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1.
BMC Cancer ; 19(1): 101, 2019 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-30674294

RESUMO

Following publication of the original article [1], we have been notified that the tagging of one of the author names was done incorrectly in the XML version of the paper. The online and pdf versions of this paper are not affected by the change. Original and corrected tagging can be seen below. The original article has been corrected.

2.
FASEB J ; 32(6): 3085-3095, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29405095

RESUMO

Based on genetic models with mutation or deletion of core clock genes, circadian disruption has been implicated in the pathophysiology of metabolic disorders. Thus, we examined whether circadian desynchronization in response to shift work-type schedules is sufficient to compromise metabolic homeostasis and whether inflammatory mediators provide a key link in the mechanism by which alterations of circadian timekeeping contribute to diet-induced metabolic dysregulation. In high-fat diet (HFD)-fed mice, exposure to chronic shifts of the light-dark cycle (12 h advance every 5 d): 1) disrupts photoentrainment of circadian behavior and modulates the period of spleen and macrophage clock gene rhythms; 2) potentiates HFD-induced adipose tissue infiltration and activation of proinflammatory M1 macrophages; 3) amplifies macrophage proinflammatory cytokine expression in adipose tissue and bone marrow-derived macrophages; and 4) exacerbates diet-induced increases in body weight, insulin resistance, and glucose intolerance in the absence of changes in total daily food intake. Thus, complete disruption of circadian rhythmicity or clock gene function as transcription factors is not requisite to the link between circadian and metabolic phenotypes. These findings suggest that macrophage proinflammatory activation and inflammatory signaling are key processes in the physiologic cascade by which dysregulation of circadian rhythmicity exacerbates diet-induced systemic insulin resistance and glucose intolerance.-Kim, S.-M., Neuendorff, N., Alaniz, R. C., Sun, Y., Chapkin, R. S., Earnest, D. J. Shift work cycle-induced alterations of circadian rhythms potentiate the effects of high-fat diet on inflammation and metabolism.


Assuntos
Tecido Adiposo/metabolismo , Ritmo Circadiano/efeitos dos fármacos , Gorduras na Dieta/efeitos adversos , Macrófagos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tecido Adiposo/patologia , Animais , Citocinas/genética , Citocinas/metabolismo , Gorduras na Dieta/farmacologia , Feminino , Inflamação/induzido quimicamente , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Macrófagos/patologia , Masculino , Camundongos , Camundongos Transgênicos
3.
BMC Cancer ; 18(1): 43, 2018 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-29316898

RESUMO

BACKGROUND: The circadian clock is the basis for biological time keeping in eukaryotic organisms. The clock mechanism relies on biochemical signaling pathways to detect environmental stimuli and to regulate the expression of clock-controlled genes throughout the body. MAPK signaling pathways function in both circadian input and output pathways in mammals depending on the tissue; however, little is known about the role of p38 MAPK, an established tumor suppressor, in the mammalian circadian system. Increased expression and activity of p38 MAPK is correlated with poor prognosis in cancer, including glioblastoma multiforme; however, the toxicity of p38 MAPK inhibitors limits their clinical use. Here, we test if timed application of the specific p38 MAPK inhibitor VX-745 reduces glioma cell invasive properties in vitro. METHODS: The levels and rhythmic accumulation of active phosphorylated p38 MAPK in different cell lines were determined by western blots. Rhythmic luciferase activity from clock gene luciferase reporter cells lines was used to test the effect of p38 MAPK inhibition on clock properties as determined using the damped sine fit and Levenberg-Marquardt algorithm. Nonlinear regression and Akaike's information criteria were used to establish rhythmicity. Boyden chamber assays were used to measure glioma cell invasiveness following time-of-day-specific treatment with VX-745. Significant differences were established using t-tests. RESULTS: We demonstrate the activity of p38 MAPK cycles under control of the clock in mouse fibroblast and SCN cell lines. The levels of phosphorylated p38 MAPK were significantly reduced in clock-deficient cells, indicating that the circadian clock plays an important role in activation of this pathway. Inhibition of p38 MAPK activity with VX-745 led to cell-type-specific period changes in the molecular clock. In addition, phosphorylated p38 MAPK levels were rhythmic in HA glial cells, and high and arrhythmic in invasive IM3 glioma cells. We show that inhibition of p38 MAPK activity in IM3 cells at the time of day when the levels are normally low in HA cells under control of the circadian clock, significantly reduced IM3 invasiveness. CONCLUSIONS: Glioma treatment with p38 MAPK inhibitors may be more effective and less toxic if administered at the appropriate time of the day.


