The circadian gene Rev-erbα improves cellular bioenergetics and provides preconditioning for protection against oxidative stress

Abstract

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 (Ser55/59 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) vs 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) vs WT. This allowed the SD cells to survive 1h incubation with 500 μM H2O2 as well as 24hrs 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.

REV‐ERB agonism suppresses osteoclastogenesis and prevents ovariectomy‐induced bone loss partially via FABP4 upregulation

“…Moreover, the level of ROS decreased with SR9009 treatment in a concentration‐dependent manner; this effect was accompanied by down‐regulation of the NADPH oxidases gp91phox, p47phox, p40phox, and p22phox. This finding is in agreement with a previous study showing that REV‐ERBα protected cells from oxidative stress (14). Gp91phox, p67phox, p47phox, p40phox, and p22phox are important regulatory subunits of the ROS system (33).…”

“…In addition, the skeletal muscle of NR1D1 knockout mice displayed reduced mitochondrial content and oxidative function (13). According to Sengupta et al (14), REV‐ERBα also protects cells against oxygen stress. Mouse lung fibroblasts transfected with a degradation‐resistant REV‐ERBα [Ser (55/59) to Asp, SD] showed a 4‐fold reduction in mitophagy.…”

NR1D1 modulates synovial inflammation and bone destruction in rheumatoid arthritis

“…Sengupta and colleagues demonstrated that NR1D1 controls cellular homeostasis and reduces oxidative stress. Also, embryonic fibroblasts from C57Bl/6-SD transgenic mice showed increased mRNA levels of the downstream antioxidants HO-1, Mn superoxide dismutase, and catalase compared with wild-type mice 31 . Furthermore, Woldt and co-workers reported that NR1D1 deficiency in skeletal muscle resulted in reduced mitochondrial content and oxidative activity, whereas its overexpression in muscle or pharmacological activation in vivo increased exercise capacity 12 .…”

Circadian rhythms in mitochondrial respiration

“…A study in athletes found that plasma levels of glutathione and catalase are higher in the evening compared to the morning, suggesting higher capacity to cope with oxidative stress ( Ammar et al 2015 ). Mechanistic studies in mouse MEFs revealed increased catalase levels upon constitutive overexpression of Reverba , suggesting a direct link to the molecular clock ( Sengupta et al 2016 ). In a study of mouse liver, glutathione expression was rhythmic and peaked at the beginning of the active phase ( Xu et al .…”