Sengupta Shaon, Yang Guang, O'Donnell John C, Hinson Maurice D, McCormack Shana E, Falk Marni J, La Ping, Robinson Michael B, Williams Monica L, Yohannes Mekdes T, Polyak Erzsebet, Nakamaru-Ogiso Eiko, Dennery Phyllis A
Division of Neonatology, Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA; University of Pennsylvania, Philadelphia, PA 19104, USA.
Division of Neonatology, Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA.
Free Radic Biol Med. 2016 Apr;93:177-89. doi: 10.1016/j.freeradbiomed.2016.02.004. Epub 2016 Feb 5.
Diurnal oscillations in the expression of antioxidant genes imply that protection against oxidative stress is circadian-gated. We hypothesized that stabilization of the core circadian gene Rev-erbα (Nr1d1) improves cellular bioenergetics and protects against nutrient deprivation and oxidative stress. Compared to WT, mouse lung fibroblasts (MLG) stably transfected with a degradation resistant Rev-erbα (Ser(55/59) to Asp; hence referred to as SD) had 40% higher protein content, 1.5-fold higher mitochondrial area (confocal microscopy), doubled oxidative phosphorylation by high-resolution respirometry (Oroboros) and were resistant to glucose deprivation for 24h. This resulted from a 4-fold reduction in mitophagy (L3CB co-localized with MitoTracker Red) versus WT. Although PGC1α protein expression was comparable between SD and WT MLG cells, the role of mitochondrial biogenesis in explaining increased mitochondrial mass in SD cells was less clear. Embryonic fibroblasts (MEF) from C57Bl/6-SD transgenic mice, had a 9-fold induction of FoxO1 mRNA and increased mRNA of downstream antioxidant targets heme oxygenase-1 (HO-1), Mn superoxide dismutase and catalase (1.5, 2 fold and 2 fold respectively) versus WT. This allowed the SD cells to survive 1h incubation with 500 µM H2O2 as well as 24h of exposure to 95% O2 and remain attached whereas most WT cells did not. These observations establish a mechanistic link between the metabolic functions of Rev-erbα with mitochondrial homeostasis and protection against oxidative stress.
抗氧化基因表达的昼夜振荡表明，针对氧化应激的保护作用是由昼夜节律控制的。我们假设核心昼夜节律基因Rev-erbα（Nr1d1）的稳定化可改善细胞生物能量代谢，并抵御营养剥夺和氧化应激。与野生型相比，稳定转染了抗降解Rev-erbα（Ser(55/59)突变为Asp；因此称为SD）的小鼠肺成纤维细胞（MLG）蛋白质含量高40%，线粒体面积高1.5倍（共聚焦显微镜观察），通过高分辨率呼吸测定法（Oroboros）测定的氧化磷酸化增加了一倍，并且对24小时的葡萄糖剥夺具有抗性。这是由于与野生型相比，线粒体自噬减少了4倍（L3CB与MitoTracker Red共定位）。尽管SD和野生型MLG细胞之间的PGC1α蛋白表达相当，但线粒体生物发生在解释SD细胞中线粒体质量增加方面的作用尚不清楚。与野生型相比，C57Bl/6-SD转基因小鼠的胚胎成纤维细胞（MEF）中FoxO1 mRNA诱导了9倍，下游抗氧化靶点血红素加氧酶-1（HO-1）、锰超氧化物歧化酶和过氧化氢酶的mRNA分别增加了1.5倍、2倍和2倍。这使得SD细胞在500µM H2O2中孵育1小时以及在95% O2中暴露24小时后仍能存活并保持附着状态，而大多数野生型细胞则不能。这些观察结果建立了Rev-erbα的代谢功能与线粒体稳态以及抗氧化应激保护之间的机制联系。