Assuntos
Proteínas CLOCK/genética , Relógios Circadianos/genética , Glioblastoma/tratamento farmacológico , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Animais , Linhagem da Célula/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Luciferases , Camundongos , Invasividade Neoplásica/genética , Fosforilação , Piridazinas/administração & dosagem , Pirimidinas/administração & dosagem , Transdução de Sinais/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genética
4.
J Biol Chem ; 289(23): 16374-88, 2014 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-24770415

RESUMO

The circadian clockworks gate macrophage inflammatory responses. Given the association between clock dysregulation and metabolic disorders, we conducted experiments to determine the extent to which over-nutrition modulates macrophage clock function and whether macrophage circadian dysregulation is a key factor linking over-nutrition to macrophage proinflammatory activation, adipose tissue inflammation, and systemic insulin resistance. Our results demonstrate that 1) macrophages from high fat diet-fed mice are marked by dysregulation of the molecular clockworks in conjunction with increased proinflammatory activation, 2) global disruption of the clock genes Period1 (Per1) and Per2 recapitulates this amplified macrophage proinflammatory activation, 3) adoptive transfer of Per1/2-disrupted bone marrow cells into wild-type mice potentiates high fat diet-induced adipose and liver tissue inflammation and systemic insulin resistance, and 4) Per1/2-disrupted macrophages similarly exacerbate inflammatory responses and decrease insulin sensitivity in co-cultured adipocytes in vitro. Furthermore, PPARγ levels are decreased in Per1/2-disrupted macrophages and PPARγ2 overexpression ameliorates Per1/2 disruption-associated macrophage proinflammatory activation, suggesting that this transcription factor may link the molecular clockworks to signaling pathways regulating macrophage polarization. Thus, macrophage circadian clock dysregulation is a key process in the physiological cascade by which diet-induced obesity triggers macrophage proinflammatory activation, adipose tissue inflammation, and insulin resistance.


Assuntos
Células da Medula Óssea/metabolismo , Dieta Hiperlipídica , Inflamação/metabolismo , Resistência à Insulina , Proteínas Circadianas Period/metabolismo , Adipócitos/metabolismo , Animais , Técnicas de Cocultura , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , PPAR gama/metabolismo
5.
Eur J Neurosci ; 33(8): 1533-40, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21366728

RESUMO

In the mammalian circadian system, cell-autonomous clocks in the suprachiasmatic nuclei (SCN) are distinguished from those in other brain regions and peripheral tissues by the capacity to generate coordinated rhythms and drive oscillations in other cells. To further establish in vitro models for distinguishing the functional properties of SCN and peripheral oscillators, we developed immortalized cell lines derived from fibroblasts and the SCN anlage of mPer2 (Luc) knockin mice. Circadian rhythms in luminescence driven by the mPER2::LUC fusion protein were observed in cultures of mPer2 (Luc) SCN cells and in serum-shocked or SCN2.2-co-cultured mPer2 (Luc) fibroblasts. SCN mPer2 (Luc) cells generated self-sustained circadian oscillations that persisted for at least four cycles with periodicities of ≈24 h. Immortalized fibroblasts only showed circadian rhythms of mPER2::LUC expression in response to serum shock or when co-cultured with SCN2.2 cells. Circadian oscillations of luminescence in mPer2 (Luc) fibroblasts decayed after 3-4 cycles in serum-shocked cultures but robustly persisted for 6-7 cycles in the presence of SCN2.2 cells. In the co-culture model, the circadian behavior of mPer2 (Luc) fibroblasts was dependent on the integrity of the molecular clockworks in co-cultured SCN cells as persistent rhythmicity was not observed in the presence of immortalized SCN cells derived from mice with targeted disruption of Per1 and Per2 (Per1(ldc) /Per2 (ldc) ). Because immortalized mPer2 (Luc) SCN cells and fibroblasts retain their indigenous circadian properties, these in vitro models will be valuable for real-time comparisons of clock gene rhythms in SCN and peripheral oscillators and identifying the diffusible signals that mediate the distinctive pacemaking function of the SCN.


