Hidenori Shirai

CLOCK is involved in the circadian transactivation of peroxisome-proliferator-activated receptor α (PPARα) in mice

PPARα (peroxisome-proliferator-activated receptor α) is a member of the nuclear receptor superfamily of ligand-activated transcription factors that regulate the expression of genes associated with lipid metabolism. In the present study, we show that circadian expression of mouse PPARα mRNA requires the basic helix–loop–helix PAS (Per-Arnt-Sim) protein CLOCK, a core component of the negative-feedback loop that drives circadian oscillators in mammals. The circadian expression of PPARα mRNA was abolished in the liver of homozygous Clock mutant mice. Using wild-type and Clock-deficient fibroblasts derived from homozygous Clock mutant mice, we showed that the circadian expression of PPARα mRNA is regulated by the peripheral oscillators in a CLOCK-dependent manner. Transient transfection and EMSAs (electrophoretic mobility-shift assays) revealed that the CLOCK–BMAL1 (brain and muscle Arnt-like protein 1) heterodimer transactivates the PPARα gene via an E-box-rich region located in the second intron. This region contained two perfect E-boxes and four E-box-like motifs within 90 bases. ChIP (chromatin immunoprecipitation) also showed that CLOCK associates with this E-box-rich region in vivo. Circadian expression of PPARα mRNA was intact in the liver of insulin-dependent diabetic and of adrenalectomized mice, suggesting that endogenous insulin and glucocorticoids are not essential for the rhythmic expression of the PPARα gene. These results suggested that CLOCK plays an important role in lipid homoeostasis by regulating the transcription of a key protein, PPARα.

CLOCK is involved in obesity‐induced disordered fibrinolysis in ob/ob mice by regulating PAI‐1 gene expression

Summary.  Background: An increased level of obesity‐induced plasma plasminogen activator inhibitor‐1 (PAI‐1) is considered a risk factor for cardiovascular disease. Aim: The present study investigates whether the circadian clock component CLOCK is involved in obesity‐induced PAI‐1 elevation. Methods: We examined plasma PAI‐1 and mRNA expression levels in tissues from leptin‐deficient obese and diabetic ob/ob mice lacking functional CLOCK protein. Results: Our results demonstrated that plasma PAI‐1 levels were augmented in a circadian manner in accordance with the mRNA expression levels in ob/ob mice. Surprisingly, a Clock mutation normalized the plasma PAI‐1 concentrations in accordance with the mRNA levels in the heart, lung and liver of ob/ob mice, but significantly increased PAI‐1 mRNA levels in adipose tissue by inducing adipocyte hypertrophy in ob/ob mice. The Clock mutation also normalized tissue PAI‐1 antigen levels in the liver but not in the adipose tissue of ob/ob mice. Conclusion: These observations suggest that CLOCK is involved in obesity‐induced disordered fibrinolysis by regulating PAI‐1 gene expression in a tissue‐dependent manner. Furthermore, it appears that obesity‐induced PAI‐1 production in adipose tissue is not closely related to systemic PAI‐1 increases in vivo.

Radiosensitization effect of poly(ADP‐ribose) polymerase inhibition in cells exposed to low and high liner energy transfer radiation

