Regulation of p53 Expression by NF-kB in ER Stress Figure 7. Knockdown of p53 protects cells against BFA-induced cell death. Down-regulation of p53 expression by p53 RNAi during ER stress. (A) MCF-7 cells were cotransfect with pcDNA3.1/pSuper or pcDNA3.1/pSuper-p53 RNAi plasmid by Lipofectammine 2000. The stable transfectants were selected by G418, and then expression of p53 was analyzed by immunoblotting (upper panel). The cell growth rate of MCF-7, MCF-7-pSuper, MCF-7-pSuper-p53-1, and MCF-7-pSuper-p53-2 cells were performed by MTT assay (lower panel). Furthermore, western blot analyses of the expression levels of p53 in MCF-7 cells were subjected to tunicamycin treatment (lower panel). (B) The morphologic changes after a 48-hour 2.5 mg/ml and 5 mg/ml tunicamycin treatment of MCF-7 cells. The cells were followed by photography under phase-contrast magnification. (C) shRNA-mediated knockdown of p53 protects MCF-7 cells from Tunicamycin- or Brefeldin A-induced cell death. The stable transfectants were incubated with Tunicamycin or Brefeldin A in 10% FBS–supplemented DMEM for 48 hr. Cell viability was measured by the MTT assay. Columns, mean of three independent experiments; bars, SD (*, P,0.05; **, P,0.01, Student’s t test). doi:10.1371/journal.pone.0039120.g007 p53 are tightly regulated by several posttranslational mechanisms, such as protein stability, phosphorylation, and subcellular local- ization. Previous studies have demonstrated that phosphorylation of p53 at S315 and S376 is required for its nuclear export and degradation of p53 is mediated by Hdm2 within 3 h of ER stress treatment [26]. In our study we observed p53 degradation occurred after treating with tunicamycin or brefeldin A in the period of 3 to 6 h. After 12 h treatment of brefeldin A, however, p53 was significantly induced by ER stress. Based on this phenomenon, there appears to be degradation of p53 in the early period of ER stress, however, when adaptation fails, prolonged and excessive ER stress induces expression of genes encoding mediation of cell death, such as CHOP and p53. This represents a mechanism of last resort among multicellular organisms to dispense with dysfunctional cells. Previously, UPR has shown that GSK3b is involved in the cytosolic localization of p53 during ER stress, which prevents p53- dependent apoptosis during early ER stress [26,30]. In addition to p53, GSK3b regulates the activation of caspase-2 during ER stress in leukemia [38]. GSK3b is required for the induction of long- chain acyl-CoA synthetase 3 (ACSL3) and lipid accumulation in response to ER stress [39]. On the other hand, previous study also indicated that GSK3b has been shown to affect NF-kB and STAT family members in the inflammatory process [40,41]. In addition, GSK3b also participates in regulation of CHOP expression during ER stress in neuronal cells [42]. Therefore, we tried to co-incubate brefeldin A and the GSK3b inhibitor, lithium chloride, with MCF-7 cells, and found lithium chloride did not affect p53 expression in response to ER stress. The result indicates that GSK3b signal pathway is not involved in induction of p53 expression during ER stress. When normal mammalian cells are subjected to stress signals (e.g. hypoxia, radiation, DNA damage or chemotherapeutic drug) p53 is activated. With regard to p53 modifications, phosphorylation has been studied most intensively and has been proposed to play a critical role in the stabilization and activation of the tumor suppressor. Multiple serine (6, 9, 15, 20, 33, 37, 46, 315, 371, 376, 378, and 392) and three threonine residues (18, 55, and 81) have been reported to undergo phosphorylation in response to diverse stresses. We therefore investigated the role of p53 phosphorylation on five key serine residues (Ser6, Ser15, Ser20, Ser37, and Ser46) for p53 activation. The result showed that phosphorylation of p53 at ser15 was induced in response to ER stress. Many studies have exhibited ser15 phosphorylation of p53 in cells exposed to ionizing radiation, UV irradiation and chemotherapy drugs [32,43]. p53 phosphor- ylation on S15 and other sites has been linked to apoptosis by chemotherapeutic drugs and chemopreventive agents. For exam- ple, induction of p53 phosphorylation on ser15 is caused by Topo PLoS ONE | www.plosone.org 9 July 2012 | Volume 7 | Issue 7 | e39120