Naoki Ohkura

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 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.

Up-regulation of the expression of leucine-rich α2-glycoprotein in hepatocytes by the mediators of acute-phase response

Abstract Leucine-rich α2-glycoprotein (LRG) is a plasma protein in which leucine-rich repeats (LRRs) were first discovered. Although the physiological function of LRG is not known, increases in the serum level of LRG have been reported in various diseases. In this study, we found that LRG was induced by recombinant human IL-6 in human hepatoma HepG2 cells. The induction of LRG by IL-6 was up-regulated synergistically with either IL-1β or TNFα in a pattern similar to those for type 1 acute-phase proteins. We also found that lipopolysaccharide (LPS) administered intraperitoneally to mice enhanced dose-dependently the expression of LRG mRNA in the liver as well as those for mouse major acute-phase proteins. These results strongly suggest that LRG was a secretory type 1 acute-phase protein whose expression was up-regulated by the mediator of acute-phase response.

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.

Circadian clock molecules CLOCK and CRYs modulate fibrinolytic activity by regulating the PAI-1 gene expression

Summary.  Disruptions of circadian rhythms are associated with the development of many disorders. However, whether a disruption of the circadian clock can cause anomalies of the hemostatic balance remains unknown. The present study examines coagulation and fibrinolytic activities in circadian clock mutants, a homozygous Clock mutant and Cry1/Cry2 double knockout (Cry1/2‐deficient) mice. The euglobulin clot lysis time (ELT) showed circadian variations that peaked at 21:00 (early night) in wild‐type mice, suggesting that fibrinolytic activity is lowest at this time. The ELT was continuously reduced in Clock mutants, while the ELT was significantly increased and did not differ between day and night (9:00 and 21:00) in Cry1/2‐deficient mice. The prothrombin time (PT) and activated partial prothrombin time (APTT) were constant in all genotypes. To identify which factors cause the loss of ELT rhythm, we measured fibrinolytic parameters in Clock mutant and Cry1/2‐deficient mice. The robust circadian fluctuation of plasma plasminogen activator inhibitor 1 (PAI‐1) that peaked at early night was damped to trough levels in Clock mutant mice. On the other hand, PAI‐1 levels in Cry1/2‐deficient mice remained equivalent to the peak levels of those in wild‐type mice at both 9:00 and 21:00. Circadian changes in plasma PAI‐1 levels seemed to be regulated at the level of gene expression, because the plasma PAI‐1 levels in Clock mutant and Cry1/2‐deficient mice were closely correlated with the level of PAI‐1 mRNA transcript in these mice. Plasma plasminogen and hepatic mRNA levels were not rhythmic in wild‐type mice, and continuously higher in Clock mutant than in wild‐type or Cry1/2‐deficient mice. In contrast, the activity and mRNA levels of tissue type plasminogen activator (t‐PA), plasma levels and mRNA levels of plasminogen, and plasma levels of α2 plasmin inhibitor (α2PI) in all genotypes were constant throughout the day. Coagulation parameters such as factor VII, factor X, prothrombin and fibrinogen remained constant throughout the day, and were not affected by clock gene mutations. These results suggest that circadian clock molecules play an important role in hemostatic balance by regulating the fibrinolytic systems.

Involvement of circadian clock gene Clock in diabetes-induced circadian augmentation of plasminogen activator inhibitor-1 (PAI-1) expression in the mouse heart.

Diabetes is associated with an excess risk of cardiac events, and one of the risk factors for infarction is the elevated‐levels of plasminogen activator inhibitor‐1 (PAI‐1). To evaluate how the molecular clock mechanism is involved in the diabetes‐induced circadian augmentation of PAI‐1 gene expression, we examined the expression profiles of PAI‐1 mRNA in the hearts of Clock mutant mice with streptozotocin‐induced diabetes. Circadian expression of PAI‐1 mRNA was blunted to low levels under both normal and diabetic conditions in Clock mutant mice, although the expression rhythm was augmented in diabetic wild‐type (WT) mice. Furthermore, plasma PAI‐1 levels became significantly higher in WT mice than in Clock mutant mice after STZ administration. Our results suggested that the circadian clock component, CLOCK, is involved in the diabetes‐induced circadian augmentation of PAI‐1 expression in the mouse heart.

PERIOD2 is a circadian negative regulator of PAI-1 gene expression in mice.

