Regulation of p53 Expression by NF-kB in ER Stress Figure 2. Induction of p53 expression is regulated at the transcriptional level during ER stress. (A) Induction of p53 expression by BFA was inhibited by protein synthesis inhibitor cycloheximide in MCF-7 cells. The cells were exposed with 1 mg/ml brefeldin A in the presence of the various concentrations of cycloheximide for 24 hr. The expression of p53 and b-actin were analyzed by Western blotting. (B) The p53 mRNA expression was inhibited by actinomycin D during ER stress. MCF-7 cells were treated 1 mg/ml brefeldin A with or without 20 mg/ml actinomycin D for 24 hr and harvested for total RNA isolation. The mRNA level of p53, GRP78 and GAPDH were determined by RT-PCR. (C) The effect of ER stress on p53 mRNA expression, with MCF-7 cells treated with 1 mg/ml brefeldin A in the dose- and time-dependent manner. The total RNA was isolated and then subjected to RT-PCR analysis. The p53 mRNA expression level was quantified densitometrically. (D) The level of p53 mRNA in response to ER stress was determined by real-time RT-PCR. MCF-7 cells were treated with 1mg/ml brefeldin A or 5 mg/ml tunicamycin for 24 hr and harvested for total RNA isolation. Real-time RT–PCR was performed as described in Materials and methods. Columns, mean of three independent experiments; bars, SD (**, P,0.01, Student’s t test). doi:10.1371/journal.pone.0039120.g002 including ser6, ser15, ser20, ser37, and ser46 [31,32], MCF-7 cells exposed to 50 J/m2 of UV light were used to serve as a positive control for this experiment. As shown in the Fig. 3C, phosphor- ylation of p53 was observed at ser15, one of the most important p53 phosphorylation sites responsible for p53 transactivation function, thereby suggesting that ER stress-induced p53 protein is functional and is very likely transcriptionally active. To test transactivation ability of p53 protein, MCF-7 cells were trans- fected with a p53 reporter vector encoding a luciferase reporter gene under the control of a p53-responsive element. In the luciferase assay, tunicamycin or brefeldin A treatment both were shown to activate the expression of luciferase reporter (Fig. 3D), demonstrating that ER stress-induced p53 protein is transcrip- tionally active. Higher p53-depedent transcriptional activity is induced in the cells that undergo ER stress. Induction of p53 expression by ER stress is coupled with NF-kB activation NF-kB family members have been previously suggested to have a role to regulate p53 gene in response to certain types of stress [33]. Based on these findings, we hypothesized that activation of NF-kB may be the mechanism that accounts for the increased expression of p53 during ER stress. To examine this hypothesis, we performed western blotting to measure protein level of IkB-a, an inhibitor protein of NF-kB, which functions to sequester NF-kB in the cytoplasm. A marked decrease of IkBa was detected in the MCF-7 cells treated with brefeldin A (Fig. 4A), and the corresponding increase in the nuclear localization of NF-kB (p50 and p65) was also been seen in the treated cells within a similar time frame (Fig. 3A, 3B). The phosphorylation of NF-kB (p65) at serine 276 and serine 311 has been previously suggested to be important for NF-kB transcriptional activation [34,35]. We therefore examined the phosphorylation status of p65 using antibodies specifically against phosph-Ser276 or phosph-Ser311 on p65 protein. By the analyses of western blotting, we observed that upon brefeldin A stimulation, the phosphorylation levels of p65 at serine 276 and serine 311 were enhanced (Fig. 4B), suggestive of the functionality of p65 that acts as a transcription factor during the process of ER stress. In addition, we also performed a luciferase reporter assay to further assess the ability of activated NF-kB to drive target gene expression. For this purpose, we transfected MCF-7 cells with a NF-kB reporter plasmid that was constructed by numerous copies of consensus NF-kB-binding sequence and a luciferase reporter gene, and then challenged these transfectants with brefeldin A. Utilizing a luciferase assay, we demonstrated that NF-kB transcriptional activity was significantly increased in the MCF-7 cells following the brefeldin A treatment (Fig. 4C). Furthermore, we performed an electrophoretic mobility shift assay (EMSA) to analyze binding activity of NF-kB on p53 promoter using the nuclear extracts isolated from MCF-7 cells and a labeled double-stranded probe that bears the consensus NF-kB- binding sequence of p53 promoter. As shown in the left panel of Fig. 4D, NF-kB and DNA binding activity was markedly increased, particularly at the time point of 12 and 24 hour post- treatment. The upper gel-shifted band was further depleted by anti-NF-kB p50 and p65 antibodies to the binding reactions. The PLoS ONE | www.plosone.org 5 July 2012 | Volume 7 | Issue 7 | e39120