Katsutaka Oishi

Genome-wide Expression Analysis of Mouse Liver Reveals CLOCK-regulated Circadian Output Genes

CLOCK is a positive component of a transcription/translation-based negative feedback loop of the central circadian oscillator in the suprachiasmatic nucleus in mammals. To examine CLOCK-regulated circadian transcription in peripheral tissues, we performed microarray analyses using liver RNA isolated from Clock mutant mice. We also compared expression profiles with those of Cryptochromes (Cry1 and Cry2) double knockout mice. We identified more than 100 genes that fluctuated from day to night and of which expression levels were decreased in Clock mutant mice. In Cry-deficient mice, the expression levels of most CLOCK-regulated genes were elevated to the upper range of normal oscillation. Most of the screened genes had a CLOCK/BMAL1 binding site (E box) in the 5′-flanking region. We found that CLOCK was absolutely concerned with the circadian transcription of one type of liver genes (such as DBP, TEF, and Usp2) and partially with another (such as mPer1, mPer2, mDec1, Nocturnin, P450 oxidoreductase, and FKBP51) because the latter were damped but remained rhythmic in the mutant mice. Our results showed that CLOCK and CRY proteins are involved in the transcriptional regulation of many circadian output genes in the mouse liver. In addition to being a core component of the negative feedback loop that drives the circadian oscillator, CLOCK also appears to be involved in various physiological functions such as cell cycle, lipid metabolism, immune functions, and proteolysis in peripheral tissues.

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.

Functional CLOCK is not involved in the entrainment of peripheral clocks to the restricted feeding: entrainable expression of mPer2 and BMAL1 mRNAs in the heart of Clock mutant mice on Jcl:ICR background

The mammalian circadian timing system consists of a central pacemaker in brain hypothalamus and damping oscillators in most peripheral tissues. To investigate the mechanism that controls circadian rhythms in the mammalian peripheral tissues, we examined the expression rhythm of mPer2, BMAL1, albumin D-site binding protein (DBP), and Rev-erbalpha mRNAs in the heart of homozygous Clock mutant mice on Jcl:ICR background under the temporal feeding restriction. Unexpectedly, the restricted feeding (RF) shifted the circadian phase of both mPer2 and BMAL1 mRNA expressions in the heart not only of wild-type mice but also of Clock mutant mice. Furthermore, in the Clock mutant mice, the amplitude of the circadian expression of mPer2 and BMAL1 mRNAs was dramatically increased by the RF. These data indicate that functional CLOCK is not required for an entrainment of peripheral clocks to RF. On the other hand, the expression levels of DBP and Rev-erbalpha mRNAs were blunted in Clock mutant mice not only under ad libitum but also under RF conditions. Thus, it seems that the rhythmic expression of Rev-erbalpha is not involved in the RF-induced circadian expression of BMAL1 mRNA, although REV-ERBalpha has been identified as a major regulator of BMAL1 transcription. Thus, the entraining mechanism of peripheral tissues to the RF seems to be different from that to the central clock in the suprachiasmatic nucleus.

Thrombomodulin Is a Clock-controlled Gene in Vascular Endothelial Cells

Cardiovascular diseases are closely related to circadian rhythm, which is under the control of an internal biological clock mechanism. Although a biological clock exists not only in the hypothalamus but also in each peripheral tissue, the biological relevance of the peripheral clock remains to be elucidated. In this study we searched for clock-controlled genes in vascular endothelial cells using microarray technology. The expression of a total of 229 genes was up-regulated by CLOCK/BMAL2. Among the genes that we identified, we examined the thrombomodulin (TM) gene further, because TM is an integral membrane glycoprotein that is expressed primarily in vascular endothelial cells and plays a major role in the regulation of intravascular coagulation. TM mRNA and protein expression showed a clear circadian oscillation in the mouse lung and heart. Reporter analyses, gel shift assays, and chromatin immunoprecipitation analyses using the TM promoter revealed that a heterodimer of CLOCK and BMAL2 binds directly to the E-box of the TM promoter, resulting in TM promoter transactivation. Indeed, the oscillation of TM gene expression was abolished in clock mutant mice, suggesting that TM expression is regulated by the clock gene in vivo. Finally, the phase of circadian oscillation of TM mRNA expression was altered by temporal feeding restriction, suggesting TM gene expression is regulated by the peripheral clock system. In conclusion, these data suggest that the peripheral clock in vascular endothelial cells regulates TM gene expression and that the oscillation of TM expression may contribute to the circadian variation of cardiovascular events.

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.

