PER2 Protein Research Articles

<p>Chronic chemotherapy with paclitaxel nanoparticles induced apoptosis in lung cancer in vitro and in vivo</p>

AimPaclitaxel (PTX) is an effective antitumor drug. Previous research demonstrated that paclitaxel nanoparticles (PTX-NPs) exhibited the greatest antitumor effect at 15 hours after light onset (15 HALO), but the mechanism in chronic chemotherapy is still unknown. In our study, we investigated whether PTX-NPs regulated Period2 (Per2) during chronic chemotherapy to induce apoptosis in vivo and in vitro.MethodsTo improve the antitumor effect and reduce organ damage induced by PTX treatment, PTX-NPs were prepared using a film dispersion method. Then, A549 cells were treated with PTX-NPs at 0, 5, 10, 15, and 20 HALO. An annexin/PI V-FITC apoptosis kit was measured for apoptosis, and PI was analyzed for cell cycle. The relative mechanism was detected by RT-PCR and Western blotting. Tumor volume and weight were measured to evaluate the antitumor effect of the PTX-NPs, and H&E staining was performed to assess organ damage.ResultsCell cycle analysis demonstrated that PTX-NPs blocked cell cycle in G2 phase and that the ratio of cell death was significantly increased in A549 cells, while the ratios of cells in G2 phase and of apoptotic cells were highest at 15 HALO. Evaluation of in vivo antitumor activity revealed that PTX-NPs inhibited tumor growth and decreased tumor weight at 15 HALO. RT-PCR and Western blotting demonstrated that PTX-NPs upregulated Per2 mRNA and protein expression, and the highest Per2 expression was observed at 15 HALO in vivo and in vitro. Meanwhile, Bax mRNA and protein expression was upregulated, while Bcl-2 mRNA and protein expression was downregulated after PTX-NPs treatment in vivo. Moreover, H&E staining revealed that PTX-NPs reduced liver damage at 15 HALO.ConclusionPTX-NPs exhibited the most effective antitumor activity and reduced liver damage at 15 HALO through upregulation of Per2 expression to induce apoptosis in vivo and in vitro.

Smad3 Suppresses Epithelial Cell Migration and Proliferation via the Clock Gene Dec1, Which Negatively Regulates the Expression of Clock Genes Dec2 and Per1

Smad3 has circadian expression; however, whether Smad3 affects the expression of clock genes is poorly understood. Here, we investigated the regulatory mechanisms between Smad3 and the clock genes Dec1, Dec2, and Per1. In Smad3 knockout mice, the amplitude of locomotor activity was decreased, and Dec1 expression was decreased in the suprachiasmatic nucleus, liver, kidney, and tongue compared with control mice. Conversely, Dec2 and Per1 expression was increased compared with that of control mice. In Smad3 knockout mice, immunohistochemical staining revealed that Dec1 expression decreased, whereas Dec2 and Per1 expression increased in the endothelial cells of the kidney and liver. In NIH3T3 cells, Smad3 overexpression increased Dec1 expression, but decreased Dec2 and Per1 expression. In a wound-healing experiment that used Smad3 knockout mice, Dec1 expression decreased in the basal cells of squamous epithelium, promoting wound healing of the mucosa. Finally, the migration and proliferation of Smad3 knockdown squamous carcinoma cells was suppressed by Dec1 overexpression but was promoted by Dec2 overexpression. Dec1 overexpression decreased E-cadherin and proliferating cell nuclear antigen expression, whereas these expression levels were increased by Dec2 overexpression. These results suggest Smad3 is relevant to circadian rhythm and regulates cell migration and proliferation through Dec1, Dec2, and Per1 expression.

Analyses of BMAL1 and PER2 Oscillations in a Model of Breast Cancer Progression Reveal Changes With Malignancy.