Assuntos
Relógios Biológicos/fisiologia , Linhagem Celular , Ritmo Circadiano/fisiologia , Proteínas Circadianas Period/metabolismo , Núcleo Supraquiasmático/fisiologia , Animais , Arginina Vasopressina/genética , Arginina Vasopressina/metabolismo , Técnicas de Cocultura , Feminino , Fibroblastos/citologia , Fibroblastos/fisiologia , Técnicas de Introdução de Genes , Camundongos , Proteínas Circadianas Period/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Núcleo Supraquiasmático/citologia , Peptídeo Intestinal Vasoativo/genética , Peptídeo Intestinal Vasoativo/metabolismo
6.
Eur J Neurosci ; 30(5): 869-76, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19712092

RESUMO

The master circadian pacemaker located within the suprachiasmatic nucleus (SCN) of the mammalian brain controls system-level rhythms in animal physiology. Specific SCN outputs synchronize circadian physiological rhythms in other brain regions. Within the SCN, communication among neural cells provides for the coordination of autonomous cellular oscillations into ensemble rhythms. ATP is a neural transmitter involved in local communication among astrocytes and between astrocytes and neurons. Using a luciferin-luciferase chemiluminescence assay, we have demonstrated that ATP levels fluctuate rhythmically within both SCN2.2 cell cultures and the rat SCN in vivo. SCN2.2 cells generated circadian oscillations in both the production and extracellular accumulation of ATP. Circadian fluctuations in ATP accumulation persisted with an average period (tau) of 23.7 h in untreated as well as vehicle-treated and forskolin-treated SCN2.2 cells, indicating that treatment with an inductive stimulus is not necessary to propagate these rhythms. ATP levels in the rat SCN in vivo were marked by rhythmic variation during exposure to 12 h of light and 12 h of dark or constant darkness, with peak accumulation occurring during the latter half of the dark phase or subjective night. Primary cultures of cortical astrocytes similarly expressed circadian oscillations in extracellular ATP accumulation that persisted for multiple cycles with periods of about 23 h. These results suggest that circadian oscillations in extracellular ATP levels represent a physiological output of the mammalian cellular clock, common to the SCN pacemaker and astrocytes from at least some brain regions, and thus may provide a mechanism for clock control of gliotransmission between astrocytes and to neurons.


Assuntos
Trifosfato de Adenosina/metabolismo , Astrócitos/metabolismo , Ritmo Circadiano/fisiologia , Neurônios/metabolismo , Núcleo Supraquiasmático/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Medições Luminescentes , Microdiálise , Rede Nervosa/metabolismo , Fotoperíodo , Ratos , Ratos Sprague-Dawley , Núcleo Supraquiasmático/citologia , Transmissão Sináptica , Fatores de Tempo
7.
Sci Rep ; 9(1): 8909, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31222133

