ROS Stress Resets Circadian Clocks to Coordinate Pro-Survival Signals

<div><p>Dysfunction of circadian clocks exacerbates various diseases, in part likely due to impaired stress resistance. It is unclear how circadian clock system responds toward critical stresses, to evoke life-protective adaptation. We identified a reactive oxygen species (ROS), H₂O₂ -responsive circadian pathway in mammals. Near-lethal doses of ROS-induced critical oxidative stress (cOS) at the branch point of life and death resets circadian clocks, synergistically evoking protective responses for cell survival. The cOS-triggered clock resetting and pro-survival responses are mediated by transcription factor, central clock-regulatory BMAL1 and heat shock stress-responsive (HSR) HSF1. Casein kinase II (CK2) –mediated phosphorylation regulates dimerization and function of BMAL1 and HSF1 to control the cOS-evoked responses. The core cOS-responsive transcriptome includes CK2-regulated crosstalk between the circadian, HSR, NF-kappa-B-mediated anti-apoptotic, and Nrf2-mediated anti-oxidant pathways. This novel circadian-adaptive signaling system likely plays fundamental protective roles in various ROS-inducible disorders, diseases, and death.</p> </div

Circadian adaptive signaling responsive to the critical ROS stress.

<p>(A) The schematic figure shows resetting of the circadian clock by near-lethal doses of ROS (cOS) at the life-death boundary. (B) The schematic figure shows the core circadian signaling system for adaptation to critical ROS stress for cell survival. This signaling system is composed of CK2-orchestrated mutual crosstalk between circadian, HSR, apoptotic, and Nrf2-mediated anti-oxidant pathways. The component genes identified by PathVisio are shown as rectangles with red indicating up-regulation and pale green for unchanged expression at 4 h post cOS-pulse.</p

Circadian/HSR systems are indispensable for cOS-evoked responses.

<p>(A) HSF1 or BMAL1 deficiency abolishes cOS-synchronized circadian Per2 rhythms and HSE-driven acute surge. Wild-type (Wild) (a,d), BMAL1^−/− (b,d), and HSF1^−/− (c,d) MEFs transfected with the expression vector for Per2-Luc and HSE-SLR were OS-pulsed. Acute (a-c) and circadian (d) profiles were monitored by real-time bioluminescence assay. Relative (RLU) or normalized (deviation from moving average) profiles are shown (n = 4). (B) Each relative cell survival score 1 week after H₂O₂ treatment is shown. The score ++++ indicates 90–100% viable (negative control level), + indicates 25–50% viable, − indicates less than 25% viable (in this case, less than 5% viable). Annexin V/PI-FACS at 12 h post cOS-pulse reveals drastic apoptosis of BMAL1^−/− and HSF1^−/− MEFs in contrast to WT. </p

Critical oxidative stress (cOS) at the branch point of life and death resets circadian clocks.

<p>(A) NIH-3T3:Per2-Luc /HSE-SLR were cOS-pulsed by treatment with an optimal dose of H₂O₂ (5 mM, 10 min) to reset clocks. Circadian Per2-Luc/HSE-SLR profiles were monitored by real-time dual-color bioluminescence assay. Relative (RLU; <i>a</i>) and normalized (detrended; deviation from moving average; b) profiles are shown (n = 5). (B) Annexin V/PI-FACS for NIH-3T3 cells after 12 h of various OS doses revealed the critical dose (5 mM, 10 min) for cell survivability. Numerical values indicate the percent of cells belonging to the 4 divided regions. </p

cOS-responsive circadian transcriptome regulated by CK2-signaling.

<p>(A) Expression profile of the core cOS-evoked circadian transcriptome for cell survival. Microarray analysis was performed to profile regulated genes in NIH-3T3:Per2-Luc with/without cOS-pulse. Functionally relevant genes for cOS-evoked responses were listed by annotation clustering and exploring biological pathways. A heatmap of the cOS-responsive genes encoding CK2, circadian, HSR, apoptosis, and anti-oxidant–related proteins is shown. Gradient representation from brightest red to brightest green indicates relatively high to low levels of gene expression. The values for the heatmap are shown in Table S2. (B-G) CK2-mediated BMAL1/HSF1 phosphorylation regulates anti-apoptotic/oxidant pathways after cOS-pulse. BMAL1^−/− MEFs harboring BMAL1-WT or BMAL1-S90A (B,D,F) and HSF1^−/− MEFs harboring HSF1-WT or HSF1-T142A (C,E,G) were cOS-pulsed. (B,C) (a) Normalized profiles for caspase-3/7-active cell numbers as monitored by live cell time-lapse imaging using CellEvent™ Caspase-3/7 Green (Movie S3 and S4). (b) Representative confocal images of fixed cells with DAPI-visualized nuclei post cOS-pulse are shown. (D,E) Cells transiently expressing NF-kappa-B-driven Luc were cOS-pulsed. Normalized profiles of NF-kappa-B-Luc are shown (n = 4). (F,G) Cells transiently expressing ARE-Luc, the Nrf2-mediated anti-oxidant reporter, were cOS-pulsed. Normalized profiles of ARE-Luc are shown (n = 4).</p

CK2-mediated BMAL1/HSF1 phosphorylation controls cOS-evoked responses.

<p>BMAL1^−/− MEFs harboring BMAL1-WT or BMAL1-S90A (A,C) and HSF1^−/− MEFs harboring HSF1-WT or HSF1-T142A (B,D) were cOS-pulsed. (A,B) Acute (a,b) and circadian (c) Per2-Luc/HSE-SLR profiles are shown as determined by real-time bioluminescence assay (n = 4). (C,D) Annexin V/PI-FACS at 8 h post cOS-pulse reveals reduced survival of BMAL1-S90A and HSF1-T142A cells.</p