An increased level of obesity-induced plasma plasminogen activator inhibitor-1 (PAI-1) is considered a risk factor for cardiovascular disease. To determine whether the circadian clock component PERIOD2 (PER2) is involved in the regulation of PAI-1 gene expression, we performed transient transfection assays in vitro, and generated transgenic (Tg) mice overexpressing PER2. We then compared PAI-1 expression in Tg and wild-type (WT) mice with or without obesity induced by a high-fat/high-sucrose diet. PER2 suppressed CLOCK:BMAL1- and CLOCK:BMAL2-dependent transactivation of the PAI-1 promoter in vitro. Furthermore, nuclear translocation is dispensable for PER2 to suppress CLOCK:BMAL1-dependent transactivation of the PAI-1 promoter, because functional loss of the nuclear localization domain did not affect either the interaction with BMAL1 or the suppressive role of PER2. The diurnal expression of clock and clock-controlled genes was disrupted in a gene-specific manner, whereas that of PAI-1 mRNA was significantly damped in the hearts of PER2 Tg mice fed with a normal diet. Obesity-induced plasma PAI-1 increase was significantly suppressed in Tg mice in accordance with cardiac PAI-1 mRNA levels, whereas body weight gain and changes in metabolic parameters were identical between WT and Tg mice. Endogenous PAI-1 gene expression induced by transforming growth factor-beta1 was significantly attenuated in embryonic fibroblasts derived from Tg mice compared with those from WT mice. Our results demonstrated that PER2 represses PAI-1 gene transcription in a BMAL1/2-dependent manner. The present findings also suggest that PER2 attenuates obesity-induced hypofibrinolysis by downregulating PAI-1 expression independently of metabolic disorders.

Ketogenic Diet Disrupts the Circadian Clock and Increases Hypofibrinolytic Risk by Inducing Expression of Plasminogen Activator Inhibitor-1

Objectives—Metabolic disorders such as diabetes and obesity are considered risk factors for cardiovascular diseases by increasing levels of blood plasminogen activator inhibitor-1 (PAI-1). Ketogenic diets (KDs) have been used as an approach to weight loss in both obese and nonobese individuals. We examined circadian changes in plasma PAI-1 and its mRNA expression levels in tissues from mice fed with a KD (KD mice), to evaluate its effects on fibrinolytic functions. Methods and Results—Two weeks on the kDa increased plasma levels of free fatty acids and ketones accompanied by hypoglycemia in mice. Plasma PAI-1 concentrations were extremely elevated in accordance with mRNA expression levels in the heart and liver, but not in the kidneys of KD mice. Circadian expression of PAI-1 mRNA was phase-advanced for 4.7, 7.9, and 7.8 hours in the heart, kidney, and adipose tissues, respectively, as well as that of circadian genes mPer2 and DBP in KD mice, suggesting that peripheral clocks were phase-advanced by ketosis despite feeding ad libitum under a periodic light-dark cycle. The circadian clock that regulates behavioral activity rhythms was also phase-advanced, and its free-running period was significantly shortened in KD mice. Conclusions—Our findings suggest that ketogenic status increases hypofibrinolytic risk by inducing abnormal circadian expression of PAI-1.

Hypocoagulable State of Human Preovulatory Ovarian Follicular Fluid: Role of Sulfated Proteoglycan and Tissue Factor Pathway Inhibitor in the Fluid

Abstract Ovulation accompanied by tissue damage can cause an increase in the level of tissue factor (TF) in the follicular fluid, triggering the extrinsic coagulation pathway. However, follicular fluid must block fibrin formation and maintain fluidity until the release of the oocyte at ovulation. The combination of sulfated proteoglycan, antithrombin, and TF pathway inhibitor (TFPI) appears to play a critical role in the hypocoagulability of human follicular fluid. When compared with plasma, folicular fluid differs markedly in the levels of a number of important coagulation proteins. Principal among these are 15-fold, 13-fold, and 3.7-fold increases in free TFPI, thrombin-antithrombin complex, and TF, respectively. The excessively prolonged activated partial thromboplastin time (APTT) and prothrombin time (PT) of human ovarian follicular fluid appear to be primarily due to high concentrations of sulfated proteoglycans, which accelerate the inactivation of thrombin and the anti-Xa activity of TFPI. Thus, heparitinase treatment shortened the clotting times of follicular fluid and reduced the inhibition of thrombin by the proteoglycan fraction combined with a fraction containing antithrombin. The remaining prolongation of APTT and PT may be caused by high levels of free TFPI in follicular fluid, which were confirmed by Northern blotting analysis, demonstrating TFPI mRNA expression by granulosa cells.

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.