From an epidemiological standpoint, disruptions to circadian rhythms have been shown to contribute to the development of various disease pathologies, including breast cancer. However, it is unclear how altered circadian rhythms are related to malignant transformations at the molecular level. In this article, a series of isogenic breast cancer cells representing disease progression was used to investigate the expression patterns of core circadian clock proteins BMAL1 and PER2. Our model is indicative of 4 stages of breast cancer and includes the following cells: MCF10A (non-malignant), MCF10AT.Cl2 (pre-malignant), MCF10Ca1h (well-differentiated, malignant), and MCF10Ca1a (poorly differentiated, malignant). While studies of circadian rhythms in cancer typically use low-resolution reverse transcription polymerase chain reaction assays, we also employed luciferase reporters BMAL1:Luc and PER2:Luc in real-time luminometry experiments. We found that across all 4 cancer stages, PER2 showed relatively stable oscillations compared with BMAL1. Period estimation using both wavelet-based and damped-sine-fitting methods showed that the periods are distributed over a wide circadian range and there is no clear progression in mean period as cancer severity progresses. Additionally, we used the K-nearest neighbors algorithm to classify the recordings according to cancer line, and found that cancer stages were largely differentiated from one another. Taken together, our data support that there are circadian discrepancies between normal and malignant cells, but it is difficult and insufficient to singularly use period evaluations to differentiate them. Future studies should employ other progressive disease models to determine whether these findings are representative across cancer types or are specific to this series.

Sleep/Wake Disruption in a Mouse Model of BLOC-1 Deficiency

Mice lacking a functional Biogenesis of Lysosome-related Organelles Complex 1 (BLOC-1), such as those of the pallid line, display cognitive and behavioural impairments reminiscent of those presented by individuals with intellectual and developmental disabilities. Although disturbances in the sleep/wake cycle are commonly lamented by these individuals, the underlying mechanisms, including the possible role of the circadian timing system, are still unknown. In this paper, we have explored sleep/circadian malfunctions and underlying mechanisms in BLOC-1-deficient pallid mice. These mutants exhibited less sleep behaviour in the beginning of the resting phase than wild-type mice with a more broken sleeping pattern in normal light-dark conditions. Furthermore, the strength of the activity rhythms in the mutants were reduced with significantly more fragmentation and lower precision than in age-matched controls. These symptoms were accompanied by an abnormal preference for the open arm in the elevated plus maze in the day and poor performance in the novel object recognition at night. At the level of the central circadian clock (the suprachiasmatic nucleus, SCN), loss of BLOC-1 caused subtle morphological changes including a larger SCN and increased expression of the relative levels of the clock gene Per2 product during the day but did not affect the neuronal activity rhythms. In the hippocampus, the pallid mice presented with anomalies in the cytoarchitecture of the Dentate Gyrus granule cells, but not in CA1 pyramidal neurones, along with altered PER2 protein levels as well as reduced pCREB/tCREB ratio during the day. Our findings suggest that lack of BLOC-1 in mice disrupts the sleep/wake cycle and performance in behavioural tests associated with specific alterations in cytoarchitecture and protein expression.

Effect of acute sleep deprivation on learning and memory and hippocampal circadian gene expression after sevoflurane anesthesia in rats

Objective To investigate the role of clock gene Bmal1, Per2 and Egr1 expression in learning and memory undergoing sevoflurane anesthesia after acute sleep deprivation. Methods 72 male SD rats were equally divided into four groups using a random number table (n=18) : normal control group (group Control), do not do any processing; sleep deprivation group (group SD), acute sleep deprivation for 96 h; sevoflurane group (group Sev), suffering 2.5% sevoflurane for 3 h; sleep deprivation+sevoflurane group (group SD+Sev), 96 h sleep deprivation followed by 3 h 2.5% sevoflurane inhalation. The Morris water maze, for spatial memory acquisition test, was used to measure the time percent of target quadrant and numbers of platform-site crossovers before sleep deprivation (T0) and at 1 d (T1), 3 d (T2), 7 d (T3) after inhalation anesthesia. Rats were sacrificed after spatial memory acquisition test. Brain hippocampus samples were obtained for determination of Bmal1, Per2 and Egr1 expression by Western blot, and neuron morphology was observed by the Nissl staining. Results Compared with Control group, the percentage of time in target quadrant and the numbers of platform-site crossovers were significantly decreased at T1 and T2 in Sev group (P<0.05); and compared with Control group, the percentage of time in target quadrant and the numbers of platform-site crossovers were also significantly decreased at T1, T2 and T3 in SD group rats (P<0.05). Compared with Sev group rats (the percentages of time in target quadrant: T1: (32.37±1.36)%; T2: (30.91±1.26)%; T3: (33.78±2.20)%; the numbers of platform-site crossovers: T1: (4.55±0.39); T2: (3.11±0.37); T3: (3.95±0.34)), the percentages of time in target quadrant (T1: (27.20±1.42)%; T2: (28.19±1.04)%; T3: (30.06±1.22)%) and the numbers of platform-site crossovers (T1: (3.11±0.46); T2: (3.30±0.38); T3: ( 3.20±0.39)) in SD+Sev group rats were significantly decreased at T1, T2 and T3 (all P<0.05). Compared with control group, the levels of hippocampal proteins Bmal1, Per2 and Egr1 were significantly reduced at T1 in Sev group (P<0.05). Compared with control group, the level of hippocampal protein Per2 was significantly increased, but the levels of hippocampal proteins Bmal1 and Egr1 were significantly decreased at T1 and T2 in SD group (P<0.01). Compared with Sev group, the levels of hippocampal proteins Bmal1 and Egr1 were significantly reduced at T1, T2 and T3 in SD+Sev group (P<0.01), and the protein level of hippocampal Per2 was significantly decreased at T1, but then increased at T2 and T3 in SD+Sev group (P<0.01). The hippocampal Nissl staining in CA1 at T2 revealed that there were irregular distribution of pyramidal neurons existed in Sev group, and vacuolar degeneration with vague outlines of pyramidal neurons in SD group, while pyramidal neuron atrophy and few number of Nissl bodies, compared with control group, were observed in SD+Sev group. Conclusion Acute sleep deprivation following with sevoflurane anesthesia resulted in hippocampal memory impairment, which was associated with abnormal expression of hippocampal Bmal1, Per2 and Egr1. Key words: Sleep deprivation; Inhalation anesthesia; Sevoflurane; Learning and memory; Clock gene