Poly(ADP‐ribose) polymerase (PARP)‐1 promotes base excision repair and DNA strand break repair. Inhibitors of PARP enhance the cytotoxic effects of γ‐irradiation and X‐irradiation. We investigated the impact of PARP inhibition on the responses to γ‐irradiation (low liner energy transfer [LET] radiation) and carbon‐ion irradiation (high LET radiation) in the human pancreatic cancer cell line MIA PaCa‐2. Cell survival was assessed by colony formation assay after combination treatment with the PARP inhibitor AZD2281 and single fraction γ‐irradiation and carbon‐ion irradiation (13 and 70 keV/μm [LET 13 and LET 70]). The DNA damage response (DDR) was assessed by pulse field gel electrophoresis, western blotting and flow cytometry. Treatment with a PARP inhibitor enhanced the cytotoxic effect of γ‐irradiation and LET 13 and LET 70 carbon‐ion irradiation. Moreover, the radiosensitization effect was greater for LET 70 than for LET 13 irradiation. Prolonged and increased levels of γ‐H2AX were observed both after γ‐irradiation and carbon‐ion irradiation in the presence of the PARP inhibitor. Enhanced level of phosphorylated‐p53 (Ser‐15) was observed after γ‐irradiation but not after carbon‐ion irradiation. PARP inhibitor treatment induced S phase arrest and enhanced subsequent G2/M arrest both after γ‐irradiation and carbon‐ion irradiation. These results suggest that the induction of S phase arrest through an enhanced DDR and a local delay in DNA double strand break processing by PARP inhibition caused sensitization to γ‐irradiation and carbon‐ion irradiation. Taken together, PARP inhibitors might be applicable to a wide therapeutic range of LET radiation through their effects on the DDR. (Cancer Sci 2012; 103: 1045–1050)

Circadian variations in coagulation and fibrinolytic factors among four different strains of mice.

This study examined circadian variation in coagulation and fibrinolytic parameters among Jcl:ICR, C3H/HeN, BALB/cA, and C57BL/6J strains of mice. Plasma plasminogen activator inhibitor 1 (PAI‐1) levels fluctuated in a circadian manner and peaked in accordance with the mRNA levels at the start of the active phase in all strains. Fibrinogen mRNA levels peaked at the start of rest periods in all strains, although plasma fibrinogen levels remained constant. Strain differences in plasma antithrombin (AT) activity and protein C (PC) levels were then identified. Plasma AT activity was circadian rhythmic only in Jcl:ICR, but not in other strains, although the mRNA levels remained constant in all strains. Levels of plasma PC and its mRNA fluctuated in a circadian manner only in Jcl:ICR mice, whereas those of plasma prothrombin, factor X, factor VII, prothrombin time (PT), and activated partial thrombin time (APTT) remained constant in all strains. These results suggest that genetic heterogeneity underlies phenotypic variations in the circadian rhythmicity of blood coagulation and fibrinolysis. The circadian onset of thrombotic events might be due in part to the rhythmic gene expression of coagulation and fibrinolytic factors. The present study provides fundamental information about mouse strains that will help to understand the circadian variation in blood coagulation and fibrinolysis.

Pparα is involved in photoentrainment of the circadian clock

We found that bezafibrate, a ligand of peroxisome proliferator-activated receptor &agr; (PPAR&agr;), advances the active phase of mice under light–dark (LD) conditions in a photoperiod-dependent manner. Bezafibrate gradually advanced the activity onset that consequently almost completely reversed the active phase from the dark to the light period under a long photoperiod (18 h of light and 6 h of darkness: LD 18 : 6). The activity onset was not changed under a short photoperiod (LD 8 : 16) or under constant illumination. These observations suggest that PPAR&agr; is involved in entrainment of the circadian clock to environmental LD conditions.

Identification of the circadian clock‐regulated E‐box element in the mouse plasminogen activator inhibitor‐1 gene

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Parg deficiency confers radio-sensitization through enhanced cell death in mouse ES cells exposed to various forms of ionizing radiation

