Quantitative Analyses of Circadian Gene Expression in Mammalian Cell Cultures

The central circadian pacemaker is located in the hypothalamus of mammals, but essentially the same oscillating system operates in peripheral tissues and even in immortalized cell lines. Using luciferase reporters that allow automated monitoring of circadian gene expression in mammalian fibroblasts, we report the collection and analysis of precise rhythmic data from these cells. We use these methods to analyze signaling pathways of peripheral tissues by studying the responses of Rat-1 fibroblasts to ten different compounds. To quantify these rhythms, which show significant variation and large non-stationarities (damping and baseline drifting), we developed a new fast Fourier transform–nonlinear least squares analysis procedure that specifically optimizes the quantification of amplitude for circadian rhythm data. This enhanced analysis method successfully distinguishes among the ten signaling compounds for their rhythm-inducing properties. We pursued detailed analyses of the responses to two of these compounds that induced the highest amplitude rhythms in fibroblasts, forskolin (an activator of adenylyl cyclase), and dexamethasone (an agonist of glucocorticoid receptors). Our quantitative analyses clearly indicate that the synchronization mechanisms by the cAMP and glucocorticoid pathways are different, implying that actions of different genes stimulated by these pathways lead to distinctive programs of circadian synchronization

Analysis of DNA-Damage Response to ionizing radiation in serum-shock synchronized human fibroblasts.

Many aspects of cellular physiology, including cellular response to genotoxic stress, are related to the circadian rhythmicity induced by the molecular clock. The current study investigated if the cellular response to DNA damage is in relation to endogenous expression levels of the PER2 protein, a key component of the molecular regulatory system that confers rhythmicity in mammalian cells. Human normal fibroblasts (CCD-34Lu) were subjected to serum shock to induce circadian oscillations of the PER2 protein and then irradiated with \u3b3- rays at times corresponding to the trough and peak expression of the PER2 protein. To better examine cellular response to DNA damage, the experiments performed in this study were carried out in non-proliferating CCD-34Lu fibroblasts in order to maintain the cell and circadian cycles separated while they were being exposed to genotoxic stress. Study results demonstrated that clonogenic cell survival, double-strand break repair kinetics, and TP53 protein levels were affected in the cells irradiated at the trough than in those irradiated at peak expression of the PER2 protein

EVOLUTION OF THE CIRCADIAN CLOCK IN EXTREME ENVIRONMENT: LESSONS FROM CAVEFISH.

Evolution has been strongly influenced by the daily cycles of temperature and light imposed by the rotation of the Earth. Fascinating demonstrations of this are seen in extreme environments such as caves where some animals have remained completely isolated from the day-night cycle for millions of years. Most of these species show convergent evolution, sharing a range of striking physical properties such as eye loss. One fundamental issue is whether “hypogean” species retain a functional circadian clock. This highly conserved, physiological timing mechanism allows organisms to anticipate daily environmental changes and is synchronized primarily by light. The Somalian cavefish, Phreatichthys andruzzii does possess a circadian clock that is entrained by a daily regular feeding time but strikingly, not by light. Under constant conditions the P. andruzzii clock oscillates with an extremely long period and also lacks normal temperature compensation. We document multiple mutations affecting a light-induced clock gene, Period2 as well as the genes encoding the extra-retinal photoreceptors Melanopsin (Opn4m2) and TMT-opsin. Remarkably, we show that ectopic expression of zebrafish homologs of these opsins rescues light induced clock gene expression in P. andruzzii cells. Thus, by studying this natural mutant we provide direct evidence for a peripheral light-sensing function of extra-retinal opsins in vertebrates. Furthermore, the properties of this cavefish illustrate that evolution in constant darkness leads not only to anatomical changes but also to loss of gene function linked with the detection and anticipation of the day-night cycle

Asynchronous Rhythm of Steroidogenic Factor 1 and Period Homolog 2 mRNA Expression in Mouse Y1 Adrenocorticol Tumor Cells

The relationship between the expression of Steroidogenic factor 1 (Sf1) and the circadian-related gene, period homolog 2 (Per2), in the adrenal cortex is still unknown. We show here that in Y1 adrenocortical tumor cells, expression of steroidogenic-related genes such as P450scc mRNA and Sf1 mRNA were asynchronous with Per2 mRNA. SF1 promoter analyses showed that the E-box element functions in a rhythmic pattern. Rhythmic expression of Upstream factor 1 mRNA, correlated well with Sf1 mRNA expression. We propose that tumorigenesis of adrenocortical lesions cause disruption of synchronous expression of steroidogenic-related and circadian-related genes

