Aline Gréchez-Cassiau

Effects of Chronic Jet Lag on Tumor Progression in Mice

Frequent transmeridian flights or predominant work at night can increase cancer risk. Altered circadian rhythms also predict for poor survival in cancer patients, whereas physical destruction of the suprachiasmatic nuclei (SCN), the hypothalamic circadian pacemaker, accelerates tumor growth in mice. Here we tested the effect of functional disruption of circadian system on tumor progression in a novel experimental model of chronic jet lag. B6D2F1 mice were synchronized with 12 hours of light and 12 hours of darkness or underwent repeat 8-hour advances of the light/dark cycle every 2 days before inoculation of Glasgow osteosarcoma. The 24-hour changes were assessed for plasma corticosterone, clock protein mPER1 expression in the SCN, and mRNA expression of clock genes mPer2 and mRev-erbα in liver and tumor. Time series were analyzed by spectral analysis and/or Cosinor. Differences were compared with analysis of variance (ANOVA). The 24-hour rest/activity cycle was ablated, and the rhythms of body temperature, serum corticosterone, and mPER1 protein expression in the SCN were markedly altered in jet-lagged mice as compared with controls (ANOVA, P < 0.001 for corticosterone and P = 0.01 for mPER1). Tumor grew faster in the jet-lagged animals as compared with controls (ANOVA, P < 0.001), whereas exposure to constant light or darkness had no effect (ANOVA, P = 0.66 and P = 0.8, respectively). The expression of mPer2 and mRev-erbα mRNAs in controls showed significant circadian rhythms in the liver (P = 0.006 and P = 0.003, respectively, Cosinor) and in the tumor (P = 0.04 and P < 0.001). Both rhythms were suppressed in the liver (P = 0.2 and P = 0.1, respectively, Cosinor) and in the tumor (P = 0.5) of jet-lagged mice. Altered environmental conditions can disrupt circadian clock molecular coordination in peripheral organs including tumors and play a significant role in malignant progression.

The circadian clock component BMAL1 is a critical regulator of p21WAF1/CIP1 expression and hepatocyte proliferation.

Most living organisms show circadian (∼24 h) rhythms in physiology and behavior. These oscillations are generated by endogenous circadian clocks, present in virtually all cells where they control key biological processes. Although circadian gating of mitosis has been reported for many years in some peripheral tissues, the underlying molecular mechanisms have remained poorly understood. Here we show that the cell cycle inhibitor p21WAF1/CIP1 is rhythmically expressed in mouse peripheral organs. This rhythmic pattern of mRNA and protein expression was recapitulated in vitro in serum-shocked differentiated skeletal muscle cells. p21WAF1/CIP1 circadian expression is dramatically increased and no longer rhythmic in clock-deficient Bmal1–/– knock-out mice. Biochemical and genetic data show that oscillation of p21WAF1/CIP1 gene transcription is regulated by the antagonistic activities of the orphan nuclear receptors REV-ERBα/β and RORα4/γ, which are core clock regulators. Importantly, p21WAF1/CIP1 overexpressing Bmal1–/– primary hepatocytes exhibit a decreased proliferation rate. This phenotype could be reversed using small interfering RNA-mediated knockdown of p21WAF1/CIP1. These data establish a novel molecular link between clock and cell cycle genes and suggest that the G1 progression phase is a target of the circadian clock during liver cell proliferation.

The nuclear receptor REV-ERBα is required for the daily balance of carbohydrate and lipid metabolism

