Unidad de Investigación Médica en Bioquímica, HE, Centro Médico Nacional Siglo XXI. IMSS., Av. Cuauhtémoc 330, Col Doctores, Del. Cuauhtémoc, México, DF, Mexico.
Mol Biol Rep. 2013 Aug;40(8):4947-58. doi: 10.1007/s11033-013-2595-2. Epub 2013 May 9.
Pancreatic β-cell death in type 2 diabetes has been related to p53 subcellular localisation and phosphorylation. However, the mechanisms by which p53 is phosphorylated and its activation in response to oxidative stress remain poorly understood. Therefore, the aim of this study was to investigate mitochondrial p53 phosphorylation, its subcellular localisation and its relationship with apoptotic induction in RINm5F cells cultured under high glucose conditions. Our results show that p53 phosphorylation in the mitochondrial fraction was greater at ser392 than at ser15. This increased phosphorylation correlated with an increase in reactive oxygen species, a decrease in the Bcl-2/Bax ratio, a release of cytochrome c and an increase in the rate of apoptosis. We also observed a decline in ERK 1/2 phosphorylation over time, which is an indicator of cell proliferation. To identify the kinase responsible for phosphorylating p53, p38 mitogen-activated protein kinase (MAPK) activation was analysed. We found that high glucose induced an increase in p38 MAPK phosphorylation in the mitochondria after 24-72 h. Moreover, the phosphorylation of p53 (ser392) by p38 MAPK in mitochondria was confirmed by colocalisation studies with confocal microscopy. The addition of a specific p38 MAPK inhibitor (SB203580) to the culture medium during high glucose treatment blocked p53 mobilisation to the mitochondria and phosphorylation; thus, the release of cytochrome c and the apoptosis rate in RINm5F cells decreased. These results suggest that mitochondrial p53 phosphorylation by p38 MAPK plays an important role in RINm5F cell death under high glucose conditions.
2 型糖尿病中的胰腺 β 细胞死亡与 p53 亚细胞定位和磷酸化有关。然而，p53 如何被磷酸化及其对氧化应激的激活机制仍知之甚少。因此，本研究旨在探讨高糖培养条件下 RINm5F 细胞中线粒体 p53 的磷酸化、亚细胞定位及其与凋亡诱导的关系。我们的结果表明，线粒体部分 p53 的丝氨酸 392 磷酸化比丝氨酸 15 磷酸化更为显著。这种磷酸化的增加与活性氧的增加、Bcl-2/Bax 比值的降低、细胞色素 c 的释放以及凋亡率的增加有关。我们还观察到 ERK 1/2 磷酸化随时间的推移而下降，这是细胞增殖的一个指标。为了确定磷酸化 p53 的激酶，分析了丝裂原活化蛋白激酶（MAPK）的 p38 的激活。我们发现，高糖在 24-72 小时后诱导线粒体中 p38 MAPK 的磷酸化增加。此外，通过共聚焦显微镜的共定位研究证实了 p38 MAPK 在线粒体中对 p53（丝氨酸 392）的磷酸化。在高糖处理期间将特定的 p38 MAPK 抑制剂（SB203580）添加到培养基中可以阻止 p53 向线粒体的迁移和磷酸化；因此，RINm5F 细胞中线粒体中细胞色素 c 的释放和凋亡率降低。这些结果表明，p38 MAPK 介导的线粒体 p53 磷酸化在高糖条件下 RINm5F 细胞死亡中起重要作用。
