Aims: The response to oxidative stress and inflammation varies with diurnal rhythms. Nevertheless, it is not known whether circadian genes are regulated by these stimuli. We evaluated whether Rev-erba, a key circadian gene, was regulated by oxidative stress and/or inflammation in vitro and in a mouse model. Results: A unique sequence consisting of overlapping AP-1 and nuclear factor kappa B (NFjB) consensus sequences was identified on the mouse Rev-erba promoter. This sequence mediates Rev-erba promoter activity and transcription in response to oxidative stress and inflammation. This region serves as an NrF2 platform both to receive oxidative stress signals and to activate Rev-erba, as well as an NFjB-binding site to repress Rev-erba with inflammatory stimuli. The amplitude of the rhythmicity of Rev-erba was altered by pre-exposure to hyperoxia or disruption of NFjB in a cell culture model of circadian simulation. Oxidative stress overcame the inhibitory effect of NFjB binding on Rev-erba transcription. This was confirmed in neonatal mice exposed to hyperoxia, where hyperoxiainduced lung Rev-erba transcription was further increased with NFjB disruption. Interestingly, this effect was not observed in similarly exposed adult mice. Innovation: These data provide novel mechanistic insights into how key circadian genes are regulated by oxidative stress and inflammation in the neonatal lung. Conclusion: Rev-erba transcription and circadian oscillation are susceptible to oxidative stress and inflammation in the neonate. Due to Rev-erba's role in cellular metabolism, this could contribute to lung cellular function and injury from inflammation and oxidative stress. Antioxid. Redox Signal. 21, 17-32.

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.

Hyperoxic exposure can disrupt alveolarization by inhibiting cell growth; however, it is not fully understood how this is mediated. The transcription factor CCAAT/enhancer binding protein-␣ (C/EBP␣) is highly expressed in the lung and plays a role in cell proliferation and differentiation in many tissues. After 72 h of hyperoxia, C/EBP␣ expression was significantly enhanced in the lungs of newborn mice. The increased C/EBP␣ protein was predominantly located in alveolar type II cells. Silencing of C/EBP␣ with a transpulmonary injection of C/EBP␣ small interfering RNA (siRNA) prior to hyperoxic exposure reduced expression of markers of type I cell and differentiation typically observed after hyperoxia but did not rescue the altered lung morphology at 72 h. Nevertheless, when C/EBP␣ hyperoxia-exposed siRNA-injected mice were allowed to recover for 2 wk in room air, lung epithelial cell proliferation was increased and lung morphology was restored compared with hyperoxia-exposed control siRNAinjected mice. These data suggest that C/EBP␣ is an important regulator of postnatal alveolar epithelial cell proliferation and differentiation during injury and repair.CCAAT/enhancer binding protein-␣; developing lung injury; small interfering RNA; recovery POSTNATAL LUNG DEVELOPMENT involves a series of coordinated events, including active cell proliferation and differentiation, to promote proper alveolar formation. Parenchymal lung cells, fibroblasts, endothelial cells, and epithelial type II cells have distinct growth patterns and specific proliferation rates, beginning in the neonatal period (9). Imbalance of growth factors, inflammatory insults, and oxidative stress (reviewed in Ref. 16) could alter these processes, resulting in impaired lung development. Hyperoxic exposure is well known for disrupting alveolarization in the developing lung, because it inhibits the growth of epithelial, fibroblast, and endothelial cells forming the alveoli (5,19). Several studies have demonstrated that hyperoxia results in alveolar growth arrest, induction of cell cycle inhibitory proteins (13), altered surfactant protein (SP) production (reviewed in Ref. 4), and disrupted extracellular matrix signaling (1). The effect of hyperoxia on the proliferation of specific lung cell types is not well defined.The transcription factor CCAAT/enhancer binding protein (C/EBP) family consists of six isoforms. C/EBP␣ is highly expressed in the lung (6) and plays a role in cell proliferation and differentiation in various tissues (17,18), as do C/EBP␤ and C/EBP␦. Given that the newborn lung proliferates and differentiates rapidly, we hypothesized that C/EBP␣ regulates neonatal lung cell proliferation in hyperoxia and during recovery.Here, we demonstrate that C/EBP␣ expression was enhanced in lungs of newborn mice after 72 h of hyperoxia. The increased C/EBP␣ protein was predominantly localized to alveolar type II cells, where altered proliferation was observed. Silencing of C/EBP␣ with a single injection of C/EBP␣ small interfering RNA (siRNA) prior to expo...

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