Casein kinase 1-dependent phosphorylation of familial advanced sleep phase syndrome-associated residues controls PERIOD 2 stability

The mammalian circadian clock component PERIOD2 (PER2) plays a critical role in circadian rhythm entrainment. Recently, a missense mutation at a putative phosphorylation site in hPER2, Ser-662, was identified in patients that suffer from familial advanced sleep phase syndrome (FASPS). Patients with FASPS display abnormal sleep-wake patterns characterized by a lifelong pattern of sleep onset in the early evening and offset in the early morning. Although the phosphorylation of PER2 is strongly implied from functional studies, it has not been possible to study the site-specific phosphorylation of PER2 on Ser-662, and the biochemical functions of this residue are unclear. Here, we used phospho-specific antibodies to show that PER2 is phosphorylated on Ser-662 and flanking casein kinase (CK) sites in vivo. The phosphorylation of PER2 was carried out by the combined activities of casein kinase 1δ (CK1 δ) and casein kinase 1ε (CK1ε) and was antagonized by protein phosphatase 1. PER2 phosphorylation was rapidly induced in response to circadian entrainment of mammalian cell lines and occurred in both cytosolic and nuclear compartments. Importantly, we found that the pool of Ser-662-phosphorylated PER2 proteins was more stable than the pool of total PER2 molecules, implying that the FASPS phosphorylation cluster antagonizes PER2 degradation. Consistent with this idea, a Ser-662→Ala mutation that abrogated PER2 phosphorylation significantly reduced its half-life, whereas a phosphomimetic Ser-662→Asp substitution led to an elevation in half-life. Our combined findings provide new insights into PER2 regulation and the biochemical basis of FASPS.     

Pro-inflammatory cytokines IL-6 and CCL2 suppress expression of circadian gene Period2 in mammary epithelial cells

Chronic inflammation is known to contribute to tumor initiation and cancer progression. In breast tissue, the core circadian gene Period (PER)2 plays a critical role in mammary gland development and possesses tumor suppressor function. Interleukin (IL)-6 and C-C motif chemokine ligand (CCL) 2 are among the most abundant cytokines in the inflammatory microenvironment. We found that acute stimulation by IL-6/CCL2 reduced PER2 expression in non-tumorigenic breast epithelial cells. Longer term exposure to IL-6/CCL2 suppressed PER2 to an even lower level. IL-6 activated STAT3/NFκB p50 signaling to recruit HDAC1 to the PER2 promoter. CCL2 activated the PI3K/AKT pathway to promote ELK-1 cytoplasm-to-nucleus translocation, recruit HDAC1 to the proximal PER2 promoter and facilitate DNMT3-EZH2-PER2 promoter association. Ectopic expression of PER2 inhibited IL-6 or CCL2 induced mammosphere forming ability and reduced sphere size indicating that PER2 repression in breast epithelial cells can be crucial to activate tumorigenesis in an inflammatory microenvironment. The diminished expression of PER2 can be observed over a time scale of hours to weeks following IL-6/CCL2 stimulation suggesting that PER2 suppression occurs in the early stage of the interaction between an inflammatory microenvironment and normal breast epithelial cells. These data show new mechanisms by which mammary cells interact with a cancerous microenvironment and provide additional evidence that PER2 expression contributes to breast tumorigenesis.     

Control of mammalian circadian rhythm by CKIepsilon-regulated proteasome-mediated PER2 degradation

The mammalian circadian regulatory proteins PER1 and PER2 undergo a daily cycle of accumulation followed by phosphorylation and degradation. Although phosphorylation-regulated proteolysis of these inhibitors is postulated to be essential for the function of the clock, inhibition of this process has not yet been shown to alter mammalian circadian rhythm. We have developed a cell-based model of PER2 degradation. Murine PER2 (mPER2) hyperphosphorylation induced by the cell-permeable protein phosphatase inhibitor calyculin A is rapidly followed by ubiquitination and degradation by the 26S proteasome. Proteasome-mediated degradation is critically important in the circadian clock, as proteasome inhibitors cause a significant lengthening of the circadian period in Rat-1 cells. CKIepsilon (casein kinase Iepsilon) has been postulated to prime PER2 for degradation. Supporting this idea, CKIepsilon inhibition also causes a significant lengthening of circadian period in synchronized Rat-1 cells. CKIepsilon inhibition also slows the degradation of PER2 in cells. CKIepsilon-mediated phosphorylation of PER2 recruits the ubiquitin ligase adapter protein beta-TrCP to a specific site, and dominant negative beta-TrCP blocks phosphorylation-dependent degradation of mPER2. These results provide a biochemical mechanism and functional relevance for the observed phosphorylation-degradation cycle of mammalian PER2. Cell culture-based biochemical assays combined with measurement of cell-based rhythm complement genetic studies to elucidate basic mechanisms controlling the mammalian clock.     