Mutations of clock genes can lead to diabetes and obesity. REV‐ERBα, a nuclear receptor involved in the circadian clockwork, has been shown to control lipid metabolism. To gain insight into the role of REV‐ERBα in energy homeostasis in vivo, we explored daily metabolism of carbohydrates and lipids in chow‐fed, unfed, or high‐fat‐fed Rev‐erbα−/− mice and their wild‐type littermates. Chow‐fed Rev‐erbα−/− mice displayed increased adiposity (2.5‐fold) and mild hyperglycemia (∼10%) without insulin resistance. Indirect calorimetry indicates that chow‐fed Rev‐erbα−/− mice utilize more fatty acids during daytime. A 24‐h nonfeeding period in Rev‐erbα−/− animals favors further fatty acid mobilization at the expense of glycogen utilization and gluconeogenesis, without triggering hypoglycemia and hypothermia. High‐fat feeding in Rev‐erbα−/− mice amplified metabolic disturbances, including expression of lipogenic factors. Lipoprotein lipase (Lpl) gene, critical in lipid utilization/storage, is triggered in liver at night and constitutively up‐regulated (∼ 2‐fold) in muscle and adipose tissue of Rev‐erbα−/− mice. We show that CLOCK, up‐regulated (2‐fold) at night in Rev‐erbα−/− mice, can transactivate Lpl. Thus, overexpression of Lpl facilitates muscle fatty acid utilization and contributes to fat overload. This study demonstrates the importance of clock‐driven Lpl expression in energy balance and highlights circadian disruption as a potential cause for the metabolic syndrome.—Delezie, J., Dumont, S., Dardente, H., Oudart, H., Gréchez‐Cassiau, A., Klosen, P., Teboul, M., Delaunay, F., Pévet, P., Challet, E. The nuclear receptor REV‐ERBα is required for the daily balance of carbohydrate and lipid metabolism. FASEB J. 26, 3321–3335 (2012). www.fasebj.org

Minireview: The Nuclear Hormone Receptor Family Round the Clock

Daily rhythms in behavior and physiology are observed in most organisms. These rhythms are controlled by internal self-sustained circadian ( approximately 24 h) clocks, which are present in virtually all cells. The 24-h oscillations are generated by a molecular mechanism entrained by external or internal time cues and which, in turn, regulate rhythmic outputs. In mammals, the circadian system comprises a master clock located in the hypothalamus that is directly entrained by the light-dark cycle and which coordinates the phases of local clocks in the periphery in order to ensure optimal timing of the physiology. Nuclear receptors (NRs) form a large family of transcription factors that include both ligand-inducible and orphan receptors. These NRs are key regulators of major biological processes such as reproduction, development, cell growth and death, inflammation, immunity, and metabolic homeostasis. Recent observations indicate that several NR signaling pathways play a critical role in central and peripheral circadian clocks. The REV-ERB/retinoid-related orphan receptor orphan NR subfamily regulates the expression of core clock genes and contributes to the robustness of the clock mechanism. Glucocorticoid and retinoic acid receptors are involved in the resetting of peripheral clocks. Several other NRs such as peroxisome proliferator-activated receptor-alpha, short heterodimer partner, and constitutive androstane receptor act as molecular links between clock genes and specific rhythmic metabolic outputs. The expanding functional links between NRs and circadian clocks open novel perspectives for understanding the hormonal regulation of the mammalian circadian system as well as for exploring the role of circadian clocks in the pathogenesis of NR-related diseases such as cancer and metabolic syndrome.

Kruppel-like factor KLF10 is a link between the circadian clock and metabolism in liver.

ABSTRACT The circadian timing system coordinates many aspects of mammalian physiology and behavior in synchrony with the external light/dark cycle. These rhythms are driven by endogenous molecular clocks present in most body cells. Many clock outputs are transcriptional regulators, suggesting that clock genes primarily control physiology through indirect pathways. Here, we show that Krüppel-like factor 10 (KLF10) displays a robust circadian expression pattern in wild-type mouse liver but not in clock-deficient Bmal1 knockout mice. Consistently, the Klf10 promoter recruited the BMAL1 core clock protein and was transactivated by the CLOCK-BMAL1 heterodimer through a conserved E-box response element. Profiling the liver transcriptome from Klf10−/− mice identified 158 regulated genes with significant enrichment for transcripts involved in lipid and carbohydrate metabolism. Importantly, approximately 56% of these metabolic genes are clock controlled. Male Klf10−/− mice displayed postprandial and fasting hyperglycemia, a phenotype accompanied by a significant time-of-day-dependent upregulation of the gluconeogenic gene Pepck and increased hepatic glucose production. Consistently, functional data showed that the proximal Pepck promoter is repressed directly by KLF10. Klf10−/− females were normoglycemic but displayed higher plasma triglycerides. Correspondingly, rhythmic gene expression of components of the lipogenic pathway, including Srebp1c, Fas, and Elovl6, was altered in females. Collectively, these data establish KLF10 as a required circadian transcriptional regulator that links the molecular clock to energy metabolism in the liver.