Rapid Response and Slow Recovery of the H3K4me3 Epigenomic Marker in the Liver after Light-mediated Phase Advances of the Circadian Clock.

Mammalian tissues display circadian rhythms in transcription, translation, and histone modifications. Here we asked how an advance of the light-dark cycle alters daily rhythms in the liver epigenome at the H3K4me3 (trimethylation of lysine 4 on histone 3) modification, which is found at active and poised gene promoters. H3K4me3 levels were first measured at 4 time points (zeitgeber time [ZT] 3, 8, 15, and 20) during a normal 12L:12D light-dark cycle. Peak levels were observed during the early dark phase at ZT15 and dropped to low levels around lights-on (ZT0) between ZT20 and ZT3. A 6-h phase advance at ZT18 (new lights-on after only 6 h of darkness) led to a transient extension of peak H3K4me3 levels. Although locomotor activity reentrained within a week after the phase advance, H3K4me3 rhythms failed to do so, with peak levels remaining in the light phase at the 1-week recovery time point. Eight weekly phase advances, with 1-week recovery times between each phase advance, further disrupted the H3K4me3 rhythms. Finally, we used the mPer2Luc knockin mouse to determine whether the phase advance also disrupted Per2 protein expression. Similar to the results from the histone work, we found both a rapid response to the phase advance and a delayed recovery, the latter in sync with H3K4me3 levels. A model to explain these results is offered.

Mutations in blind cavefish target the light-regulated circadian clock gene, period 2

Light represents the principal signal driving circadian clock entrainment. However, how light influences the evolution of the clock remains poorly understood. The cavefish Phreatichthys andruzzii represents a fascinating model to explore how evolution under extreme aphotic conditions shapes the circadian clock, since in this species the clock is unresponsive to light. We have previously demonstrated that loss-of-function mutations targeting non-visual opsins contribute in part to this blind clock phenotype. Here, we have compared orthologs of two core clock genes that play a key role in photic entrainment, cry1a and per2, in both zebrafish and P. andruzzii. We encountered aberrantly spliced variants for the P. andruzzii per2 transcript. The most abundant transcript encodes a truncated protein lacking the C-terminal Cry binding domain and incorporating an intronic, transposon-derived coding sequence. We demonstrate that the transposon insertion leads to a predominantly cytoplasmic localization of the cavefish Per2 protein in contrast to the zebrafish ortholog which is distributed in both the nucleus and cytoplasm. Thus, it seems that during evolution in complete darkness, the photic entrainment pathway of the circadian clock has been subject to mutation at multiple levels, extending from opsin photoreceptors to nuclear effectors.