Photoperiodic regulation of PER1 and PER2 protein expression in rat peripheral tissues

Circadian oscillations in biological variables in mammals are controlled by a central pacemaker in the suprachiasmatic nuclei (SCN) of the hypothalamus which coordinates circadian oscillators in peripheral tissues. The molecular clockwork responsible for this rhythmicity consists of several clock genes and their corresponding proteins that compose interactive feedback loops. In the SCN, two of the genes, Per1 and Per2, show circadian rhythmicity in their expression and protein production. This SCN rhythmicity is modified by the length of daylight, i.e. the photoperiod. The aim of the present study was to find out whether profiles of PER1 and PER2 proteins in peripheral organs are also affected by the photoperiod. Rats were maintained under a long photoperiod with 16 h of light and 8 h of darkness per day (LD 16:8) and under a short, LD 8:16, photoperiod. The PER1 and PER2 daily profiles were measured in peripheral organs by Western blotting. The photoperiod affected significantly the PER1 profile in livers and the PER2 profile in lungs and hearts. In lungs, PER2 in the cytoplasmic, but not in the nuclear fraction, was affected significantly. The effect of the photoperiod on PER1 profiles in peripheral organs appears to differ from that in the SCN.     

Glucocorticoids and Stress-Induced Changes in the Expression of PERIOD1 in the Rat Forebrain

The secretion of glucocorticoids in mammals is under circadian control, but glucocorticoids themselves are also implicated in modulating circadian clock gene expression. We have shown that the expression of the circadian clock protein PER1 in the forebrain is modulated by stress, and that this effect is associated with changes in plasma corticosterone levels, suggesting a possible role for glucocorticoids in the mediation of stress-induced changes in the expression of PER1 in the brain. To study this, we assessed the effects of adrenalectomy and of pretreatment with the glucocorticoid receptor antagonist, mifepristone, on the expression of PER1 in select limbic and hypothalamic regions following acute exposure to a neurogenic stressor, restraint, or a systemic stressor, 2-Deoxy-D-glucose (2DG) in rats. Acute restraint suppressed PER1 expression in the oval nucleus of the bed nucleus of the stria terminalis (BNSTov) and the central nucleus of the amygdala (CEAl), whereas 2DG increased PER1 in both regions. Both stressors increased PER1 expression in the paraventricular (PVN) and dorsomedial (DMH) nuclei of the hypothalamus, and the piriform cortex (Pi). Adrenalectomy and pretreatment with mifepristone reversed the effects of both stressors on PER1 expression in the BNSTov and CEAl, and blocked their effects in the DMH. In contrast, both treatments enhanced the effects of restraint and 2DG on PER1 levels in the PVN. Stress-induced PER1 expression in the Pi was unaffected by either treatment. PER1 expression in the suprachiasmatic nucleus, the master circadian clock, was not altered by either exposure to stress or by the glucocorticoid manipulations. Together, the results demonstrate a key role for glucocorticoid signaling in stress-induced changes in PER1 expression in the brain.     

Kinetics of doubletime kinase-dependent degradation of the Drosophila period protein

Robust circadian oscillations of the proteins PERIOD (PER) and TIMELESS (TIM) are hallmarks of a functional clock in the fruit fly Drosophila melanogaster. Early morning phosphorylation of PER by the kinase Doubletime (DBT) and subsequent PER turnover is an essential step in the functioning of the Drosophila circadian clock. Here using time-lapse fluorescence microscopy we study PER stability in the presence of DBT and its short, long, arrhythmic, and inactive mutants in S2 cells. We observe robust PER degradation in a DBT allele-specific manner. With the exception of doubletime-short (DBT(S)), all mutants produce differential PER degradation profiles that show direct correspondence with their respective Drosophila behavioral phenotypes. The kinetics of PER degradation with DBT(S) in cell culture resembles that with wild-type DBT and posits that, in flies DBT(S) likely does not modulate the clock by simply affecting PER degradation kinetics. For all the other tested DBT alleles, the study provides a simple model in which the changes in Drosophila behavioral rhythms can be explained solely by changes in the rate of PER degradation.     