Poly(ADP-ribose) glycohydrolase (Parg) is the main enzyme involved in poly(ADP-ribose) degradation. Here, the effects of Parg deficiency on sensitivity to low and high linear-energy-transfer (LET) radiation were investigated in mouse embryonic stem (ES) cells. Mouse Parg(-/-) and poly(ADP-ribose) polymerase-1 deficient (Parp-1(-/-)) ES cells were used and responses to low and high LET radiation were assessed by clonogenic survival and biochemical and biological analysis methods. Parg(-/-) cells were more sensitive to γ-irradiation than Parp-1(-/-) cells. Transient accumulation of poly(ADP-ribose) was enhanced in Parg(-/-) cells. Augmented levels of phosphorylated H2AX (γ-H2AX) from early phase were observed in Parg(-/-) ES cells. The induction level of p53 phophorylation at ser18 was similar in wild-type and Parp-1(-/-) cells and apoptotic cell death process was mainly observed in the both genotypes. These results suggested that the enhanced sensitivity of Parg(-/-) ES cells to γ-irradiation involved defective repair of DNA double strand breaks. The effects of Parg and Parp-1 deficiency on the ES cell response to carbon-ion irradiation (LET13 and 70 keV/μm) and Fe-ion irradiation (200 keV/μm) were also examined. Parg(-/-) cells were more sensitive to LET 70 keV/μm carbon-ion irradiation than Parp-1(-/-) cells. Enhanced apoptotic cell death also accompanied augmented levels of γ-H2AX in a biphasic manner peaked at 1 and 24h. The induction level of p53 phophorylation at ser18 was not different between wild-type and Parg(-/-) cells. The augmented level of poly(ADP-ribose) accumulation was noted after carbon-ion irradiation compared to γ-irradiation even in the wild-type cells. An enhanced poly(ADP-ribose) accumulation was further observed in Parg(-/-) cells. Both Parg(-/-) cells and Parp-1(-/-) cells did not show sensitization to Fe-ion irradiation. Parg deficiency sensitizes mouse ES cells to a wide therapeutic range of LET radiation through the effects on DNA double strand break repair responses and enhanced cell death.

Circadian expression of clock genes is maintained in the liver of Vitamin A-deficient mice

In mammals, circadian oscillators exist not only in the central clock of the suprachiasmatic nucleus (SCN) but also in peripheral tissues such as the liver, heart and kidneys. Peripheral clocks are entrained to the SCN clock by both neural and humoral signals. Vitamin A might be one candidate that synchronizes peripheral clocks by activating its ligand-dependent nuclear receptors in mammals. The present study examines the effect of a Vitamin A deficiency on the circadian expression of clock genes in the mouse liver. Serum Vitamin A levels remained constant throughout the day in control mice, and were significantly reduced in Vitamin A-deficient mice. Northern blots showed that circadian expression of the clock genes mPer1, mPer2, Clock, and BMAL1, and of the clock-controlled output gene D-site binding protein (DBP), was maintained in Vitamin A-deficient mice. Our results suggest that dietary Vitamin A is not essential for generating circadian rhythms of peripheral clocks in mammals.

Differential circadian expression of endothelin-1 mRNA in the rat suprachiasmatic nucleus and peripheral tissues.

The vasoconstrictor endothelin-1 (ET-1) is implicated in normal neuronal functions. Here we show the circadian expression of ET-1 mRNA in the rat suprachiasmatic nucleus (SCN) that is considered to be the location of the central circadian pacemaker, as well as in peripheral tissues including the brain, heart, and lungs. The expression of ET-1 in the SCN oscillated with a peak at Zeitgeber time (ZT) 4 under light-dark conditions. A significant number of cells in the SCN was stained with ET-1 probe during circadian time (CT) 6, but there was no significant staining at CT18 by mRNA in situ hybridization. The circadian rhythm of ET-1 mRNA in the whole brain also oscillated, but peaked at ZT20. Endothelin-1 expression in the lungs and heart peaked at ZT12 and ZT20, respectively. The results are the first description of the circadian expression of ET-1 mRNA. The diversity of rhythmic expressions among the SCN, whole brain, lungs and heart suggests that ET-1 has different functions in these tissues.

PPARα is a potential therapeutic drug target of circadian rhythm sleep disorders

O1P-JØ8 Genetic and epigenetic analyses in monozygotic twins with Rett syndrome with extreme differences in early development Chunshu Yang1, Kazushi Endoh1, Masaki Soutome1, Noboru Adachi2, Masayuki Sasaki3, Takeo Kubota1 1 Epigenetic Medicine, University of Yamanashi, Yamanashi, Japan; 2 Forensic Medicine, University of Yamanashi, Yamanashi, Japan; 3 Child Neurology, Musashi Hospital, National Center of Neurology and Psychiatry, Tokyo, Japan