Two Ck1δ transcripts regulated by m6A methylation code for two antagonistic kinases in the control of the circadian clock

The N6-methylation of internal adenosines (m6A) in mRNA has been quantified and localized throughout the transcriptome. However, the physiological significance of m6A in most highly methylated mRNAs is unknown. It was demonstrated previously that the circadian clock, based on transcription-translation negative feedback loops, is sensitive to the general inhibition of m6A. Here, we show that the Casein Kinase 1 Delta mRNA (Ck1δ), coding for a critical kinase in the control of circadian rhythms, cellular growth, and survival, is negatively regulated by m6A. Inhibition of Ck1δ mRNA methylation leads to increased translation of two alternatively spliced CK1δ isoforms, CK1δ1 and CK1δ2, uncharacterized until now. The expression ratio between these isoforms is tissue-specific, CK1δ1 and CK1δ2 have different kinase activities, and they cooperate in the phosphorylation of the circadian clock protein PER2. While CK1δ1 accelerates the circadian clock by promoting the decay of PER2 proteins, CK1δ2 slows it down by stabilizing PER2 via increased phosphorylation at a key residue on PER2 protein. These observations challenge the previously established model of PER2 phosphorylation and, given the multiple functions and targets of CK1δ, the existence of two isoforms calls for a re-evaluation of past research when CK1δ1 and CK1δ2 were simply CK1δ.

Renal Mechanism for Regulation of Blood Pressure by the Circadian Clock Protein PER1

Work from our lab suggests that the clock protein PER1 is critical for maintaining the circadian rhythm of blood pressure (BP). BP is highest during the active phase of the day whereas during the rest period BP dips at least 10%. Individuals that do not experience the dip are termed “non‐dippers.” Non‐dipping hypertension is associated with an increased risk of cardiovascular disease. The mechanisms underlying non‐dipping hypertension are not understood. Male C57BL/6J Per1 KO mice develop non‐dipping hypertension on a high‐salt diet plus mineralocorticoid treatment (HS/DOCP). Sodium (Na) handling by the kidney has large effects on blood volume and BP. Excretion of Na also demonstrates a circadian pattern with a peak of excretion during active periods. We hypothesized that Per1 contributes to circadian regulation of BP via a renal Na handling‐dependent mechanism. To test this hypothesis, we treated WT and Per1 KO mice housed in metabolic cages with a normal salt diet or HS/DOCP. We measured changes in timed urinary Na excretion using flame photometry. The night:day ratio of Na in the urine of Per1 KO mice was decreased compared to WT (4x vs. 7x, P&lt;0.001, n=6 per group). ELISA was used to measure the fold increase of urinary endothelin‐1 (ET‐1) in response to HS/DOCP treatment. Per1 KO mice exhibited a higher fold increase of ET‐1 compared to WT with HS/DOCP (5.9x vs 3.6x, P&lt;0.01, n=9 WT, 11 KO). Using real‐time quantitative PCR, we determined that the expression of Na handling genes was altered in the KO mice on HS/DOCP. ET‐1 was upregulated in the cortex of Per1 KO mice on HS/DOCP treatment (P&lt;0.01, n=6 per group). This upregulation was not present in WT mice on HS/DOCP. Nitric oxide synthase 1 (NOS1), a negative regulator of Na reabsorption, was significantly downregulated in Per1 KO medulla on HS/DOCP (P&lt;0.05, n=6 per group). Preliminary data from the first distal‐nephron specific Per1 KO mice also shows an inappropriate increase in expression of the epithelial Na channel α subunit (αENaC) and the Na chloride cotransporter (NCC) relative to control mice. These results support our hypothesis that Per1 mediates control of circadian BP rhythms via the regulation of renal Na transport genes, specifically in the distal nephron of the kidney. These findings have implications for understanding the etiology of non‐dipping hypertension and the subsequent development of novel therapies for this dangerous pathophysiological condition.Support or Funding InformationR01DK109570; R21AG052861; American Heart Association Grant‐in‐aid; Gatorade Trust Fund, Division of Nephrology, Department of Medicine, University of FloridaThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cosinor Analysis Reveals Non‐dipping Hypertension in Mixed Background Strain Mice Lacking the Circadian Clock Protein PER1