Proliferative glioblastoma cancer cells exhibit persisting temporal control of metabolism and display differential temporal drug susceptibility in chemotherapy

Even in immortalized cell lines, circadian clocks regulate physiological processes in a time-dependent manner, driving transcriptional and metabolic rhythms, the latter being able to persist without transcription. Circadian rhythm disruptions in modern life (shiftwork, jetlag, etc.) may lead to higher cancer risk. Here, we investigated whether the human glioblastoma T98G cells maintained quiescent or under proliferation keep a functional clock and whether cells display differential time responses to bortezomib chemotherapy. In arrested cultures, mRNAs for clock (Per1, Rev-erbα) and glycerophospholipid (GPL)-synthesizing enzyme genes, 32 P-GPL labeling, and enzyme activities exhibited circadian rhythmicity; oscillations were also found in the redox state/peroxiredoxin oxidation. In proliferating cells, rhythms of gene expression were lost or their periodicity shortened whereas the redox and GPL metabolisms continued to fluctuate with a similar periodicity as under arrest. Cell viability significantly changed over time after bortezomib treatment; however, this rhythmicity and the redox cycles were altered after Bmal1 knock-down, indicating cross-talk between the transcriptional and the metabolic oscillators. An intrinsic metabolic clock continues to function in proliferating cells, controlling diverse metabolisms and highlighting differential states of tumor suitability for more efficient, time-dependent chemotherapy when the redox state is high and GPL metabolism low.Fil: Wagner, Paula Micaela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: Sosa Alderete, Lucas Gastón. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Gorne, Lucas Damián. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto Multidisciplinario de Biología Vegetal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto Multidisciplinario de Biología Vegetal; ArgentinaFil: Gaveglio, Virginia Lucía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaFil: Salvador, Gabriela Alejandra. Universidad Nacional de Córdoba; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaFil: Pasquaré, Susana Juana. Universidad Nacional del Sur; ArgentinaFil: Guido, Mario Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentin

Microgravity influences circadian clock oscillation in human keratinocytes

Microgravity and sudden changes of gravitational forces exert numerous effects on tissues, organs and apparatus. Responses to these forces variably applied to cells indicate the existence of mechan- otransduction pathways able to modulate transcription. Oscillation of circadian clocks similarly influences many cellular and metabolic processes. Here we hypothesized that signals derived from changes of gravitational forces applied to epidermal cells might influence their physiology in har- mony with the oscillation of the molecular clock. In this study, we describe amplified oscillations of Bmal1 circadian clock gene in human keratinocytes exposed to short simulated microgravity and to rapid variation of gravitational forces. We found that exposure to microgravity enhances the amplitude of the Bmal1 feedback loop sustained by an apparently lower variability of Rev- erba transcription, while recovery from microgravity is characterized by increased amplitude of Bmal1 expression and elongation of the oscillatory periods of Bmal1 and Rev-erba. These data high- light the existence of integrated signaling network connecting mechanosensitive pathways to circa- dian gene regulation

Characterization of the Circadian Properties of Runt-related Transcription Factor 2 (Runx2) and its Role in the Suprachiasmatic Nucleus

Circadian rhythms orchestrate physiological, behavioral and cognitive processes in order to anticipate and adapt organisms to key environmental cues. These endogenously driven oscillations are generated by a network of interlocked auto-regulatory transcriptional-translational feedback loops driven forward by the Bmal1/Clock heterodimer transcription factor. Given the ubiquitous and dynamic quality of circadian rhythms, the identification of factors involved in the coordination and regulation of the endogenous oscillations is central in broadening our understanding of biological timing systems. In an examination of gene expression in the mammalian central circadian pacemaker, the hypothalamic suprachiasmatic nucleus (SCN), revealed a previously unreported rhythmic expression of runt-related transcription factor 2 (Runx2). The goal of the research described in the present dissertation was to determine the mechanistic basis of rhythmic Runx2 expression as well as determine whether Runx2 interacts with the core molecular machinery of the circadian clock. First, rhythmic gene expression Runx2 in the SCN, olfactory blub (OB) and paraventricular nucleus (PVN) of adult mice was demonstrated using quantitative real-time polymerase chain reaction and immunohistochemistry served to show rhythmic Runx2 protein expression in the SCN. Second, using SCN tissues from Bmal-/- mice or by transiently silencing BMAL1 expression in vitro it was shown that Runx2 expression is dependent on a functional core molecular clock network, namely the transcriptional activity of Bmal1. Chromatin immunoprecipitation was used to demonstrate that BMAL1 directly interacted with its putative binding sites within the RUNX2 promoter suggesting that the core clock machinery directly regulates its rhythmic gene expression. Next, Runx2 haplodeficient mice and transient silencing of RUNX2 expression in vitro revealed that decreases in Runx2 expression leads to a dampened amplitude of rhythmic Bmal1 expression and a lengthening of the period of molecular as well as running wheel behavioral rhythms. Finally, RUNX2 was found to interact with a putative binding site identified in the BMAL1 promoter suggesting that the influence of RUNX2 on the amplitude of BMAL1 expression was at least in part based on direct regulation at the gene level. Together, these findings serve to establish the foundational framework of the reciprocal relationship between the Runx2 transcription factor and the molecular network underlying circadian rhythms