生物钟基因PER2在结直肠癌中的表达及意义

目的:探讨PER2基因表达水平和结直肠癌发生发展的关系。方法:收集203例在上海市第一人民医院接受根治性肠切除术结直肠癌患者的标本,通过实时定量PCR和免疫组织化学技术检测PER2在肿瘤组织和邻近正常组织中的表达水平,并对PER2的表达与患者的病理资料和临床预后的相关性进行统计学分析。结果:PER2在结直肠患者肿瘤组织的表达较正常组织减少(P0.001)。与PER2阳性患者相比,PER2阴性的结直肠癌患者有远处转移(P=0.026)、AJCC分期为IV期(P=0.011)的比例更高。结论:PER2基因在结直肠癌患者中存在低表达现象,其对于患者的AJCC分期评价以及了解患者有无远处转移有一定的参考价值。     

Rhythmic expression of circadian clock genes in human leukocytes and beard hair follicle cells

Evaluating individual circadian rhythm traits is crucial for understanding the human biological clock system. The present study reports characterization of physiological and molecular parameters in 13 healthy male subjects under a constant routine condition, where interfering factors were kept to minimum. We measured hormonal secretion levels and examined temporal expression profiles of circadian clock genes in peripheral leukocytes and beard hair follicle cells. All 13 subjects had prominent daily rhythms in melatonin and cortisol secretion. Significant circadian rhythmicity was found for PER1 in 9 subjects, PER2 in 3 subjects, PER3 in all 13 subjects, and BMAL1 in 8 subjects in leukocytes. Additionally, significant circadian rhythmicity was found for PER1 in 5 of 8 subjects tested, PER2 in 2 subjects, PER3 in 6 subjects, and BMAL1 in 3 subjects in beard hair follicle cells. The phase of PER1 and PER3 rhythms in leukocytes correlated significantly with that of physiological rhythms. Our results demonstrate that leukocytes and beard hair follicle cells possess an endogenous circadian clock and suggest that PER1 and PER3 expression would be appropriate biomarkers and hair follicle cells could be a useful tissue source for the evaluation of biological clock traits in individuals.     

Involvement of stress kinase mitogen-activated protein kinase kinase 7 in regulation of mammalian circadian clock

The stress kinase mitogen-activated protein kinase kinase 7 (MKK7) is a specific activator of c-Jun N-terminal kinase (JNK), which controls various physiological processes, such as cell proliferation, apoptosis, differentiation, and migration. Here we show that genetic inactivation of MKK7 resulted in an extended period of oscillation in circadian gene expression in mouse embryonic fibroblasts. Exogenous expression in cultured mammalian cells of an MKK7-JNK fusion protein that functions as a constitutively active form of JNK induced phosphorylation of PER2, an essential circadian component. Furthermore, JNK interacted with PER2 at both the exogenous and endogenous levels, and MKK7-mediated JNK activation increased the half-life of PER2 protein by inhibiting its ubiquitination. Notably, the PER2 protein stabilization induced by MKK7-JNK fusion protein reduced the degradation of PER2 induced by casein kinase 1ε. Taken together, our results support a novel function for the stress kinase MKK7 as a regulator of the circadian clock in mammalian cells at steady state.     

