dimethylaminohydrolase (DDAH), the L-arginine analogue asymmetric dimethylarginine (ADMA) degrading enzyme, in FA-induced cell death in lung epithelial cells has not been investigated. In this study, we assessed the effect of FA on DDAH expression and endoplasmic reticulum (ER) stress in A549 cells. We also investigated the preventive effect of DDAH overexpression on ER stress and apoptosis in FA-induced cell death. FA decreased viability in A549 cells and decreased DDAH1 and DDAH2 mRNA and protein expression in a time-dependent manner (>4h). This coincided with increased phosphorylation of the ER stress proteins IRE1α, PERK, and eIF-2α, as well as increased expression of pro-apoptotic proteins such as Bax, C/EPB homologous protein (CHOP), cleaved PARP, and cleaved caspase-3, but decreased expression of the anti-apoptotic protein Bcl-2. ADMA treatment mimicked the effect of FA. Overexpression of DDAH1, but not DDAH2, prevented FA-induced decreases in cell viability, phosphorylation of IRE1α, PERK, and eIF2α, and expression of CHOP. Effects of DDAH1 overexpression, but not DDAH2 overexpression, restored FA-induced increases in Bax, CHOP, cleaved PARP, cleaved caspase-3 and decreases in Bcl-2. In conclusion, FA induces apoptosis of lung epithelial cells via a decrease of DDAH 1 through ER stress.Food and chemical toxicology: an international journal published for the British Industrial Biological Research Association 10/2013; 62. DOI:10.1016/j.fct.2013.10.014
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ABSTRACT: Endoplasmic reticulum stress plays a critical role to restore the homeostasis of protein production in eukaryotic cells. This vital process is hence involved in many types of diseases including COPD. PERK, one branch in the ER stress signaling pathways, has been reported to activate NRF2 signaling pathway, a known protective response to COPD. Based on this scientific rationale, we aimed to identify PERK activators as a mechanism to achieve NRF2 activation. In this report, we describe a phenotypic screening assay to identify PERK activators. This assay measures phosphorylation of GFP-tagged eIF2α upon PERK activation via a cell-based LanthaScreen technology. To obtain a robust assay with sufficient signal to background and low variation, multiple parameters were optimized including GFP-tagged eIF2α BacMam concentration, cell density and serum concentration. The assay was validated by a tool compound, Thapsigargin, which induces phosphorylation of eIF2α. In our assay, this compound showed maximal signal window of approximately 2.5-fold with a pEC50 of 8.0, consistent with literature reports. To identify novel PERK activators through phosphorylation of eIF2α, a focused set of 8,400 compounds was screened in this assay at 10 µM. A number of hits were identified and validated. The molecular mechanisms for several selected hits were further characterized in terms of PERK activation and effects on PERK downstream components. Specificity of these compounds in activating PERK was demonstrated with a PERK specific inhibitor and in PERK knockout mouse embryonic fibroblast (MEF) cells. In addition, these hits showed NRF2-dependent anti-oxidant gene induction. In summary, our phenotypic screening assay is demonstrated to be able to identify PERK specific activators. The identified PERK activators could potentially be used as chemical probes to further investigate this pathway as well as the link between PERK activation and NRF2 pathway activation.PLoS ONE 03/2015; 10(3):e0119738. DOI:10.1371/journal.pone.0119738
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ABSTRACT: The endoplasmic reticulum (ER) plays a major role in regulating synthesis, folding, and orderly transport of proteins. It is also essentially involved in various cellular signaling processes, primarily by its function as a dynamic Ca(2+) store. Compared to the cytosol, oxidizing conditions are found in the ER that allow oxidation of cysteine residues in nascent polypeptide chains to form intramolecular disulfide bonds. However, compounds and enzymes such as PDI that catalyze disulfide bonds become reduced and have to be reoxidized for further catalytic cycles. A number of enzymes, among them products of the ERO1 gene, appear to provide oxidizing equivalents, and oxygen appears to be the final oxidant in aerobic living organisms. Thus, protein oxidation in the ER is connected with generation of reactive oxygen species (ROS). Changes in the redox state and the presence of ROS also affect the Ca(2+) homeostasis by modulating the functionality of ER-based channels and buffering chaperones. In addition, a close relationship exists between oxidative stress and ER stress, which both may activate signaling events leading to a rebalance of folding capacity and folding demand or to cell death. Thus, redox homeostasis appears to be a prerequisite for proper functioning of the ER.Antioxidants and Redox Signaling 09/2006; 8(9-10):1391-418. DOI:10.1089/ars.2006.8.1391