用于治疗呼吸窘迫个体的补充氧(高氧)通过增强毒性活性氧(ROS)的产生和抑制线粒体呼吸而引起细胞损伤。生殖器形态发生的抑制剂(SMG-1)激酶在高氧时被激活,并通过磷酸化丝氨酸15上的肿瘤抑制剂p53来促进细胞存活。这里,我们研究了SMG-1和p53是否削弱了高氧期间出现的进行性ROS产生和线粒体呼吸下降的恶性循环。
使用RNA干扰将人肺腺癌A549和H1299或结肠癌HCT116细胞去除SMG-1,UPF-1或p53,然后暴露于室内空气(21%的氧气)或高氧(95%的氧气)。免疫印迹用于评估蛋白质表达;海马生物分析仪用于评估细胞呼吸;并且流式细胞术用于评估用线粒体或氧化还原敏感染料染色的细胞的荧光强度。
高氧增加线粒体和细胞质ROS,抑制线粒体呼吸,而不改变线粒体质量或膜电位。SMG-1或其辅因子的耗尽,UPF1显着增强了高氧诱导的线粒体,而不是胞质ROS丰度。它们不影响线粒体质量,膜电位,或高氧诱导的线粒体呼吸缺陷。A549细胞中p53的遗传耗竭和H1299或HCT116细胞中p53基因的消融表明,SMG-1通过激活p53影响线粒体ROS。
我们的发现表明,高氧不会促进进行性线粒体ROS和功能障碍的恶性循环,因为SMG-1-p53信号传导减弱线粒体ROS的产生而不保留呼吸。这表明抗氧化剂疗法在高氧期间钝化ROS的产生可能不足以恢复细胞呼吸。
Supplemental oxygen (hyperoxia) used to treat individuals in respiratory distress causes cell injury by enhancing the production of toxic reactive oxygen species (ROS) and inhibiting mitochondrial respiration. The suppressor of morphogenesis of genitalia (SMG-1) kinase is activated during hyperoxia and promotes cell survival by phosphorylating the tumor suppressor p53 on serine 15. Here, we investigate whether SMG-1 and p53 blunt this vicious cycle of progressive ROS production and decline in mitochondrial respiration seen during hyperoxia.
Human lung adenocarcinoma A549 and H1299 or colon carcinoma HCT116 cells were depleted of SMG-1, UPF-1, or p53 using RNA interference, and then exposed to room air (21% oxygen) or hyperoxia (95% oxygen). Immunoblotting was used to evaluate protein expression; a Seahorse Bioanalyzer was used to assess cellular respiration; and flow cytometry was used to evaluate fluorescence intensity of cells stained with mitochondrial or redox sensitive dyes.
Hyperoxia increased mitochondrial and cytoplasmic ROS and suppressed mitochondrial respiration without changing mitochondrial mass or membrane potential. Depletion of SMG-1 or its cofactor, UPF1, significantly enhanced hyperoxia-induced mitochondrial but not cytosolic ROS abundance. They did not affect mitochondrial mass, membrane potential, or hyperoxia-induced deficits in mitochondrial respiration. Genetic depletion of p53 in A549 cells and ablation of the p53 gene in H1299 or HCT116 cells revealed that SMG-1 influences mitochondrial ROS through activation of p53.
Our findings show that hyperoxia does not promote a vicious cycle of progressive mitochondrial ROS and dysfunction because SMG-1-p53 signaling attenuates production of mitochondrial ROS without preserving respiration. This suggests antioxidant therapies that blunt ROS production during hyperoxia may not suffice to restore cellular respiration.