Circadian expression of FGF21 is induced by PPARα activation in the mouse liver

Peroxisome proliferator‐activated receptor α (PPARα) is a nuclear receptor that regulates the expression of genes associated with lipid metabolism. Recent studies have suggested that the expression of PPARα‐dependent fibroblast growth factor 21 (FGF21) plays important roles in adaptation to fasting, such as lipolysis and ketogenesis. We found that a nighttime injection of bezafibrate, a ligand of PPARα, effectively induced FGF21 expression, whereas a daytime injection did not affect it. Furthermore, bezafibrate‐induced circadian FGF21 expression was abolished in PPARα‐deficient mice. These observations suggest that bezafibrate‐induced circadian FGF21 expression is due to circadian variations in the responsiveness of the PPARα system in the liver.

Effect of feeding on peripheral circadian rhythms and behaviour in mammals

Although feeding time is a dominant cue for circadian rhythms in mammalian peripheral tissue, the effect of feeding and fasting on circadian gene expression and behaviour is unknown. Here we report that fasting does not affect the phase of rhythmic mRNA expression levels of the clock genes, mPer1, mPer2 and of the clock controlled gene, mDBP. However, the levels of each of these genes were significantly altered in different ways and recovered by feeding. We also found that feeding enhances phase‐shifting to a new light‐dark cycle of rhythmic mPer2 mRNA expression in the heart. Furthermore, feeding enhances the phase‐shifting to new light‐dark cycle of behaviour more than fasting. Our data indicate that feeding is an important cue for circadian behaviour rhythms as well as for the photo‐entrainment of peripheral clock gene expression.

CLOCK Regulates Circadian Rhythms of Hepatic Glycogen Synthesis through Transcriptional Activation of Gys2

Hepatic glycogen content is important for glucose homeostasis and exhibits robust circadian rhythms that peak at the end of the active phase in mammals. The activities of the rate-limiting enzymes for glycogenesis and glycogenolysis also show circadian rhythms, and the balance between them forms the circadian rhythm of the hepatic glycogen content. However, no direct evidence has yet implicated the circadian clock in the regulation of glycogen metabolism at the molecular level. We show here that a Clock gene mutation damps the circadian rhythm of the hepatic glycogen content, as well as the circadian mRNA and protein expression of Gys2 (glycogen synthase 2), which is the rate-limiting enzyme of glycogenesis in the liver. Transient reporter assays revealed that CLOCK drives the transcriptional activation of Gys2 via two tandemly located E-boxes. Chromatin immunoprecipitation assays of liver tissues revealed that CLOCK binds to these E-box elements in vivo, and real time reporter assays showed that these elements are sufficient for circadian Gys2 expression in vitro. Thus, CLOCK regulates the circadian rhythms of hepatic glycogen synthesis through transcriptional activation of Gys2.

Clock mutation affects circadian regulation of circulating blood cells

Background Although the number of circulating immune cells is subject to high-amplitude circadian rhythms, the underlying mechanisms are not fully understood. Methods To determine whether intact CLOCK protein is required for the circadian changes in peripheral blood cells, we examined circulating white (WBC) and red (RBC) blood cells in homozygous Clock mutant mice. Results Daytime increases in total WBC and lymphocytes were suppressed and slightly phase-delayed along with plasma corticosterone levels in Clock mutant mice. The peak RBC rhythm was significantly reduced and phase-advanced in the Clock mutants. Anatomical examination revealed hemoglobin-rich, swollen red spleens in Clock mutant mice, suggesting RBC accumulation. Conclusion Our results suggest that endogenous clock-regulated circadian corticosterone secretion from the adrenal gland is involved in the effect of a Clock mutation on daily profiles of circulating WBC. However, intact CLOCK seems unnecessary for generating the rhythm of corticosterone secretion in mice. Our results also suggest that CLOCK is involved in discharge of RBC from the spleen.

Plasminogen activator inhibitor-1 and the circadian clock in metabolic disorders.

Plasma PAI-1 levels robustly fluctuate in a circadian manner and consequently contribute to hypofibrinolysis during the early morning. The circadian expression of PAI-1 gene is thought to be directly regulated by the circadian clock proteins such as CLOCK and BMAL1/BMAL2 which drive the endogenous biological clock. Plasma PAI-1 levels are increased in the beginning of the active phase in both diurnal humans and in nocturnal rodents, suggesting that the rhythmic PAI-1 expression is commonly indispensable for organisms. A series of our recent studies revealed that circadian clock proteins are important for hypofibrinolysis induced by metabolic disorders such as obesity and diabetes.