How nuclear receptors tell time.

Most organisms adapt their behavior and physiology to the daily changes in their environment through internal ( approximately 24 h) circadian clocks. In mammals, this time-keeping system is organized hierarchically, with a master clock located in the suprachiasmatic nuclei of the hypothalamus that is reset by light, and that, in turn, coordinates the oscillation of local clocks found in all cells. Central and peripheral clocks control, in a highly tissue-specific manner, hundreds of target genes, resulting in the circadian regulation of most physiological processes. A great deal of knowledge has accumulated during the last decade regarding the molecular basis of mammalian circadian clocks. These studies have collectively demonstrated how a set of clock genes and their protein products interact together in complex feedback transcriptional/translational loops to generate 24-h oscillations at the molecular, cellular, and organism levels. In recent years, a number of nuclear receptors (NRs) have been implicated as important regulators of the mammalian clock mechanism. REV-ERB and retinoid-related orphan receptor NRs regulate directly the core feedback loop and increase its robustness. The glucocorticoid receptor mediates the synchronizing effect of glucocorticoid hormones on peripheral clocks. Other NR family members, including the orphan NR EAR2, peroxisome proliferator activated receptors-alpha/gamma, estrogen receptor-alpha, and retinoic acid receptors, are also linked to the clockwork mechanism. These findings together establish nuclear hormone receptor signaling as an integral part of the circadian timing system.

Hydroxyindole-O-methyltransferase in the Chicken Retina: Immunocytochemical- Localization and Daily Rhythm of mRNA

In the vertebrate retina and pineal gland, melatonin production displays diurnal variations with high levels at night. Hydroxyindole‐O‐methyltransferase (HIOMT, EC 2.1.1.4) catalyses the last step of melatonin biosynthesis. In the present study, a cDNA encoding chicken HIOMT was used to examine the effects of environmental lighting on HIOMT mRNA expression in the chicken retina. A day/night rhythm of HIOMT mRNA level was observed, with an average 5‐fold increase during the night. Light strongly suppressed the night‐time rise in HIOMT mRNA concentration while darkness prevented its daytime fall. An antibody directed against chicken HIOMT was used for immunocytochemical identification of retinal melatoninergic cells. HIOMT immunoreactivity could be observed in rods as well as in cones. However, the lowest levels of HIOMT immunoreactivity were always observed in the accessory cones of double cones. A few HIOMT‐positive cell bodies could also be observed in the inner nuclear layer. Altogether, these data indicate that HIOMT gene expression in the retina is organized on a daily basis as a direct response to light, and that the different types of photoreceptors may not be equally involved in melatonin production.

Altered Stra13 and Dec2 circadian gene expression in hypoxic cells.

The circadian system regulates rhythmically most of the mammalian physiology in synchrony with the environmental light/dark cycle. Alteration of circadian clock gene expression has been associated with tumour progression but the molecular links between the two mechanisms remain poorly defined. Here we show that Stra13 and Dec2, two circadian transcriptional regulators which play a crucial role in cell proliferation and apoptosis are overexpressed and no longer rhythmic in serum shocked fibroblasts treated with CoCl(2,) a substitute of hypoxia. This effect is associated with a loss of circadian expression of the clock genes Rev-erbalpha and Bmal1, and the clock-controlled gene Dbp. Consistently, cotransfection assays demonstrate that STRA13 and DEC2 both antagonize CLOCK:BMAL1 dependent transactivation of the Rev-erbalpha and Dbp promoters. Using a transplantable osteosarcoma tumour model, we show that hypoxia is associated with altered circadian expression of Stra13, Dec2, Rev-erbalpha, Bmal1 and Dbp in vivo. These observations collectively support the notion that overexpression of Stra13 and Dec2 links hypoxia signalling to altered circadian clock gene expression.