Cardiovascular disease is the leading cause of death among Americans. Hypertension is the leading risk factor for cardiovascular disease, thus blood pressure (BP) regulation is imperative for maintaining cardiovascular health. BP exhibits a circadian rhythm that is regulated through many mechanisms. Humans experience a BP rhythm resulting in a 10–20% dip in BP during their resting period. Individuals who fail to maintain this dip, termed non‐dippers, are associated with worse cardiovascular outcomes. The circadian clock mechanism, which includes the transcription factors PERIOD, BMAL1, CLOCK, and CRYPTOCHROME, is necessary to maintain this rhythm. Past studies in our lab have shown that global loss of PER1 leads to non‐dipping hypertension in male C57BL/6 mice in response to high salt diet plus an aldosterone analogue desoxycorticosterone pivalate‐salt (DOCP) (Solocinski et al. 2017). The goal of this study was to determine the BP phenotype of Per1 knockout (KO) mice on a salt‐sensitive 129/sv/s1s4 background strain. 129/sv/s1s4 mice were implanted with radiotelemetry devices and maintained in a normal 12‐hour light/dark period. BP was monitored continuously while the mice were subjected to a control diet, a high salt (HS) diet, or a HS diet plus treatment with an aldosterone analogue DOCP. BP rhythms were analyzed through the program Cosinor (www.circadian.org) to determine the mesor, amplitude, and acrophase. When evaluating a cosine wave, the mesor is the midline between the peak and the trough, the amplitude is the measure of the mesor to the peak, and the acrophase is the time point of the peak. These values were then used to discern whether the mice maintained a BP rhythm. The 129/sv/s1s4 Per1 KO experienced a 9‐mmHg increase in BP in response to HS/DOCP (P&lt;0.01, n=8). In contrast, the wild type (WT) mice (n=5) did not experience a significant increase in BP after HS/DOCP. Furthermore, the KO mice had a 2.4‐mmHg decrease in amplitude (P=0.01), whereas the WT mice did not exhibit a significant decrease in amplitude. The WT mice on control diet exhibited a 12.8% dip in BP during their inactive period compared to their active period, which insignificantly decreased to an 11.5% dip on HS/DOCP diet. However, the KO mice on control diet had an 11% dip, which decreased to 5.4% dip on HS/DOCP diet (P&lt;0.05, n=5–8). These data demonstrate that loss of Per1 in male 129/sv/s1s4 mice leads to a non‐dipping phenotype in response to HS/DOCP treatment. Importantly, these findings represent the reproducibility of the non‐dipping hypertension phenotype we previously observed in C57BL/6 mice in a distinct strain on a mixed background.Support or Funding InformationAmerican Physiological Society Undergraduate Summer Fellowship Program; American Society of Nephrology Foundation for Kidney ResearchThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Co‐regulation of Renal Function Genes by the Circadian Clock Protein PER1 and Aldosterone

Hypertension is the leading cause of cardiovascular disease. A subset of hypertensive patients experience salt‐sensitive hypertension, which causes elevations in blood pressure (BP) after high salt intake. With large increases in dietary salt worldwide, the prevalence of hypertension has reached 30%. BP follows a circadian rhythm. The circadian clock is a highly conserved mechanism that allows organisms to adapt to the light/dark cycles of our environment. The molecular clock is comprised of core transcription proteins, such as the clock protein PER1. Further, the renin angiotensin aldosterone system (RAAS) is an important homeostatic mechanism to manage sodium and BP homeostasis. Previous results from our lab show that the circadian clock and RAAS both contribute to sodium and BP homeostasis. Based on these studies, we hypothesized that aldosterone and Per1 coordinately regulate genes that contribute to kidney function. We tested this hypothesis first by using a cell culture model from the renal cortical collecting duct. Cells were treated with aldosterone and/or siRNA to knockdown down PER1 expression. Using an unbiased microarray approach, we determined PER1 and aldosterone‐dependent changes in gene expression. Several hundred genes exhibited significant changes in expression in response to hormone treatment and PER1 knockdown. For the first time, we identified secreted frizzled related protein 1 (sFRP1), Claudin‐1, and Rhesus glycoprotein C (Rhcg) as target genes of aldosterone and PER1. The expression of sFRP1 demonstrated a 1.5‐fold increase in response to aldosterone (p&lt;0.05, n=4), which was not affected in the absence of PER1, indicating that aldosterone and PER1 independently regulate sFRP1. In contrast, the 2.9‐fold (p&lt;0.05, n=4) and 1.9‐fold (p&lt;0.05, n=3) induction of Rhcg and Claudin‐1, respectively, by aldosterone was decreased significantly in the absence of PER1, suggesting that PER1 contributes to aldosterone‐mediated regulation of these genes. The ET‐1 gene is of particular interest due to its known role in the regulation of renal salt handling and BP. In the microarray study, knockdown of PER1 augmented the aldosterone response of ET‐1 mRNA by 3‐fold (p&lt;0.05, n=4). Importantly, a similar effect was observed in the kidneys of Per1 knockout mice treated with a high salt diet plus an aldosterone analogue: loss of Per1 augmented the response of ET‐1 by 50% (p&lt;0.01, n=6) compared to no change in WT mice. Interrogation of the database CircaDB revealed that Claudin‐1 and ET‐1 are both expressed with a significant circadian rhythm in the kidney (p&lt;0.001). Together these data demonstrate that multiple genes critical to renal function are co‐regulated by the circadian clock PER1 and RAAS. These data may have important implications for understanding the underlying mechanisms of, and designing novel treatments for, salt‐sensitive hypertension in humans.Support or Funding InformationR01DK109570; R21AG052861; American Heart Association Grant‐in‐aid; Gatorade Trust Fund, Division of Nephrology, Department of Medicine, University of Florida; NF/SG VAMCThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Circadian Rhythms and Clock Genes in Reproduction: Insights From Behavior and the Female Rabbit's Brain.