RESUMO

Proinflammatory signaling cascades have been implicated in the mechanism by which high fat diet (HFD) and saturated fatty acids (SFA) modulate fundamental circadian properties of peripheral clocks. Because the cytokines TNFα and IL-6 are key signals in HFD- and SFA-induced proinflammatory responses that ultimately lead to systemic insulin resistance, the present study examined the roles of these cytokines in the feedback modulation of peripheral circadian clocks by the proinflammatory SFA, palmitate. IL-6 and TNFα secretion in Bmal1-dLuc fibroblast cultures was increased during palmitate treatment although the time course and amplitude of the inductive response differed between these cytokines. Similar to the time-dependent phase shifts observed in response to palmitate, treatment with IL-6 or with the low dose (0.1 ng/ml) of TNFα at hour 12 (i.e., after forskolin synchronization) induced phase advances of fibroblast Bmal1-dLuc rhythms. In complementary experiments, treatment with neutralizing antibodies against these proinflammatory cytokines or their receptors to inhibit of IL-6- or TNFα-mediated signaling repressed palmitate-induced phase shifts of the fibroblast clock. These studies suggest that TNFα, IL-6 and other proinflammatory cytokines may mediate the feedback modulation of peripheral circadian clocks by SFA-induced inflammatory signaling.


Assuntos
Relógios Circadianos/genética , Citocinas/fisiologia , Ácidos Graxos/farmacologia , Mediadores da Inflamação/fisiologia , Anticorpos Neutralizantes/imunologia , Ritmo Circadiano/efeitos dos fármacos , Citocinas/imunologia , Humanos , Mediadores da Inflamação/imunologia , Palmitatos/farmacologia , Proteínas Recombinantes/farmacologia , Transdução de Sinais/efeitos dos fármacos
8.
Alcohol Clin Exp Res ; 32(3): 544-52, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18215209

RESUMO

BACKGROUND: In rats, alcohol exposure during the period of rapid brain growth produces long-term changes in the free-running period, photoentrainment and phase-shifting responses of the circadian rhythm in wheel-running behavior. To determine whether these alterations in circadian behavior are associated with permanent damage to the circadian timekeeping mechanism or reconfiguration of its molecular components, we examined the long-term effects of neonatal alcohol exposure on clock gene rhythms in the pacemaker located in the suprachiasmatic nucleus (SCN) and in other brain or peripheral tissues of adult rats. METHODS: Artificially reared male rat pups were exposed to alcohol (4.5 g/kg/d) or isocaloric milk formula (gastrostomy control; GC) on postnatal days 4 to 9. At 3 months of age, animals were exposed to constant darkness and then SCN, cerebellum, and liver tissue were harvested at 6-hour intervals for subsequent analysis of Period1 (Per1), Per2, Cryptochrome1 (Cry1), Bmal1, and Rev-erbalpha mRNA levels by quantitative PCR. RESULTS: In the SCN, cerebellum and liver, Per1, Per2, Cry1, Bmal1, and Rev-erbalpha expression oscillated with a similar amplitude (peak-to-trough differences of 2- to 9-fold) and phase in the suckle control (SC) and GC groups. These clock gene rhythms in control animals were marked by peak expression of Per1, Per2, Cry1, and Rev-erbalpha during the subjective day and of Bmal1 during the subjective night. The EtOH group was distinguished by altered rhythms in the expression of specific clock genes within the SCN, cerebellum and liver. In EtOH-treated rats, the SCN rhythm in Cry1 expression was strongly damped and the Per2 rhythms in the cerebellum and liver were phase-advanced such that peak expression occurred during the mid-subjective day. CONCLUSIONS: These results demonstrate alcohol exposure during the brain growth spurt alters the circadian regulation of some molecular components of the clock mechanism in the rat SCN, cerebellum, and liver. The observed alterations in the temporal configuration of essential "gears" of the molecular clockworks may play a role in the long-term effects of neonatal alcohol exposure on the regulation of circadian behavior.


