Contribution of FSH and triiodothyronine to the development of circadian clocks during granulosa cell maturation

The involvement of FSH and triiodothyronine (T(3)) in circadian clocks was investigated using immature granulosa cells of ovaries during the progress of cell maturation. Granulosa cells were prepared from preantral follicles of mouse Period2 (Per2)-dLuc reporter gene transgenic rats injected subcutaneously with the synthetic nonsteroidal estrogen diethylstilbestrol. Analysis of the cellular clock of the immature granulosa cells was performed partly using a serum-free culture system. Several bioluminescence oscillations of Per2-dLuc promoter activity were generated in the presence of FSH + fetal bovine serum, but not in the presence of either FSH or serum. As revealed by bioluminescence recording and analysis of clock gene expression, the granulosa cells lack the functional cellular clock at the immature stage, although Lhr was greatly expressed during the period of cell maturation. The granulosa cells gained a strong circadian rhythm of bioluminescence during stimulation with FSH, whereas LH reset the cellular clock of matured granulosa cells. During strong circadian rhythms of clock genes, the Star gene showed significant expression in matured granulosa cells. In contrast, T(3) showed an inhibitory effect on the development of the functional cellular clock during the period of cell maturation. These results indicate that FSH provides a cue for the development of the functional cellular clock of the immature granulosa cells, and T(3) blocks the development of the cellular clock.     

Intercellular coupling confers robustness against mutations in the SCN circadian clock network

Molecular mechanisms of the mammalian circadian clock have been studied primarily by genetic perturbation and behavioral analysis. Here, we used bioluminescence imaging to monitor Per2 gene expression in tissues and cells from clock mutant mice. We discovered that Per1 and Cry1 are required for sustained rhythms in peripheral tissues and cells, and in neurons dissociated from the suprachiasmatic nuclei (SCN). Per2 is also required for sustained rhythms, whereas Cry2 and Per3 deficiencies cause only period length defects. However, oscillator network interactions in the SCN can compensate for Per1 or Cry1 deficiency, preserving sustained rhythmicity in mutant SCN slices and behavior. Thus, behavior does not necessarily reflect cell-autonomous clock phenotypes. Our studies reveal previously unappreciated requirements for Per1, Per2, and Cry1 in sustaining cellular circadian rhythmicity and demonstrate that SCN intercellular coupling is essential not only to synchronize component cellular oscillators but also for robustness against genetic perturbations.     

Daily expression of clock genes in whole blood cells in healthy subjects and a patient with circadian rhythm sleep disorder

In recent years, circadian rhythm sleep disorders in humans have been increasing. Clinical features characteristic of this disorder are well known, but the specific causes remain unknown. However, various derangements of circadian expression of the clock gene are a probable cause of this disease. We have attempted to elucidate the relationship between the expression of the clock genes in whole blood cells and the clinical features characteristic of this disorder. In this study, we indicate the daily expression of clock genes period (Per) 1, 2, 3, Bmal1, and Clock in whole blood cells in 12 healthy male subjects. The peak phase of Per1, Per2, and Per3 appeared in the early morning, whereas that of Bmal1 and Clock appeared in the midnight hours. Furthermore, in one patient case with circadian rhythm sleep disorder, we observed variations of the peak phase in clock genes by treatments such as light therapy, exercise therapy, and medicinal therapy. This study suggested that the monitoring of human clock genes in whole blood cells, which may be functionally important for the molecular control of the circadian pacemaker as well as in suprachiasmatic nucleus, might be useful to evaluate internal synchronization.     

