Regulation of p53 Expression by NF-kB in ER Stress Figure 3. Increased p53 phosphorylation and activity by ER stress. (A) Nuclear localization of NF-kB and p53 in response to ER stress in MCF- 7 cells. The cells were treated with 1 mg/ml BFA, and the localization of NF-kB and p53 were determined by using immunofluorescence staining. (B) NF-kB and p53 nuclear localization were observed in a time-dependent manner after 0, 6, 12, and 24 h exposure to BFA in MCF-7 cells. The cells were treated with 1 mg/ml brefeldin A as time indicated, and NF-kB subunits in the cytoplasmic and nuclear fractions were analyzed by Western blotting with antibodies against NF-kB subunits p65, p50, c-Rel, and p53. Topoisomerase II and a-tubulin were used as internal markers for nuclear and cytoplasmic proteins. (C) time-dependent effect of BFA on p53 phosphorylation. MCF-7 cells were treated with 1 mg/ml brefeldin A in 10% FBS– supplemented DMEM as time indicated. The total cell lysates subjected to immunoblotting with antibodies against anti-p53, anti-p-ser6-p53, anti-p- ser15-p53, anti-p-ser20-p53, anti-p-ser37-p53, and anti-p-ser46-p53. Exposure of MCF-7 cells to 50 J/m2 of UV light served as positive control. (D) ER stress induced transcriptional up-regulation of p53. MCF-7 cells were transfected with reporter vectors encoding firefly luciferase driven by p53 promoter, pp53-TA-Luc plasmid. Transfectants were treated with tunicamycin or Brefeldin A for 24 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). doi:10.1371/journal.pone.0039120.g003 disappearance of NF-kB/DNA complex revealed that two NF-kB family members p50 and p65 were involved (Fig. 4D, right panel). To further validate that interaction between NF-kB proteins and p53 promoter indeed occur in vivo, we conducted a chromatin immunoprecipitation (CHIP) assay. Cross-linked chromatin was isolated from MCF-7 cells, immunoprecipitated by anti-p65 or anti-p50 antibodies and then used as a template in a quantitative PCR to amplify a small DNA fragment targeted to the p53 promoter. Consistent with the EMSA result, data from the CHIP assay showed that treatment of brefeldin A for 24 hours enhanced the association of both p65 and p50 proteins with p53 promoter (Fig. 4E), providing the direct evidence to support the role of NF- kB in modulation of p53 gene expression during ER stress. NF-kB activity is required for the increased expression of p53 in response to ER stress To test whether attenuation of NF-kB signaling affects the increased expression of p53 protein, we used an inhibitor Bay11- 7028 attempting to inhibit NF-kB activity. By an assessment using a NF-kB reporter assay, we showed that the concentration of the NF-kB inhibitor applied in this experiment was able to efficiently reduce NF-kB activity that was activated by brefeldin A (Fig. 5A). To further investigate the effect of Bay11-7082 on ER stress- induced p53 expression, co-treatment of Bay11-7028 with brefeldin A dramatically decreased the induction of p53 at both protein and mRNA levels (Fig. 5B, 5C). To more specifically inhibit NF-kB signaling, expression of the p65 subunit of NF-kB was knocked down by two p65 shRNAs targeting different p65 sequences respectively. MCF-7 cells were transfected with p65- shRNA plasmids and measured the brefeldin A-triggered p53 induction in these p65 knock-down cells. We observed that the increased expression of p53 was abolished in the p65-shRNA transfected cells, proving that NF-kB signaling is required for ER stress-induced p53 expression (Fig. 5D). GSK-3b does not involve in p53 expression that is induced by the prolonged ER stress Previous studies have indicated that when mild ER stress is induced, p53 protein abundance is regulated through the GSK-3b signaling transduction pathway [26,30]. To investigate whether GSK-3b is also involved in regulation of p53 expression if cells are challenged with more prolonged or intense ER stress, we incubated MCF-7 cells with brefeldin A in the absence or presence of GSK-3b inhibitor, lithium chloride (LiCl), for a time PLoS ONE | www.plosone.org 6 July 2012 | Volume 7 | Issue 7 | e39120