Assuntos
Relógios Biológicos/efeitos dos fármacos , Cerebelo/efeitos dos fármacos , Etanol/administração & dosagem , Fígado/efeitos dos fármacos , Núcleo Supraquiasmático/efeitos dos fármacos , Transativadores/genética , Fatores Etários , Animais , Animais Recém-Nascidos , Relógios Biológicos/genética , Proteínas CLOCK , Cerebelo/fisiologia , Ritmo Circadiano/efeitos dos fármacos , Ritmo Circadiano/genética , Fígado/fisiologia , Masculino , Ratos , Ratos Sprague-Dawley , Núcleo Supraquiasmático/fisiologia , Transativadores/fisiologia
9.
Physiol Genomics ; 29(3): 280-9, 2007 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-17284666

RESUMO

To screen for output signals that may distinguish the pacemaker in the mammalian suprachiasmatic nucleus (SCN) from peripheral-type oscillators in which the canonical clockworks are similarly regulated in a circadian manner, the rhythmic behavior of the transcriptome in forskolin-stimulated NIH/3T3 fibroblasts was analyzed and compared relative to SCN2.2 cells in vitro and the rat SCN. Similar to the circadian profiling of the SCN2.2 and rat SCN transcriptomes, NIH/3T3 fibroblasts exhibited circadian fluctuations in the expression of the core clock genes, Per2, Cry1, and Bmal1, and 323 functionally diverse transcripts, many of which regulate cellular communication. Overlap in rhythmic transcripts among NIH/3T3 fibroblasts, SCN2.2 cells, and the rat SCN was limited to these clock genes and four other genes that mediate fatty acid and lipid metabolism or function as nuclear factors. Compared with NIH/3T3 cells, circadian gene expression in SCN oscillators was more prevalent among genes mediating glucose metabolism and neurotransmission. Coupled with evidence for the rhythmic regulation of the inducible isoform of nitric oxide synthase (iNos) in SCN2.2 cells and the rat SCN but not in fibroblasts, studies examining the effects of a NOS inhibitor on metabolic rhythms in cocultures containing SCN2.2 cells and untreated NIH/3T3 cells suggest that the gaseous neurotransmitter nitric oxide may play a key role in SCN pacemaker function. This comparative analysis of circadian gene expression in SCN and non-SCN cells may have important implications in the selective analysis of circadian signals involved in the coupling of SCN oscillators and regulation of rhythmicity in downstream cells.


Assuntos
Ritmo Circadiano/genética , Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Células NIH 3T3 , RNA Mensageiro/análise , Núcleo Supraquiasmático/metabolismo , Animais , Células Cultivadas , Análise por Conglomerados , Fibroblastos/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Ratos
10.
EBioMedicine ; 7: 100-11, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-27322464

RESUMO

Inflammatory signaling may play a role in high-fat diet (HFD)-related circadian clock disturbances that contribute to systemic metabolic dysregulation. Therefore, palmitate, the prevalent proinflammatory saturated fatty acid (SFA) in HFD and the anti-inflammatory, poly-unsaturated fatty acid (PUFA), docosahexaenoic acid (DHA), were analyzed for effects on circadian timekeeping and inflammatory responses in peripheral clocks. Prolonged palmitate, but not DHA, exposure increased the period of fibroblast Bmal1-dLuc rhythms. Acute palmitate treatment produced phase shifts of the Bmal1-dLuc rhythm that were larger in amplitude as compared to DHA. These phase-shifting effects were time-dependent and contemporaneous with rhythmic changes in palmitate-induced inflammatory responses. Fibroblast and differentiated adipocyte clocks exhibited cell-specific differences in the time-dependent nature of palmitate-induced shifts and inflammation. DHA and other inhibitors of inflammatory signaling (AICAR, cardamonin) repressed palmitate-induced proinflammatory responses and phase shifts of the fibroblast clock, suggesting that SFA-mediated inflammatory signaling may feed back to modulate circadian timekeeping in peripheral clocks.