The clock genes Period 2 and Cryptochrome 2 differentially balance bone formation

Background: Clock genes and their protein products regulate circadian rhythms in mammals but have also been implicated in various physiological processes, including bone formation. Osteoblasts build new mineralized bone whereas osteoclasts degrade it thereby balancing bone formation. To evaluate the contribution of clock components in this process, we investigated mice mutant in clock genes for a bone volume phenotype. Methodology/Principal Findings: We found that Per2Brdm1 mutant mice as well as mice lacking Cry2-/- displayed significantly increased bone volume at 12 weeks of age, when bone turnover is high. Per2Brdm1 mutant mice showed alterations in parameters specific for osteoblasts whereas mice lacking Cry2-/- displayed changes in osteoclast specific parameters. Interestingly, inactivation of both Per2 and Cry2 genes leads to normal bone volume as observed in wild type animals. Importantly, osteoclast parameters affected due to the lack of Cry2, remained at the level seen in the Cry2-/- mutants despite the simultaneous inactivation of Per2. Conclusions/Significance: This indicates that Cry2 and Per2 affect distinct pathways in the regulation of bone volume with Cry2 influencing mostly the osteoclastic cellular component of bone and Per2 acting on osteoblast parameters

A conserved circadian function for the Neurofibromatosis 1 gene

Summary: Loss of the Neurofibromatosis 1 (Nf1) protein, neurofibromin, in Drosophila disrupts circadian rhythms of locomotor activity without impairing central clock function, suggesting effects downstream of the clock. However, the relevant cellular mechanisms are not known. Leveraging the discovery of output circuits for locomotor rhythms, we dissected cellular actions of neurofibromin in recently identified substrates. Herein, we show that neurofibromin affects the levels and cycling of calcium in multiple circadian peptidergic neurons. A prominent site of action is the pars intercerebralis (PI), the fly equivalent of the hypothalamus, with cell-autonomous effects of Nf1 in PI cells that secrete DH44. Nf1 interacts genetically with peptide signaling to affect circadian behavior. We extended these studies to mammals to demonstrate that mouse astrocytes exhibit a 24-hr rhythm of calcium levels, which is also attenuated by lack of neurofibromin. These findings establish a conserved role for neurofibromin in intracellular signaling rhythms within the nervous system. : Bai et al. show that the gene mutated in the disease Neurofibromatosis 1 is required for maintaining levels or cycling of calcium in circadian neurons in Drosophila and in mammalian cells. These effects likely account for effects of Nf1 on circadian behavior in Drosophila and may be relevant in explaining sleep phenotypes in patients. Keywords: circadian rhythms, neurofibromatosis 1, Drosophila, peptide signaling, cycling of calcium, mouse astrocyte

ZZE-Configuration of chromophore ß-153 in C-phycocyanin from Mastigocladus laminosus

The photochemistry of C-phycocyanin has been studied after denaturation in the dark. It shows an irreversible reaction which has characteristics of a Ζ,Ζ,Ε- to Z,Z,Z-isomerization of dihydrobilins. Its amplitude depends on the reaction conditions, with a maximum corresponding to 15% conversion of one of the three PC chromophores. This chromophore is suggested to be ß-153, for which recent X-ray data T. Schirmer, W. Bode, and R. Huber, J. Mol. Biol., submitted, show ring D being highly twisted out of the plane of the other rings. During unfolding, there is thus a probability of falling into the photochemically labile Z,Z,^-configuration