Figure 1.
Critical oxidative stress (cOS) at the branch point of life and death resets circadian clocks.
(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; a) 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.
Figure 2.
Circadian/HSR systems are indispensable for cOS-evoked responses.
(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.
Figure 3.
CK2-mediated phosphorylation regulates BMAL1-HSF1 binding.
(A) WT MEFs were cOS-pulsed. At the indicated time, BMAL1- and HSF-IP and lysates were analyzed by immunoblotting for BMAL1, phospho-BMAL1-S90 (pBMAL1-S90), HSF1, phospho-HSF1-T142 (pHSF1-T142), CK2alpha, CK2beta, and actin. Representative images are shown (n = 3). (B) CK2 regulates BMAL1-HSF1 binding in living cells. U2OS cells transiently expressing ELucN-HSF1 (WT or T142A lacking a CK2-phospholylation site) and ELucC-BMAL1 (WT or S90A lacking a CK2-phospholylation site) (a; construction map, b; immunoblot detection of recombinant and native proteins) were analyzed by real-time split luciferase complementation assay to detect binding between BMAL1 and HSF1. Relative bioluminescence profiles (n = 4) reveal H2O2 (treated for 10 min) dose-dependent BMAL1-HSF1 binding (c). BMAL1- and HSF-IP and lysates of U2OS cells after H2O2 (0, 1, 5 mM) treatment (0.5 h for HSF1-IP or, 4 h for BMAL1-IP) were analyzed by immunoblotting for BMAL1, pBMAL1-S90, HSF1, and pHSF1-T142. Representative images are shown (n = 3) (d). Normalized profiles (n = 4) show a significant difference between WT and the mutants: ⋆ (P<0.05); ⋆⋆ (P< 0.01); at 8 h post cOS-pulse (e). (C) BMAL1−/− MEFs harboring BMAL1-WT or BMAL1-S90A and HSF1−/− MEFs harboring HSF1-WT or HSF1-T142A were cOS-pulsed. At 3 h (30 min for P-HSF1-T142 and P-BMAL1-S90 detection), HSF- and BMAL1-IP and lysates were analyzed by immunoblotting. Representative images are shown (n = 3).
Figure 4.
CK2-mediated BMAL1/HSF1 phosphorylation controls cOS-evoked responses.
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.
Figure 5.
Microarray analysis of gene regulation by cOS-pulse.
Microarray analysis using workflow for one-color Mouse GE 4x44K v2 (Agilent Technologies; harboring 39430 mouse transcripts) was performed to identify up-regulated genes during the early stage (4h post cOS-pulse) immediate surge of Per2-Luc and late stage (20h and 32h), as compared vs. the control (only medium-change). (A) The graph shows the total number of the (≧ 2-fold) up-regulated genes at the each time point. (B) The graph shows the total number of the circadian circadian-fluctuated (≧ 2-fold) genes at the each time point. (C) The graph shows the total number of the up-regulated annotation clusters (ACs) at the each time point.
Figure 6.
cOS-responsive circadian transcriptome regulated by CK2-signaling.
(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).
Figure 7.
Circadian adaptive signaling responsive to the critical ROS stress.
(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.