Assuntos
Relógios Circadianos/efeitos dos fármacos , Ácidos Graxos Insaturados/farmacologia , Ácidos Graxos/farmacologia , Interleucina-6/genética , NF-kappa B/metabolismo , Adipócitos/classificação , Adipócitos/efeitos dos fármacos , Adipócitos/imunologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Ritmo Circadiano/efeitos dos fármacos , Ácidos Docosa-Hexaenoicos/farmacologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Ácido Palmítico/farmacologia , Fosforilação , Transdução de Sinais/efeitos dos fármacos
11.
Endocrinology ; 157(7): 2836-43, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27254002

RESUMO

Circadian clock desynchronization has been implicated in the pathophysiology of cardiovascular disease and related risk factors (eg, obesity, diabetes). Thus, we examined the extent to which circadian desynchronization exacerbates ischemic stroke outcomes and whether its detrimental effects on stroke severity and functional impairments are further modified by biological sex. Circadian entrainment of activity rhythms in all male and female rats was observed during exposure to a fixed light-dark (LD) 12:12 cycle but was severely disrupted when this LD cycle was routinely shifted (12 h advance/5 d) for approximately 7 weeks. In contrast to the regular estrous cycles in fixed LD animals, cyclicity was abolished and persistent estrus was evident in all shifted LD females. The disruption of estrous cyclicity in shifted LD females was associated with a significant increase in serum estradiol levels relative to that observed in fixed LD controls. Circadian rhythm disruption exacerbated stroke outcomes in both shifted LD male and female rats and further amplified sex differences in stroke impairments. In males, but not females, circadian disruption after exposure to the shifted LD cycle was marked by high rates of mortality. In surviving females, circadian desynchronization after exposure to shifted LD cycles produced significant increases in stroke-induced infarct volume and sensorimotor deficits with corresponding decreases in serum IGF-1 levels. These results suggest that circadian rhythm disruption associated with shift work schedules or the irregular nature of our everyday work and/or social environments may interact with other nonmodifiable risk factors such as biological sex to modulate the pathological effects of stroke.


Assuntos
Isquemia Encefálica/patologia , Ritmo Circadiano/fisiologia , Fotoperíodo , Caracteres Sexuais , Acidente Vascular Cerebral/patologia , Animais , Isquemia Encefálica/fisiopatologia , Modelos Animais de Doenças , Ciclo Estral/fisiologia , Feminino , Masculino , Atividade Motora/fisiologia , Ratos , Meio Social , Acidente Vascular Cerebral/fisiopatologia
12.
FEBS Lett ; 588(17): 3015-22, 2014 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-24928439

RESUMO

Based on their extracellular expression and targeting of the clock gene Bmal1, miR-142-3p and miR-494 were analyzed for evidence of vesicle-mediated communication between cells and intracellular functional activity. Our studies demonstrate that: miR-142-3p+miR-494 overexpression decreases endogenous BMAL1 levels, increases the period of Per2 oscillations, and increases extracellular miR-142-3p/miR-494 abundance in conditioned medium; miRNA-enriched medium increases intracellular expression of miR-142-3p and represses Bmal1 3'-UTR activity in naïve cells; and inhibitors of vesicular trafficking modulate intercellular communication of these miRNAs and ensemble Per2 rhythms. Thus, miR-142-3p and miR-494 may function as cis- and trans-acting signals contributing to local temporal coordination of cell-autonomous circadian clocks.


Assuntos
Relógios Circadianos/genética , MicroRNAs/genética , Fatores de Transcrição ARNTL/genética , Animais , Comunicação Celular , Endocitose , Exocitose , Espaço Extracelular/metabolismo , Regulação da Expressão Gênica/genética , Células HEK293 , Humanos , Camundongos , MicroRNAs/metabolismo , Células NIH 3T3
13.
PLoS One ; 8(6): e65300, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23755214