Circadian expression of clock genes in human peripheral leukocytes

In mammals, behavioral and physiological processes display 24-h rhythms that are regulated by a circadian system. In the present study, we investigated the possibility that the expression of clock genes in peripheral leukocytes can be used to assess the circadian clock system. We found that Per1 and Per2 exhibit circadian oscillations in mRNA expression in mouse peripheral leukocytes. Furthermore, the rhythms of Per1 and Per2 mRNA expression in peripheral leukocytes are severely blunted in homozygous Cry1/2 double-deficient mice that are known to have an abolished biological clock. We have examined the circadian expression of clock genes in human leukocytes and found that Per1 mRNA exhibits a robust circadian expression while Per2 and Bmal1 mRNA showed weak rhythm. These observations suggest that monitoring Per1 mRNA expression in human leukocytes may be useful for investigating the function of the circadian system in physiological and pathophysiological states.     

Refeeding after fasting elicits insulin-dependent regulation of Per2 and Rev-erbα with shifts in the liver clock

The mammalian circadian clock is known to be entrained by both a daily light-dark cycle and daily feeding cycle. However, the mechanisms of feeding-induced entrainment are not as fully understood as those of light entrainment. To elucidate the first step of entrainment of the liver clock, we identified the circadian clock gene(s) that show both phase advance and acute change of gene expression during the early term of the daytime refeeding schedule in mice. The expressions of liver Per2 and Rev-erbα genes were phase-advanced within 1 day of refeeding. Additionally, the upregulation of Per2 mRNA and down-regulation of Rev-erbα mRNA were induced within 2 hours, not only by food intake but also by insulin injection in intact mice. These expression changes by food intake were not revealed in streptozotocin-treated insulin-deficient mice, but insulin injection was able to recover the impairment of Per2 and Rev-erbα gene expression. Furthermore, we demonstrated using an ex vivo luciferase monitoring system that insulin injection during the daytime causes a phase advance of liver Per2 expression rhythm in Per2::luciferase knock-in mice. In embryonic fibroblasts from Per2::luciferase knock-in mice, insulin infusion caused an acute increase of Per2 gene expression and a similar phase advance of Per2 expression rhythm. Our results indicate that an acute change of Per2 and Rev-erbα gene expression mediated by refeeding-induced insulin secretion is a critical step mediating the early phase of feeding-induced entrainment of the liver clock.     

The Impact of HIF1α on the Per2 Circadian Rhythm in Renal Cancer Cell Lines

In mammals, the circadian rhythm central generator consists of interactions among clock genes, including Per1/2/3, Cry1/2, Bmal1, and Clock. Circadian rhythm disruption may lead to increased risk of cancer in humans, and deregulation of clock genes has been implicated in many types of cancers. Among these genes, Per2 is reported to have tumor suppressor properties, but little is known about the correlation between Per2 and HIF, which is the main target of renal cell carcinoma (RCC) therapy. In this study, the rhythmic expression of the Per2 gene was not detectable in renal cancer cell lines, with the exception of Caki-2 cells. In Caki-2 cells, HIF1α increased the amplitude of Per2 oscillation by directly binding to the HIF-binding site located on the Per2 promoter. These results indicate that HIF1α may enhance the amplitude of the Per2 circadian rhythm.     

Dissociation between circadian Per1 and neuronal and behavioral rhythms following a shifted environmental cycle

The suprachiasmatic nucleus (SCN) of the anterior hypothalamus contains a major circadian pacemaker that imposes or entrains rhythmicity on other structures by generating a circadian pattern in electrical activity. The identification of "clock genes" within the SCN and the ability to dynamically measure their rhythmicity by using transgenic animals open up new opportunities to study the relationship between molecular rhythmicity and other well-documented rhythms within the SCN. We investigated SCN circadian rhythms in Per1-luc bioluminescence, electrical activity in vitro and in vivo, as well as the behavioral activity of rats exposed to a 6-hr advance in the light-dark cycle followed by constant darkness. The data indicate large and persisting phase advances in Per1-luc bioluminescence rhythmicity, transient phase advances in SCN electrical activity in vitro, and an absence of phase advances in SCN behavioral or electrical activity measured in vivo. Surprisingly, the in vitro phase-advanced electrical rhythm returns to the phase measured in vivo when the SCN remains in situ. Our study indicates that hierarchical levels of organization within the circadian timing system influence SCN output and suggests a strong and unforeseen role of extra-SCN areas in regulating pacemaker function.     

