Melatonin Signaling Modulates Clock Genes Expression in the Mouse Retina

Previous studies have shown that retinal melatonin plays an important role in the regulation of retinal daily and circadian rhythms. Melatonin exerts its influence by binding to G-protein coupled receptors named melatonin receptor type 1 and type 2 and both receptors are present in the mouse retina. Earlier studies have shown that clock genes are rhythmically expressed in the mouse retina and melatonin signaling may be implicated in the modulation of clock gene expression in this tissue. In this study we determined the daily and circadian expression patterns of Per1, Per2, Bmal1, Dbp, Nampt and c-fos in the retina and in the photoreceptor layer (using laser capture microdissection) in C3H-f+/+ and in melatonin receptors of knockout (MT₁ and MT₂) of the same genetic background using real-time quantitative RT-PCR. Our data indicated that clock and clock-controlled genes are rhythmically expressed in the retina and in the photoreceptor layer. Removal of melatonin signaling significantly affected the pattern of expression in the retina whereas in the photoreceptor layer only the Bmal1 circadian pattern of expression was affected by melatonin signaling removal. In conclusion, our data further support the notion that melatonin signaling may be important for the regulation of clock gene expression in the inner or ganglion cells layer, but not in photoreceptors.     

Deregulated expression of circadian clock and clock-controlled cell cycle genes in chronic lymphocytic leukemia

Circadian rhythms are endogenous and self-sustained oscillations of multiple biological processes with approximately 24-h rhythmicity. Circadian genes and their protein products constitute the molecular components of the circadian oscillator that form positive/negative feedback loops and generate circadian rhythms. The circadian regulation extends from core clock genes to various clock-controlled genes that include various cell cycle genes. Aberrant expression of circadian clock genes, therefore, may lead to genomic instability and accelerated cellular proliferation potentially promoting carcinogenesis. The current study encompasses the investigation of simultaneous expression of four circadian clock genes (Bmal1, Clock, Per1 and Per2) and three clock-controlled cell cycle genes (Myc, Cyclin D1 and Wee1) at mRNA level and determination of serum melatonin levels in peripheral blood samples of 37 CLL (chronic lymphocytic leukemia) patients and equal number of age- and sex-matched healthy controls in order to indicate association between deregulated circadian clock and manifestation of CLL. Results showed significantly down-regulated expression of Bmal1, Per1, Per2 and Wee1 and significantly up-regulated expression of Myc and Cyclin D1 (P < 0.0001) in CLL patients as compared to healthy controls. When expression of these genes was compared between shift-workers and non-shift-workers within the CLL group, the expression was found more aberrant in shift-workers as compared to non-shift-workers. However, this difference was found statistically significant for Myc and Cyclin D1 only (P < 0.05). Serum melatonin levels were found significantly low (P < 0.0001) in CLL subjects as compared to healthy controls whereas melatonin levels were found still lower in shift-workers as compared to non-shift-workers within CLL group (P < 0.01). Our results suggest that aberrant expression of circadian clock genes can lead to aberrant expression of their downstream targets that are involved in cell proliferation and apoptosis and hence may result in manifestation of CLL. Moreover, shift-work and low melatonin levels may also contribute in etiology of CLL by further perturbing of circadian clock.     

Clock-controlled mir-142-3p can target its activator, Bmal1

ABSTRACT: BACKGROUND: microRNAs (miRNAs) are shown to be involved in the regulation of circadian clock. However, it remains largely unknown whether miRNAs can regulate the core clock genes (Clock and Bmal1). RESULTS: In this study, we found that mir-142-3p directly targeted the 3'UTR of human BMAL1 and mouse Bmal1. The over-expression (in 293ET and NIH3T3 cells) and knockdown (in U87MG cells) of mir-142-3p reduced and up-regulated the Bmal1/BMAL1 mRNA and protein levels, respectively. Moreover, the expression level of mir-142-3p oscillated in serum-shocked NIH3T3 cells and the results of ChIP and luciferase reporter assays suggested that the expression of mir-142-3p was directly controlled by CLOCK/BMAL1 heterodimers in NIH3T3 cells. CONCLUSIONS: Our study demonstrates that mir-142-3p can directly target the 3'UTR of Bmal1. In addition, the expression of mir-142-3p is controlled by CLOCK/BMAL1 heterodimers, suggesting a potential negative feedback loop consisting of the miRNAs and the core clock genes. These findings open new perspective for studying the molecular mechanism of circadian clock.     