RESUMO

MicroRNAs (miRNAs) are small non-coding RNAs that function as post-transcriptional modulators by regulating stability or translation of target mRNAs. Recent studies have implicated miRNAs in the regulation of mammalian circadian rhythms. To explore the role of miRNAs in the post-transcriptional modulation of core clock genes in the master circadian pacemaker, we examined miR-142-3p for evidence of circadian expression in the suprachiasmatic nuclei (SCN), regulation of its putative clock gene target Bmal1 via specific binding sites in the 3' UTR and overexpression-induced changes in the circadian rhythm of BMAL1 protein levels in SCN cells. In mice exposed to constant darkness (DD), miR-142-3p levels in the SCN were characterized by circadian rhythmicity with peak expression during early subjective day at CT 3. Mutagenesis studies indicate that two independent miRNA recognition elements located at nucleotides 1-7 and 335-357 contribute equally to miR-142-3p-induced repression of luciferase-reported Bmal1 3' UTR activity. Importantly, overexpression of miR-142-3p in immortalized SCN cells abolished circadian variation in endogenous BMAL1 protein levels in vitro. Collectively, our results suggest that miR-142-3p may play a role in the post-transcriptional modulation of Bmal1 and its oscillatory regulation in molecular feedback loops mediating SCN circadian function.


Assuntos
Fatores de Transcrição ARNTL/genética , MicroRNAs/genética , Interferência de RNA , Núcleo Supraquiasmático/metabolismo , Regiões 3' não Traduzidas , Fatores de Transcrição ARNTL/metabolismo , Animais , Sítios de Ligação , Relógios Circadianos , Ritmo Circadiano , Elementos E-Box , Retroalimentação Fisiológica , Expressão Gênica , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismo , Neurônios/metabolismo , Neurônios/fisiologia , Regiões Promotoras Genéticas , Núcleo Supraquiasmático/fisiologia , Transcrição Gênica
14.
J Biol Rhythms ; 26(5): 412-22, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21921295

RESUMO

Even though peripheral circadian oscillators in the cardiovascular system are known to exist, the daily rhythms of the cardiovascular system are mainly attributed to autonomic or hormonal inputs under the control of the central oscillator, the suprachiasmatic nucleus (SCN). In order to examine the role of peripheral oscillators in the cardiovascular system, we used a transgenic mouse where the Clock gene is specifically disrupted in cardiomyocytes. In this cardiomyocyte-specific CLOCK mutant (CCM) mouse model, the circadian input from the SCN remains intact. Both CCM and wild-type (WT) littermates displayed circadian rhythms in wheel-running behavior. However, the overall wheel-running activities were significantly lower in CCM mice compared to WT over the course of 5 weeks, indicating that CCM mice either have lower baseline physical activities or they have lower physical adaptation abilities because daily wheel running, like routine exercise, induces physical adaptation over a period of time. Upon further biochemical analysis, it was revealed that the diurnal oscillations of phosphorylation states of several kinases and protein expression of the L-type voltage-gated calcium channel (L-VGCC) α1D subunit found in WT hearts were abolished in CCM hearts, indicating that in mammalian hearts, the daily oscillations of the activities of these kinases and L-VGCCs were downstream elements of the cardiac core oscillators. However, the phosphorylation of p38 MAPK exhibited robust diurnal rhythms in both WT and CCM hearts, indicating that cardiac p38 could be under the influence of the central clock through neurohormonal signals or be part of the circadian input pathway in cardiomyocytes. Taken together, these results indicate that the cardiac core oscillators have an impact in regulating circadian rhythmicities and cardiac function.


Assuntos
Proteínas CLOCK/genética , Canais de Cálcio Tipo L/genética , Ritmo Circadiano/fisiologia , Coração/fisiologia , Atividade Motora/fisiologia , Miócitos Cardíacos/metabolismo , Proteínas Circadianas Period/fisiologia , Fatores de Transcrição ARNTL/metabolismo , Animais , Comportamento Animal/fisiologia , Canais de Cálcio Tipo L/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Resistência Física/fisiologia , Transdução de Sinais/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
15.
Toxicol Lett ; 196(1): 28-32, 2010 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-20371273

