Regulation of p53 Expression by NF-kB in ER Stress Figure 5. Regulation of p53 expression depends on NF-kB signaling pathway in ER stress. The effect of the pharmacologic inhibitor of NF- kB, Bay 11-7082, on NF-kB activity. (A) The cells were transfected with NF-kB activation reporter and the transfectants were treated 1 mg/ml Brefeldin A with or without 10 mM Bay 11-7082 for 12 h. Luciferase activities were normalized to that of cotransfected Renilla luciferase. Columns, mean of three independent experiments; bars, SD (*, P,0.05, Student’s t test). (B) Bay 11-7082 inhibits the effect of ER stress-activated p53 expression in MCF- 7 cells. Cultures of MCF-7 cells were treated with 1 mg/ml Brefeldin A in the presence of Bay 11-7082 in the dose- and time-dependent manner. The cell lysates were analyzed by immunoblotting with p53, GRP78, and b-actin antibodies. (C) The effect of Brefeldin A or combination with NF-kB inhibitor on p53 mRNA expression in MCF-7 cell line. Total RNA was isolated from MCF-7 cells and then subjected to RT-PCR analysis with p53, GRP78 and G3APDH specific primers. (D) Downregulation of NF-kB p65 by shRNA decreased p53 protein level during ER stress. Cells were transfected with NF-kB p65 shRNA plasmid, and the whole cell lysates were subjected to immunoblotting with antibodies against anti-p53, anti-p65, anti-GRP78 and b-actin. doi:10.1371/journal.pone.0039120.g005 cells trigger programmed cell death when faced with unfolded protein accumulation. ER stress can induce multiple signal pathway involving in ER stress-induced apoptosis, such as caspase12-caspase-9-caspase-3, PERk-ATF-4-CHOP, IRE-1- Figure 6. GSK3b signaling pathway does not involve in p53 expression through ER stress. Effects of GSK-3b inhibitor, lithium chloride, on p53 expression in ER stress. MCF-7 cells were incubated 1 mg/ml Brefeldin A with or without lithium chloride as time indicated. The total lysates were subjected to immunoblotting with antibodies against anti-p53, anti-GSK-3b, p-ser9-GSK-3b, and a-tubulin. doi:10.1371/journal.pone.0039120.g006 ASK1-JNK, and p53 pathways [19,36,37,23]. Here, we have demonstrated that ER stress can induce the expression of p53, and the induction is dependent on the transcription factors NF-kB. Induction of p53 expression through NF-kB by ER stress plays an important role in ER stress-induce cell death (Fig. 8). In this report, the contribution, function and regulation of p53 in ER stress-induced apoptosis in MCF-7 cells has been investigated. Although ER stress induced expression of p53 and p53 target gene have been described, the regulation of p53 gene expression was still unclear. We demonstrate that expression of p53 is induced by ER stress, and the p53 induction is dependent on the transcription factors NF-kB. We also show that p53 phosphorylation and nuclear localization is induced in response to ER stress. NF-kB activation and phosphorylation are increased by ER stress. p53 is required for ER stress-induced cell death in MCF-7 cells as demonstrated by the results from the p53-targeted siRNA experiment. These results suggest that induction of p53 expression by ER stress participates in ER stress-induced cell death. In addition, NF-kB in regulation of p53 expression evinces pro- apoptosis effects in response to ER stress. Wild-type p53 is inactivated through cytoplasmic sequestration in a subset of human tumor cells. The levels and localization of PLoS ONE | www.plosone.org 8 July 2012 | Volume 7 | Issue 7 | e39120