Proxisome proliferator-activated receptor-α mediates high-fat, diet-enhanced daily oscillation of plasminogen activator inhibitor-1 activity in mice

Acute thrombotic events frequently occur in the early morning among hyperlipidemic patients. The activity of plasminogen activator inhibitor-1 (PAI-1), a potent inhibitor of the fibrinolytic system, oscillates daily, and this is considered one mechanism that underlies the morning onset of acute thrombotic events in hyperlipidemia. Although several studies have reported the expression of the PAI-1 gene is under the control of the circadian clock system, the molecular mechanism of the circadian transactivation of PAI-1 gene under hyperlipidemic conditions remains to be elucidated. Here, the authors investigated whether hyperlipidemia induced by a high-fat diet (HFD) enhances the daily oscillation of plasma PAI-1 activity in mice. The mRNA levels of the PAI-1 gene were increased and rhythmically fluctuated with high-oscillation amplitude in the livers of wild-type mice fed with the HFD. Circadian expression of proxisome proliferator-activated receptor-α (PPARα) mRNA was also augmented as well as that of PAI-1. Chromatin immunoprecipitation showed the HFD-induced hyperlipidemia significantly increased the binding of PPARα to the PAI-1 promoter. Luciferase reporter analysis using primary hepatocytes revealed CLOCK/BMAL1-mediated PAI-1 promoter activity was synergistically enhanced by cotransfection with PPARα/retinoid X receptor-α (RXRα), and this synergistic transactivation was repressed by negative limbs of the circadian clock, PERIOD2 and CRYPTOCHROME1. As expected, HFD-induced PAI-1 mRNA expression was significantly attenuated in PPARα-null mice. These results suggest a molecular link between the circadian clock and lipid metabolism system in the regulation of PAI-1 gene expression, and provide an aid for understanding why hyperlipidemia increases the risk of acute thrombotic events in the morning.     

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.     

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.     

PROXISOME PROLIFERATOR-ACTIVATED RECEPTOR-α MEDIATES HIGH-FAT,DIET-ENHANCED DAILY OSCILLATION OF PLASMINOGEN ACTIVATOR INHIBITOR-1 ACTIVITY IN MICE

Acute thrombotic events frequently occur in the early morning among hyperlipidemic patients. The activity of plasminogen activator inhibitor-1 (PAI-1), a potent inhibitor of the fibrinolytic system, oscillates daily, and this is considered one mechanism that underlies the morning onset of acute thrombotic events in hyperlipidemia. Although several studies have reported the expression of the PAI-1 gene is under the control of the circadian clock system, the molecular mechanism of the circadian transactivation of PAI-1 gene under hyperlipidemic conditions remains to be elucidated. Here, the authors investigated whether hyperlipidemia induced by a high-fat diet (HFD) enhances the daily oscillation of plasma PAI-1 activity in mice. The mRNA levels of the PAI-1 gene were increased and rhythmically fluctuated with high-oscillation amplitude in the livers of wild-type mice fed with the HFD. Circadian expression of proxisome proliferator-activated receptor-α (PPARα) mRNA was also augmented as well as that of PAI-1. Chromatin immunoprecipitaion showed the HFD-induced hyperlipidemia significantly increased the binding of PPARα to the PAI-1 promoter. Luciferase reporter analysis using primary hepatocytes revealed CLOCK/BMAL1-mediated PAI-1 promoter activity was synergistically enhanced by cotransfection with PPARα/retinoid X receptor-α (RXRα), and this synergistic transactivation was repressed by negative limbs of the circadian clock, PERIOD2 and CRYPTOCHROME1. As expected, HFD-induced PAI-1 mRNA expression was significantly attenuated in PPARα-null mice. These results suggest a molecular link between the circadian clock and lipid metabolism system in the regulation of PAI-1 gene expression, and provide an aid for understanding why hyperlipidemia increases the risk of acute thrombotic events in the morning. (Author correspondence: ssoeda@fukuoka-u.ac.jp)     

The molecular mechanism regulating the autonomous circadian expression of Topoisomerase I in NIH3T3 cells

To identify whether Topoisomerase I (TopoI) has autonomous circadian rhythms regulated by clock genes, we tested mouse TopoI (mTopoI) promoter oscillation in NIH3T3 cells using a real-time monitoring assay and TopoI mRNA oscillations using real-time RT-PCR. Analysis of the mTopoI promoter region with Matlnspector software revealed two putative E-box (E1 and E2) and one DBP/E4BP4-binding element (D-box). Luciferase assays indicated that mTopoI gene expression was directly regulated by clock genes. The real-time monitoring assay showed that E-box and D-box response elements participate in the regulation of the circadian expression of mTopoI. Furthermore, a gel-shift assay showed that E2 is a direct target of the BMAL1/CLOCK heterodimer and DBP binds to the putative D-site. These results indicate that TopoI is expressed in an autonomous circadian rhythm in NIH3T3 cells.     

Circadian clock genes cause activation of the human PAI-1 gene promoter with 4G/5G allelic preference

Increased plasminogen activator inhibitor-1 (PAI-1) activity is associated with greater risk of myocardial infarction. PAI-1 expression is regulated by a 4G/5G promoter polymorphism. The 4G allele is associated with higher PAI-levels and greater circadian variation. Here we show that clock protein heterodimers BMAL/CLOCK cause greater activation (approximately 2-fold, P<0.05) of the 4G allele. Site-directed mutagenesis studies suggest that clock genes act on two canonical E-boxes to regulate PAI-1 promoter activity. These results identify a potential novel mechanism whereby allele-specific clock genes - mediated modulation of PAI-1 expression may contribute to circadian variation in cardiac risk.     

生物钟基因period的分子生物学

生物过程的昼夜节律是所有真核生物和部分原核生物的基本特征。自从ｐｅｒｉｏｄ基因被克隆以后,生物钟基因的研究已经取得长足的促进。本文回顾了ｐｅｒ基因及生物钟分子机制的研究历史,旨在为人类复杂行为的研究提供一条思路。     

Chronobiology of acute myocardial infarction molecular biology

The incidence of heart attacks increases in the early morning. This circadian oscillation is closely related to the function of the internal biological clock. In this article, we review the chronobiology of acute myocardial infarction (AMI) from the viewpoint of molecular biology.