Disrupting Circadian Homeostasis of Sympathetic Signaling Promotes Tumor Development in Mice

Background

Cell proliferation in all rapidly renewing mammalian tissues follows a circadian rhythm that is often disrupted in advanced-stage tumors. Epidemiologic studies have revealed a clear link between disruption of circadian rhythms and cancer development in humans. Mice lacking the circadian genes Period1 and 2 (Per) or Cryptochrome1 and 2 (Cry) are deficient in cell cycle regulation and Per2 mutant mice are cancer-prone. However, it remains unclear how circadian rhythm in cell proliferation is generated in vivo and why disruption of circadian rhythm may lead to tumorigenesis.

Methodology/Principal Findings

Mice lacking Per1 and 2, Cry1 and 2, or one copy of Bmal1, all show increased spontaneous and radiation-induced tumor development. The neoplastic growth of Per-mutant somatic cells is not controlled cell-autonomously but is dependent upon extracellular mitogenic signals. Among the circadian output pathways, the rhythmic sympathetic signaling plays a key role in the central-peripheral timing mechanism that simultaneously activates the cell cycle clock via AP1-controlled Myc induction and p53 via peripheral clock-controlled ATM activation. Jet-lag promptly desynchronizes the central clock-SNS-peripheral clock axis, abolishes the peripheral clock-dependent ATM activation, and activates myc oncogenic potential, leading to tumor development in the same organ systems in wild-type and circadian gene-mutant mice.

Conclusions/Significance

Tumor suppression in vivo is a clock-controlled physiological function. The central circadian clock paces extracellular mitogenic signals that drive peripheral clock-controlled expression of key cell cycle and tumor suppressor genes to generate a circadian rhythm in cell proliferation. Frequent disruption of circadian rhythm is an important tumor promoting factor.     

Timed high-fat diet in the evening affects the hepatic circadian clock and PPARα-mediated lipogenic gene expressions in mice

A long-term high-fat diet may result in a fatty liver. However, whether or not high-fat diets affect the hepatic circadian clock is controversial. The objective of this study is to investigate the effects of timed high-fat diet on the hepatic circadian clock and clock-controlled peroxisome proliferator-activated receptor (PPAR) α-mediated lipogenic gene expressions. Mice were orally administered high-fat milk in the evening for 4 weeks. The results showed that some hepatic clock genes, such as Clock, brain-muscle-Arnt-like 1 (Bmal1), Period 2 (Per2), and Cryptochrome 2 (Cry2) exhibited obvious changes in rhythms and/or amplitudes. Alterations in the expression of clock genes, in turn, further altered the circadian rhythm of PPARα expression. Among the PPARα target genes, cholesterol 7α-hydroxylase (CYP7A1), 3-hydroxy-3-methylglutaryl-coenzyme A reductase, low-density lipoprotein receptor, lipoprotein lipase, and diacylglycerol acyltransferase (DGAT) showed marked changes in rhythms and/or amplitudes. In particular, significant changes in the expressions of DGAT and CYP7A1 were observed. The effects of a high-fat diet on the expression of lipogenic genes in the liver were accompanied by increased hepatic cholesterol and triglyceride levels. These results suggest that timed high-fat diets at night could change the hepatic circadian expressions of clock genes Clock, Bmal1, Per2, and Cry2 and subsequently alter the circadian expression of PPARα-mediated lipogenic genes, resulting in hepatic lipid accumulation.     

The Molecular Clockwork of the Fire Ant Solenopsis invicta

The circadian clock is a core molecular mechanism that allows organisms to anticipate daily environmental changes and adapt the timing of behaviors to maximize efficiency. In social insects, the ability to maintain the appropriate temporal order is thought to improve colony efficiency and fitness. We used the newly sequenced fire ant (Solenopsis invicta) genome to characterize the first ant circadian clock. Our results reveal that the fire ant clock is similar to the clock of the honeybee, a social insect with an independent evolutionary origin of sociality. Gene trees for the eight core clock genes, period, cycle, clock, cryptochrome-m, timeout, vrille, par domain protein 1 & clockwork orange, show ant species grouping closely with honeybees and Nasonia wasps as an outgroup to the social Hymenoptera. Expression patterns for these genes suggest that the ant clock functions similar to the honeybee clock, with period and cry-m mRNA levels increasing during the night and cycle and clockwork orange mRNAs cycling approximately anti-phase to period. Gene models for five of these genes also parallel honeybee models. In particular, the single ant cryptochrome is an ortholog of the mammalian-type (cry-m), rather than Drosophila-like protein (cry-d). Additionally, we find a conserved VPIFAL C-tail region in clockwork orange shared by insects but absent in vertebrates. Overall, our characterization of the ant clock demonstrates that two social insect lineages, ants and bees, share a similar, mammalian-like circadian clock. This study represents the first characterization of clock genes in an ant and is a key step towards understanding socially-regulated plasticity in circadian rhythms by facilitating comparative studies on the organization of circadian clockwork.