Endoplasmic Reticulum Stress Stimulates p53 Expression through NF-kB Activation Wan-Chi Lin1, Yu-Chi Chuang 2 , Yung-Sheng Chang2,3, Ming-Derg Lai2,3,4, Yen-Ni Teng5, Ih-Jen Su 6 , Clay C. C. Wang 7 , Kuan-Han Lee8, Jui-Hsiang Hung 9 * 1 Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska, United States of America, 2 Department of Biochemistry and Molecular Biology, College of Medicine, National Cheng Kung University, Tainan, Taiwan, 3 Institute of Basic Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan, 4 Infectious Diseases and Signaling Research Center, College of Medicine, National Cheng Kung University, Tainan, Taiwan, 5 Department of Biological Sciences and Technology, National University of Tainan, Tainan, Taiwan, 6 National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Tainan, Taiwan, 7 Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, California, United States of America, 8 Institute of Pharmaceutical Science, Chia Nan University of Pharmacy and Science, Tainan, Taiwan, 9 Department of Biotechnology, Chia Nan University of Pharmacy and Science, Tainan, Taiwan Abstract Background: Induction of apoptosis by endoplasmic reticulum (ER) stress is implicated as the major factor in the development of multiple diseases. ER stress also appears to be a potentially useful major response to many chemotherapeutic drugs and environmental chemical compounds. A previous study has indicated that one major apoptotic regulator, p53, is significantly increased in response to ER stress, and participates in ER stress-induced apoptosis. However, the regulators of p53 expression during ER stress are still not fully understood. Principal Findings: In this report, we demonstrate that induction of p53 expression is mediated through NF-kB signaling pathways during ER stress in MCF-7 cells. Tunicamycin or brefeldin A, two ER stress inducers, increased p53 expression in MCF-7 and Hela cells. We found p53 nuclear localization, activity, and phosphorylation at serine 15 on p53 increased during ER stress. Nuclear translocation of NF-kB and activity of NF-kB were also observed during ER stress. ER stress-induced p53 expression was significantly inhibited by coincubation with the NF-kB inhibitor, Bay 11-7082 and downregulation of NF-kB p65 expression. The role of p53 in mediating Brefeldin A-induced apoptosis was also investigated. Induction of p53 expression by Brefeldin A was correlated to Brefeldin A-induced apoptosis. Furthermore, downregulation of p53 expression by p53 siRNA significantly reduced Brefeldin A-induced apoptosis in MCF-7 cells. Significance: Taken together, NF-kB activation and induction of p53 expression is essential for ER stress-induced cell death which is important for therapeutic effects of clinical cancer drugs. Our results may provide insight into the mechanism of cancer chemotherapy efficacy that is associated with induction of ER stress. Citation: Lin W-C, Chuang Y-C, Chang Y-S, Lai M-D, Teng Y-N, et al. (2012) Endoplasmic Reticulum Stress Stimulates p53 Expression through NF-kB Activation. PLoS ONE 7(7): e39120. doi:10.1371/journal.pone.0039120 Editor: Nai Sum Wong, University of Hong Kong, Hong Kong Received July 7, 2011; Accepted May 18, 2012; Published July 30, 2012 Copyright: 2012 Lin et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work has been supported by a research grant from the National Science Council on Taiwan (NSC 99-2320-B-041-001), and in part by grant CN9911 to Dr. Jui-Hsiang Hung from Chia-Nan University of Pharmacy & Science, Tainan, Taiwan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: hung86@mail.chna.edu.tw Introduction In eukaryotic cells, the endoplasmic reticulum (ER) is a dynamic membranous organelle which plays an important role in protein folding, transport, and processing. Many chemical agents, viral proteins, and adverse metabolic conditions cause protein misfold- ing or protein accumulation in the ER, leading to ER stress. Research over the past decade has also demonstrated that many physiological conditions cause ER stress, e.g. nutrient or glucose deprivation, degenerative neuronal disorders [1,2], type II diabetes [3,4], differentiation of B-cells into plasma cells [5,6], and virus infection [7–9]. In this study, tunicamycin and brefeldin A were used to induce ER stress and activate complex signaling pathways known as the unfolding protein response (UPR) and the ER- overloading response pathway (EOR) [10,11]. UPR pathway has three components in mammalian cells: basic leucine zipper transcription factor ATF6, IRE1 RNA-processing enzyme, and ER localized kinase (PERK). Previous studies have indicated that activation of NF-kB is through calcium release, reactive oxygen species production, IRE1, and PERK signal pathway during ER stress [12,13,14,15,16]. We have also characterized the NF-kB response and found that NF-kB was activated through multiple pathways, including calcium signaling and pp38 kinase [17]. Activation of NF-kB is known to regulate expression in more than 100 genes, which are involved in diverse cell processes, such as cell proliferation, differentiation, apoptosis, and inflammation and immune responses [18]. Severe or prolonged ER stress induces activation of unique pathways that lead to cell death through apoptosis. Recently, several pathways have been directly implicated in ER stress- PLoS ONE | www.plosone.org 1 July 2012 | Volume 7 | Issue 7 | e39120