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Sarah X. Zhang, Chen Chen, Yimin Zhong, Jingming Li, Michael Daneshfar, Joshua J. Wang; Dysregulated Unfolded Protein Response: a Novel Mechanism for Cigarette Smoke-induced RPE Cell Injury. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3192.
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© ARVO (1962-2015); The Authors (2016-present)
Endoplasmic reticulum (ER) stress and oxidative stress have been shown to be the major causes of apoptosis and cell death. In response to ER stress, the cell initiates a complex set of signaling pathways, which are collectively known as the unfolded protein response (UPR). The objective of this study is to examine the role and mechanism of the UPR signaling in cigarette smoke-induced RPE cell injury and its implication in age-related macular degeneration (AMD).
Human RPE (ARPE-19) cells were exposed to hydroquinone (HQ), a potent oxidant and a major component in cigarette smoke, for up to 48 h. Expression of ER stress markers and UPR components were determined by Western blot analysis, RT-PCR, and immunocytochemistry. Apoptosis and mitochondria-related apoptotic pathways were examined by TUNEL assay, activation of caspases, and expression of Bcl-2 proteins. ER stress and the UPR signaling were manipulated by chemical chaperone 4-phenylbutyrate (4-PBA), tauroursodeoxycholic acid (TUDCA), and adenoviruses expressing key UPR regulators including XBP1, GADD34, and dominant negative mutant of PERK (PERKDN).
Expression of GRP78, XBP1 splicing, phosphorylation of PERK and eIF2α were induced rapidly after HQ treatment. Nuclear levels of pro-apoptotic transcription factors ATF4 and CHOP were significantly increased, accompanied by elevated mitochondrial cytochrome c release and caspase 3 activation. Inhibition of ER stress by 4-PBA or TUDCA, or suppression of CHOP activation by Ad-GADD34 or Ad-PERKDN, markedly alleviated HQ-induced apoptosis of RPE cells. Interestingly, despite increased mRNA splicing, the protein level of active XBP1, a master regulator of the adaptive UPR that eliminates ER stress, was drastically decreased in HQ-treated cells. Further, XBP1-deficient RPE cells isolated from conditional RPE-XBP1 knockout mice expressed much lower level of anti-apoptotic protein Bcl-2, and were sensitive to HQ-induced CHOP activation and apoptosis. Conversely, over-expression of active XBP1 abolished HQ-induced CHOP expression and apoptosis in ARPE-19 cells.
Our findings provide strong evidence that cigarette smoke is sufficient to induce ER stress, and, further, dysregulates the UPR signaling, resulting in down-regulation of the protective UPR genes and activation of pro-apoptotic cascades. Enhancing the adaptive UPR function may, therefore, represent a novel therapeutic strategy to combat cigarette smoke-associated RPE injury in AMD.
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