Forskolin induces circadian gene expression of rPer1, rPer2 and dbp in mammalian rat-1 fibroblasts

Mammalian culture cells have the potential for periodicity, since high concentrations of serum can elicit the circadian expression of clock genes in rat-1 fibroblasts. However, the mechanism by which serum affects circadian gene expression remains unclear. In the present study, we incubated rat-1 cells with forskolin and successfully induced the rhythmic expression of Per1, Per2 and dbp. In the initial step of the circadian gene expression, a marked transient induction of Per1 was observed accompanied with CREB phosphorylation. Thus the present study strongly suggests that CREB activation through the cAMP/PKA pathway is involved in the generation of circadian rhythm in rat-1 cells     

Resveratrol regulates circadian clock genes in Rat-1 fibroblast cells

Circadian clocks, especially peripheral clocks, can be strongly entrained by daily feedings, but few papers have reported the effects of food components on circadian rhythm. The effects of resveratrol, a natural polyphenol, on circadian clocks of Rat-1 cells were analyzed. A dose of 100 muM resveratrol, which did not show cytotoxicity, regulated the expression of clock genes Per1, Per2, and Bmal1.     

The circadian gene per1 plays an important role in cell growth and DNA damage control in human cancer cells

The Per1 gene is a core clock factor that plays an essential role in generating circadian rhythms. Recent data reveal that major biological pathways, including those critical to cell division, are under circadian control. We report here that Per1 provides an important link between the circadian system and the cell cycle system. Overexpression of Per1 sensitized human cancer cells to DNA damage-induced apoptosis; in contrast, inhibition of Per1 in similarly treated cells blunted apoptosis. The apoptotic phenotype was associated with altered expression of key cell cycle regulators. In addition, Per1 interacted with the checkpoint proteins ATM and Chk2. Ectopic expression of Per1 in human cancer cell lines led to significant growth reduction. Finally, Per1 levels were reduced in human cancer patient samples. Our results highlight the importance of circadian regulation to fundamental cellular functions and support the hypothesis that disruption of core clock genes may lead to cancer development.     

Influence of the estrous cycle on clock gene expression in reproductive tissues: Effects of fluctuating ovarian steroid hormone levels

Circadian rhythms in physiology and behavior are known to be influenced by the estrous cycle in female rodents. The clock genes responsible for the generation of circadian oscillations are widely expressed both within the central nervous system and peripheral tissues, including those that comprise the reproductive system. To address whether the estrous cycle affects rhythms of clock gene expression in peripheral tissues, we first examined rhythms of clock gene expression (Per1, Per2, Bmal1) in reproductive (uterus, ovary) and non-reproductive (liver) tissues of cycling rats using quantitative real-time PCR (in vivo) and luminescent recording methods to measure circadian rhythms of PER2 expression in tissue explant cultures from cycling PER2::LUCIFERASE (PER2::LUC) knockin mice (ex vivo). We found significant estrous variations of clock gene expression in all three tissues in vivo, and in the uterus ex vivo. We also found that exogenous application of estrogen and progesterone altered rhythms of PER2::LUC expression in the uterus. In addition, we measured the effects of ovarian steroids on clock gene expression in a human breast cancer cell line (MCF-7 cells) as a model for endocrine cells that contain both the steroid hormone receptors and clock genes. We found that progesterone, but not estrogen, acutely up-regulated Per1, Per2, and Bmal1 expression in MCF-7 cells. Together, our findings demonstrate that the timing of the circadian clock in reproductive tissues is influenced by the estrous cycle and suggest that fluctuating steroid hormone levels may be responsible, in part, through direct effects on the timing of clock gene expression.