Acetylation of p53 Figure 5. The ability of estrogen to suppress the elevation of Acetyl p53-Lysine373 levels following GCI is lost in long-term estrogen deprived animals. (A) Representative DAB images of immediate (Imm, 7 d) and 10 week (10 W) E2 deprived animals at 24 h following ischemic reperfusion. Note that 17b2E2 attenuation of Acetyl p53-Lysine373 levels is lost in long-term E2 deprived (10 W) animals and that there is robust p53 acetylation in all 10 W animals. Magnification 40X. (B) Semi-quantitative analysis of Acetyl p53-Lysine373-positively stained cells in Imm (7 d) and long- term (10 W) E2-deprived animals, expressed as folds vs. sham in 4–5 sections. *p,0.05 vs. Sham, #p,0.05 vs. Pla or 10 W E2 group; NS = No significant difference. n = 4–5 animals per treatment group. doi:10.1371/journal.pone.0027039.g005 may be an important novel regulatory mechanism for enhancing p53 stability and activation following cerebral ischemia, leading to enhanced apoptotic cell death. Our study also shows that 17b2E2 acts in the hippocampus to decrease acetylation of p53 on Lysine373 and Lysine382 following GCI. Figure 6. Neuroprotective effects of curcumin in the hippo- campus CA1 following global ischemia. (A) Representative photomicrographs of the hippocampal CA1 region showing NeuN and TUNEL staining following 7 days of ischemic reperfusion. (B) Quantitative summary of data (means6SE, n = 6 animals per group) indicating the number of surviving neurons and apoptotic neurons per 250 mm length of medial CA1. NeuN positive pyramidal cells containing intact and round nuclei were counted as surviving cells. Scale bar = 50 mm, magnification 40X. *p,0.01 vs. sham and curcumin groups. Veh = vehicle; Cur = curcumin. doi:10.1371/journal.pone.0027039.g006 It is currently unclear how 17b2E2 is able to attenuate acetylation of p53. It is possible that the regulatory effects of 17b2E2 are mediated indirectly, through its previously reported ability to suppress NOX2 NADPH oxidase activation [36]. Along these lines, administration of a NOX2 inhibitor in the current study led to inhibition of p53 acetylation following GCI. This suggests that NOX2 activation may contribute to cerebral ischemia-induced p53 acetylation in the brain, potentially via induction of ROS. NADPH oxidase activation leads to generation of superoxide, which can be converted to highly reactive and damaging ROS such as hydroxyl ion and peroxynitrite and contribute to oxidative stress [47]. Intriguingly, a role for oxidative stress in regulating p53 acetylation is supported by previous in vitro studies which showed that oxidative stress markedly enhances p53 acetylation [29,48]. Other pro-death (Lysine317) and pro-survival (Lysine320) acet- ylation sites have also been implicated to regulate p53 function and have different biological consequences [27,30]. A significant caveat of our studies is that we only examined a couple of pro- death acetylation sites (Lysine373 and Lysine382) using the acetylated-Lysine-specific antibodies directed to the particular site of interest. It is possible that acetylation at the other known acetylation sites could change as well after cerebral ischemia and/ or 17b2E2 treatment, but further studies will be needed to address this issue. In addition to acetylation, other post-translational modifications can also influence p53 function and could be regulated by 17b2E2. For instance, studies in breast cancer cells revealed that 17b2E2 inhibited resveratrol-stimulated phosphor- ylation of Serines15, 20 and 392 of p53 and acetylation of p53 in a concentration-and time-dependent manner, and decreased apop- tosis [49]. To our knowledge, no one has examined whether 17b2E2 exerts a similar regulatory effect upon phosphorylation of p53 in the brain following cerebral ischemia. Further studies are thus needed to address this issue. Finally, it has been demonstrated that p53 can be methylated at Lysine372, which leads to enhanced stability and activity of p53 [50]. Intriguingly, we found that 17b2E2 treatment significantly attenuated methylation of p53 at Lysine372 following global cerebral ischemia, which may contrib- ute to decreasing stability and activity of p53 and to the overall PLoS ONE | www.plosone.org 8 October 2011 | Volume 6 | Issue 10 | e27039