Clock gene oscillations are necessary for a successful pregnancy and parturition, but little is known about their function during lactation, a period demanding from the mother multiple physiological and behavioral adaptations to fulfill the requirements of the offspring. First, we will focus on circadian rhythms and clock genes in reproductive tissues mainly in rodents. Disruption of circadian rhythms or proper rhythmic oscillations of clock genes provoke reproductive problems, as found in clock gene knockout mice. Then, we will focus mainly on the rabbit doe as this mammal nurses the young just once a day with circadian periodicity. This daily event synchronizes the behavior and the activity of specific brain regions critical for reproductive neuroendocrinology and maternal behavior, like the preoptic area. This region shows strong rhythms of the PER1 protein (product of the Per1 clock gene) associated with circadian nursing. Additionally, neuroendocrine cells related to milk production and ejections are also synchronized to daily nursing. A threshold of suckling is necessary to entrain once a day nursing; this process is independent of milk output as even virgin does (behaving maternally following anosmia) can display circadian nursing behavior. A timing motivational mechanism may regulate such behavior as mesolimbic dopaminergic cells are entrained by daily nursing. Finally, we will explore about the clinical importance of circadian rhythms. Indeed, women in chronic shift-work schedules show problems in their menstrual cycles and pregnancies and also have a high risk of preterm delivery, making this an important field of translational research.

Mammalian Circadian Period, But Not Phase and Amplitude, Is Robust Against Redox and Metabolic Perturbations.

Aims: Circadian rhythms permeate all levels of biology to temporally regulate cell and whole-body physiology, although the cell-autonomous mechanism that confers ∼24-h periodicity is incompletely understood. Reports describing circadian oscillations of over-oxidized peroxiredoxin abundance have suggested that redox signaling plays an important role in the timekeeping mechanism. Here, we tested the functional contribution that redox state and primary metabolism make to mammalian cellular timekeeping.Results: We found a circadian rhythm in flux through primary glucose metabolic pathways, indicating rhythmic NAD(P)H production. Using pharmacological and genetic perturbations, however, we found that timekeeping was insensitive to changes in glycolytic flux, whereas oxidative pentose phosphate pathway (PPP) inhibition and other chronic redox stressors primarily affected circadian gene expression amplitude, not periodicity. Finally, acute changes in redox state decreased PER2 protein stability, phase dependently, to alter the subsequent phase of oscillation.Innovation: Circadian rhythms in primary cellular metabolism and redox state have been proposed to play a role in the cellular timekeeping mechanism. We present experimental data testing that hypothesis.Conclusion: Circadian flux through primary metabolism is cell autonomous, driving rhythmic NAD(P)+ redox cofactor turnover and maintaining a redox balance that is permissive for circadian gene expression cycles. Redox homeostasis and PPP flux, but not glycolysis, are necessary to maintain clock amplitude, but neither redox nor glucose metabolism determines circadian period. Furthermore, cellular rhythms are sensitive to acute changes in redox balance, at least partly through regulation of PER protein. Redox and metabolic state are, thus, both inputs and outputs, but not state variables, of cellular circadian timekeeping. Antioxid. Redox Signal. 28, 507–520.

Clocking In Time to Gate Memory Processes: The Circadian Clock Is Part of the Ins and Outs of Memory.