Stress-Induced Changes in the Expression of the Clock Protein PERIOD1 in the Rat Limbic Forebrain and Hypothalamus: Role of Stress Type,Time of Day,and Predictability

Stressful events can disrupt circadian rhythms in mammals but mechanisms underlying this disruption remain largely unknown. One hypothesis is that stress alters circadian protein expression in the forebrain, leading to functional dysregulation of the brain circadian network and consequent disruption of circadian physiological and behavioral rhythms. Here we characterized the effects of several different stressors on the expression of the core clock protein, PER1 and the activity marker, FOS in select forebrain and hypothalamic nuclei in rats. We found that acute exposure to processive stressors, restraint and forced swim, elevated PER1 and FOS expression in the paraventricular and dorsomedial hypothalamic nuclei and piriform cortex but suppressed PER1 and FOS levels exclusively in the central nucleus of the amygdala (CEAl) and oval nucleus of the bed nucleus of the stria terminalis (BNSTov). Conversely, systemic stressors, interleukin-1β and 2-Deoxy-D-glucose, increased PER1 and FOS levels in all regions studied, including the CEAl and BNSTov. PER1 levels in the suprachiasmatic nucleus (SCN), the master pacemaker, were unaffected by any of the stress manipulations. The effect of stress on PER1 and FOS was modulated by time of day and, in the case of daily restraint, by predictability. These results demonstrate that the expression of PER1 in the forebrain is modulated by stress, consistent with the hypothesis that PER1 serves as a link between stress and the brain circadian network. Furthermore, the results show that the mechanisms that control PER1 and FOS expression in CEAl and BNSTov are uniquely sensitive to differences in the type of stressor. Finally, the finding that the effect of stress on PER1 parallels its effect on FOS supports the idea that Per1 functions as an immediate-early gene. Our observations point to a novel role for PER1 as a key player in the interface between stress and circadian rhythms.     

The acute and temporary modulation of PERIOD genes by hydrocortisone in healthy subjects

The physiological stress system and the circadian clock system communicate with each other at different signaling levels. The steroid hormone cortisol, the end-effector of the hypothalamus–pituitary–adrenal axis, is released in response to stress and acts as a mediator in circadian rhythms. We determined the effect of escalating cortisol doses on the expression of PERIOD genes (PER1, PER2 and PER3) in healthy subjects and analyzed whether the glucocorticoid receptor (GR) is involved in the cortisol-mediated PERIOD gene expression. Forty participants (50% males and 50% females) were randomly assigned to groups receiving a saline placebo solution or 3 mg, 6 mg, 12 mg and 24 mg of hydrocortisone. Blood was drawn every 15 min to measure quantitative gene expression of PER1, PER2 and PER3. A potential role of the GR was determined by an ex vivo study stimulating whole blood with hydrocortisone and RU486 (a GR antagonist). As a result, moderate doses of hydrocortisone produced an acute and temporary induction of PER1 and PER3 mRNA levels, whereas PER2 was not responsive to the hormone administration. The cortisol-dependent induction of PER1 was blocked by the GR antagonist in whole blood after treatment with hydrocortisone and RU486 ex vivo. In conclusion, acute pharmacological stress modulated the expression of PER1 and PER3 in whole blood temporarily in our short-term sampling design, suggesting that these circadian genes mediate stable molecular mechanisms in the periphery.     

The role of {beta}-TrCP1 and {beta}-TrCP2 in circadian rhythm generation by mediating degradation of clock protein PER2

The mammalian circadian clock proteins undergo a daily cycle of accumulation followed by phosphorylation and degradation. The mechanism by which clock proteins undergo degradation has not been fully understood. Circadian clock protein PERIOD2 (PER2) is shown to be the potential target of F-box protein beta-TrCP1, a component of ubiquitin E3 ligase. Here, we show that beta-TrCP2 as well as beta-TrCP1 target PER2 protein in vitro. We also identified beta-TrCP binding site (m2) of PER2 being recognized by both beta-TrCP1 and beta-TrCP2. Luciferase-PER2 fusion system revealed that m2 site was responsible for the stability of PER2. The role of beta-TrCP1 and beta-TrCP2 in circadian rhythm generation was analysed by real-time reporter assay revealing that siRNA-mediated suppressions of beta-TrCP1 and/or beta-TrCP2 attenuate circadian oscillations in NIH3T3 cell. beta-TrCP1-deficient mice, however, showed normal period length, light-induced phase-shift response in behaviour and normal expression of PER2, suggesting that beta-TrCP1 is dispensable for the central clock in the suprachiasmatic nucleus. Our study indicates that beta-TrCP1 and beta-TrCP2 were involved in the cell autonomous circadian rhythm generation in culture cells, although the role of beta-TrCP2 in the central clock in the suprachiasmatic nucleus remains to be elucidated.