RESUMO

Transcription factors expressing Per-Arnt-Sim (PAS) domains are key components of the mammalian circadian clockworks found in most cells and tissues. Because these transcription factors interact with other PAS genes mediating xenobiotic metabolism and because toxin responses are often marked by daily variation, we determined whether the toxin-mediated activation of the signaling pathway involving several PAS genes, the aryl hydrocarbon receptor (AhR) and AhR nuclear translocator (ARNT), fluctuates rhythmically and whether this diurnal oscillation is affected by targeted disruption of key PAS genes in the circadian clockworks, Period 1 (Per1) and Per2. Treatment with the prototypical Ahr ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), had inductive effects on a key target of AhR signaling, Cyp1A1, in both the mammary gland and liver of all animals. In wild type mice, the amplitude of this TCDD-induced Cyp1A1 expression in the mammary gland and liver was significantly greater (23-43-fold) during the night than during the daytime. However, the diurnal variation in the TCDD induction of mammary gland and liver Cyp1A1 expression was abolished in Per1(ldc), Per2(ldc) and Per1(ldc)/Per2(ldc) mutant mice, suggesting that Per1, Per2 and their timekeeping function in the circadian clockworks mediate the diurnal modulation of AhR-regulated responses to TCDD in the mammary gland and liver.


Assuntos
Ritmo Circadiano/genética , Citocromo P-450 CYP1A1/metabolismo , Dioxinas/toxicidade , Fígado/metabolismo , Glândulas Mamárias Animais/metabolismo , Proteínas Circadianas Period/fisiologia , Animais , Proteínas CLOCK , Ritmo Circadiano/efeitos dos fármacos , Feminino , Fígado/efeitos dos fármacos , Glândulas Mamárias Animais/efeitos dos fármacos , Camundongos , Mutação , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/metabolismo
16.
Alcohol ; 43(5): 387-96, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19671465

RESUMO

Neonatal alcohol exposure produces long-term changes in the suprachiasmatic nucleus (SCN) that are presumably responsible for disturbances in the light-dark regulation of circadian behavior in adult rats, including the pattern of photoentrainment, rate of re-entrainment to shifted light-dark cycles, and phase-shifting responses to light. Because SCN neurons containing vasoactive intestinal polypeptide (VIP) receive direct photic input via the retinohypothalamic tract and thus play an important role in the circadian regulation of the SCN clock mechanism by light, the present study examined the long-term effects of neonatal alcohol exposure on VIP neuronal populations within the SCN of adult rats. Male Sprague-Dawley rat pups were exposed to alcohol (EtOH; 3.0, 4.5, or 6.0 g/kg/day) or isocaloric milk formula (gastrostomy control; GC) on postnatal days 4-9 using artificial-rearing methods. At 2-3 months of age, animals from the suckle control (SC), GC, and EtOH groups were exposed to constant darkness (DD) and SCN tissue was harvested for subsequent analysis of either VIP mRNA expression by quantitative polymerase chain reaction (PCR) and in situ hybridization or of VIP-immunoreactive (ir) neurons using stereological methods. Neonatal alcohol exposure had no impact on VIP mRNA expression but dramatically altered immunostaining of neurons containing this peptide within the SCN of adult rats. The relative abundance of VIP mRNA and anatomical distribution of neurons expressing this transcript were similar among all control- and EtOH-treated groups. However, the total number and density of VIP-ir neurons within the SCN were significantly decreased by about 35% in rats exposed to alcohol at a dose of 6.0 g/kg/day relative to that observed in both control groups. These results demonstrate that VIP neuronal populations in the SCN are vulnerable to EtOH-induced insult during brain development. The observed alterations in SCN neurons containing VIP may have an impact upon clock responses to light input and thus contribute to the long-term effects of neonatal alcohol exposure on the photic regulation of circadian behavior.


Assuntos
Núcleo Supraquiasmático/efeitos dos fármacos , Peptídeo Intestinal Vasoativo/metabolismo , Animais , Animais Recém-Nascidos , Ritmo Circadiano/efeitos dos fármacos , Etanol/sangue , Etanol/farmacologia , Feminino , Masculino , Neurogênese/efeitos dos fármacos , Gravidez , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Núcleo Supraquiasmático/fisiologia
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