Learning, memory consolidation, and retrieval are processes known to be modulated by the circadian (circa: about; dies: day) system. The circadian regulation of memory performance is evolutionarily conserved, independent of the type and complexity of the learning paradigm tested, and not specific to crepuscular, nocturnal, or diurnal organisms. In mammals, long-term memory (LTM) formation is tightly coupled to de novo gene expression of plasticity-related proteins and posttranslational modifications and relies on intact cAMP/protein kinase A (PKA)/protein kinase C (PKC)/mitogen-activated protein kinase (MAPK)/cyclic adenosine monophosphate response element-binding protein (CREB) signaling. These memory-essential signaling components cycle rhythmically in the hippocampus across the day and night and are clearly molded by an intricate interplay between the circadian system and memory. Important components of the circadian timing mechanism and its plasticity are members of the Period clock gene family (Per1, Per2). Interestingly, Per1 is rhythmically expressed in mouse hippocampus. Observations suggest important and largely unexplored roles of the clock gene protein PER1 in synaptic plasticity and in the daytime-dependent modulation of learning and memory. Here, we review the latest findings on the role of the clock gene Period 1 (Per1) as a candidate molecular and mechanistic blueprint for gating the daytime dependency of memory processing.

Circadian PER2 protein oscillations do not persist in cycloheximide-treated mouse embryonic fibroblasts in culture

ABSTRACTIt is not known whether the endogenous mammalian core clock proteins sustain measurable oscillations in cells in culture where de novo translation is pharmacologically inhibited. We studied here the mammalian core clock protein PER2, which undergoes robust circadian oscillations in both abundance and phosphorylation. With a newly developed antibody that enables tracing the endogenous PER2 protein oscillations over circadian cycles with cultured mouse embryonic fibroblast cells, we provide evidence that PER2 does not persist noticeable circadian rhythms when translation is inhibited.

Salt-inducible kinase 3 regulates the mammalian circadian clock by destabilizing PER2 protein.

Salt-inducible kinase 3 (SIK3) plays a crucial role in various aspects of metabolism. In the course of investigating metabolic defects in Sik3-deficient mice (Sik3-/-), we observed that circadian rhythmicity of the metabolisms was phase-delayed. Sik3-/- mice also exhibited other circadian abnormalities, including lengthening of the period, impaired entrainment to the light-dark cycle, phase variation in locomotor activities, and aberrant physiological rhythms. Ex vivo suprachiasmatic nucleus slices from Sik3-/- mice exhibited destabilized and desynchronized molecular rhythms among individual neurons. In cultured cells, Sik3-knockdown resulted in abnormal bioluminescence rhythms. Expression levels of PER2, a clock protein, were elevated in Sik3-knockdown cells but down-regulated in Sik3-overexpressing cells, which could be attributed to a phosphorylation-dependent decrease in PER2 protein stability. This was further confirmed by PER2 accumulation in the Sik3-/- fibroblasts and liver. Collectively, SIK3 plays key roles in circadian rhythms by facilitating phosphorylation-dependent PER2 destabilization, either directly or indirectly.

CREB Protein Mediates Alcohol-Induced Circadian Disruption and Intestinal Permeability.

Alcoholic liver disease (ALD) is commonly associated with intestinal permeability. An unanswered question is why only a subset of heavy alcohol drinkers develop endotoxemia. Recent studies suggest that circadian disruption is the susceptibility factor for alcohol-induced gut leakiness to endotoxins. The circadian protein PER2 is increased after exposure to alcohol and siRNA knockdown of PER2 invitro blocks alcohol-induced intestinal barrier dysfunction. We have shown that blocking CYP2E1 (i.e., important for alcohol metabolism) with siRNA inhibits the alcohol-induced increase in PER2 and suggesting that oxidative stress may mediate alcohol-induced increase in PER2 in intestinal epithelial cells. The aim of this study was to elucidate whether a mechanism incited by alcohol-derived oxidative stress mediates the transcriptional induction of PER2 and subsequent intestinal hyperpermeability. Caco-2 cells were exposed to 0.2% alcohol with or without pretreatment with modulators of oxidative stress or PKA activity. Permeability of the Caco-2 monolayer was assessed by transepithelial electrical resistance. Protein expression was measured by Western blot and mRNA with real-time polymerase chain reaction. Wild-type C57BL/6J mice were fed with alcohol diet (29% of total calories, 4.5% v/v) for 8weeks. Western blot was used to analyze PER2 expression in mouse proximal colon tissue. Alcohol increased oxidative stress, caused Caco-2 cell monolayer dysfunction, and increased levels of the circadian clock proteins PER2 and CLOCK. These effects were mitigated by pretreatment of Caco-2 cells with an antioxidant scavenger. Alcohol-derived oxidative stress activated cAMP response element-binding (CREB) via the PKA pathway and increased PER2 mRNA and protein. Inhibiting CREB prevented the increase in PER2 and Caco-2 cell monolayer hyperpermeability. Taken together, these data suggest that strategies to reduce alcohol-induced oxidative stress may alleviate alcohol-mediated circadian disruption and intestinal leakiness, critical drivers of ALD.