Hydroxyindole-O-methyltransferase gene expression in the pineal gland of chicken embryo: development of messenger RNA levels and regulation by serum

Hydroxyindole-O-methyltransferase (HIOMT), the enzyme which catalyzes the final step of melatonin biosynthesis, constitutes a marker of the functional differentiation of pineal cells. In addition, a day/night rhythm of HIOMT mRNA concentration, previously described in the chicken pineal gland [6], would suggest that HIOMT gene transcription is one output of the circadian system that controls pineal function. The study sought to monitor the developmental expression of HIOMT mRNA in the chick pineal gland and to investigate a possible role of instructive signals in this differentiation process. RT-PCR analysis indicated that HIOMT mRNA is expressed at embryonic day 8 (E8). At E12, HIOMT mRNA became detectable on northern blots and traces of HIOMT activity could be measured. HIOMT mRNA concentration increased 100-fold between E14 and day 10 post-hatch, then levelled off. A day/night rhythm of HIOMT mRNA concentration was readily observed in the pineal gland of 2-day-old chicks. Pineal glands isolated on minimum culture medium at E11 stopped developing HIOMT gene expression. However, the addition of serum to the culture medium restored HIOMT mRNA concentration to the levels observed in vivo. The data suggest that the functional differentiation of melatoninergic cells observed during the second week of embryonic life may be controlled [correction of controled] by serum factors.

The hepatic circadian clock regulates the choline kinase α gene through the BMAL1-REV-ERBα axis

The circadian timing system adapts most of the mammalian physiology and behaviour to the 24 h light/dark cycle. This temporal coordination relies on endogenous circadian clocks present in virtually all tissues and organs and implicated in the regulation of key cellular processes including metabolism, transport and secretion. Environmental or genetic disruption of the circadian coordination causes metabolic imbalance leading for instance to fatty liver, dyslipidaemia and obesity, thereby contributing to the development of a metabolic syndrome state. In the liver, a key metabolic organ, the rhythmic regulation of lipid biosynthesis is known, yet the molecular mechanisms through which the circadian clock controls lipogenesis, in particular, that of phospholipids, is poorly characterised. In this study, we show that the wild-type mice display a rhythmic accumulation of hepatic phosphatidylcholine with a peak at ZT 22-0 while clock-deficient Bmal1-/- mice show elevated phosphatidylcholine levels in the liver associated with an atherogenic lipoprotein profile. Profiling of the mRNA expression of enzymes from the Kennedy and phosphatidylethanolamine N-methyltransferase pathways which control the production of hepatic phosphatidylcholine revealed a robust circadian pattern for Chkα while other mRNA showed low amplitude (Chkβ and Pemt) or no rhythm (Cctα and Chpt1). Chkα mRNA expression was increased and no longer rhythmic in the liver from clock-deficient Bmal1-/- mice. This change resulted in the upregulation of the CHKα protein in these animals. We further show that the robust circadian expression of Chkα is restricted to the liver and adrenal glands. Analysis of the Chkα gene promoter revealed the presence of a conserved response element for the core clock transcription factors REV-ERB and ROR. Consistent with the antiphasic phase relationship between Chkα and Rev-erbα expression, in cotransfection experiments using HepG2 cells we show that RORα4-dependent transactivation of this element is repressed by REV-ERBα· Correspondingly, Rev-erbα-/-mice displayed higher Chkα mRNA levels in liver at ZT 12. Collectively, these data establish that hepatic phosphatidylcholine is regulated by the circadian clock through a Bmal1-Rev-erbα-Chkα axis and suggest that an intact circadian timing system is important for the temporal coordination of phospholipid metabolism.