Period2 3′-UTR and microRNA-24 regulate circadian rhythms by repressing PERIOD2 protein accumulation

We previously created two PER2::LUCIFERASE (PER2::LUC) circadian reporter knockin mice that differ only in the Per2 3'-UTR region: Per2::Luc, which retains the endogenous Per2 3'-UTR and Per2::LucSV, where the endogenous Per2 3'-UTR was replaced by an SV40 late poly(A) signal. To delineate the in vivo functions of Per2 3'-UTR, we analyzed circadian rhythms of Per2::LucSV mice. Interestingly, Per2::LucSV mice displayed more than threefold stronger amplitude in bioluminescence rhythms than Per2::Luc mice, and also exhibited lengthened free-running periods (∼24.0 h), greater phase delays following light pulse, and enhanced temperature compensation relative to Per2::Luc Analysis of the Per2 3'-UTR sequence revealed that miR-24, and to a lesser degree miR-30, suppressed PER2 protein translation, and the reversal of this inhibition in Per2::LucSV augmented PER2::LUC protein level and oscillatory amplitude. Interestingly, Bmal1 mRNA and protein oscillatory amplitude as well as CRY1 protein oscillation were increased in Per2::LucSV mice, suggesting rhythmic overexpression of PER2 enhances expression of Per2 and other core clock genes. Together, these studies provide important mechanistic insights into the regulatory roles of Per2 3'-UTR, miR-24, and PER2 in Per2 expression and core clock function.

Rapid Communication: Period2 gene silencing increases the synthesis of αs-casein protein in bovine mammary epithelial cells.

(), a core clock gene, encodes a circadian rhythm protein which has been shown to control mammary metabolism in rodents. Whether regulates milk component synthesis such as α-casein protein in bovine mammary cells is unknown. Thus, we used gene silencing technology to determine if silencing could affect α-casein synthesis and cell growth in cultured primary bovine mammary epithelial cells (BMEC). The BMEC were established by enzymatic digestion of mammary tissue from mid-lactation cows. A transient-transfection technique was used to insert a small interfering RNA (siRNA) oligonucleotide specific for to inhibit transcription. Control and siRNA-transfected cells were cultured for 48 h. qRT-PCR and ELISA analysis showed that silencing enhanced the synthesis of 2 kinds of α-casein ( < 0.05) through upregulating the mRNA level of and ( < 0.01). Furthermore, the 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) results demonstrated that cell proliferation was not affected ( > 0.05). These data led us to hypothesize that PER2 protein may potentially play an important role in the control of milk protein synthesis and, hence, represents a target that can be used to regulate protein synthesis rate during lactation.

Differential Phase Arrangement of Cellular Clocks along the Tonotopic Axis of the Mouse Cochlea Ex Vivo

Topological distributions of individual cellular clocks have not been demonstrated in peripheral organs. The cochlea displays circadian patterns of core clock gene expression [1, 2]. PER2 protein is expressed in the hair cells and spiral ganglion neurons of the cochlea in the spiral ganglion neurons [1]. To investigate the topological organization of cellular oscillators inthe cochlea, we recorded circadian rhythms from mouse cochlear explants using highly sensitive real-time tracking of PER2::LUC bioluminescence. Here, we show cell-autonomous and self-sustained oscillations originating from hair cells and spiral ganglion neurons. Multi-phased cellular clocks were arranged along the length of the cochlea with oscillations initiating at the apex (low-frequency region) and traveling toward the base (high-frequency region). Phase differences of 3hr were found between cellular oscillators in the apical and middle regions and from isolated individual cochlear regions, indicating that cellular networks organize the rhythms along the tonotopic axis. This is the first demonstration of a spatiotemporal arrangement of circadian clocks at the cellular level in a peripheral organ. Cochlear rhythms were disrupted in the presence of either voltage-gated potassium channel blocker (TEA) or extracellular calcium chelator (BAPTA), demonstrating that multiple types of ion channels contribute to the maintenance of coherent rhythms. In contrast, preventing action potentials with tetrodotoxin (TTX) or interfering with cell-to-cell communication the broad-spectrum gap junction blocker (CBX [carbenoxolone]) had no influence on cochlear rhythms. These findings highlight a dynamic regulation and longitudinal distribution of cellular clocks in the cochlea.