Qing Jun Meng

Setting clock speed in mammals: the CK1 epsilon tau mutation in mice accelerates circadian pacemakers by selectively destabilizing PERIOD proteins.

The intrinsic period of circadian clocks is their defining adaptive property. To identify the biochemical mechanisms whereby casein kinase1 (CK1) determines circadian period in mammals, we created mouse null and tau mutants of Ck1 epsilon. Circadian period lengthened in CK1epsilon-/-, whereas CK1epsilon(tau/tau) shortened circadian period of behavior in vivo and suprachiasmatic nucleus firing rates in vitro, by accelerating PERIOD-dependent molecular feedback loops. CK1epsilon(tau/tau) also accelerated molecular oscillations in peripheral tissues, revealing its global role in circadian pacemaking. CK1epsilon(tau) acted by promoting degradation of both nuclear and cytoplasmic PERIOD, but not CRYPTOCHROME, proteins. Together, these whole-animal and biochemical studies explain how tau, as a gain-of-function mutation, acts at a specific circadian phase to promote degradation of PERIOD proteins and thereby accelerate the mammalian clockwork in brain and periphery.

Entrainment of disrupted circadian behavior through inhibition of casein kinase 1 (CK1) enzymes

Circadian pacemaking requires the orderly synthesis, posttranslational modification, and degradation of clock proteins. In mammals, mutations in casein kinase 1 (CK1) ε or δ can alter the circadian period, but the particular functions of the WT isoforms within the pacemaker remain unclear. We selectively targeted WT CK1ε and CK1δ using pharmacological inhibitors (PF-4800567 and PF-670462, respectively) alongside genetic knockout and knockdown to reveal that CK1 activity is essential to molecular pacemaking. Moreover, CK1δ is the principal regulator of the clock period: pharmacological inhibition of CK1δ, but not CK1ε, significantly lengthened circadian rhythms in locomotor activity in vivo and molecular oscillations in the suprachiasmatic nucleus (SCN) and peripheral tissue slices in vitro. Period lengthening mediated by CK1δ inhibition was accompanied by nuclear retention of PER2 protein both in vitro and in vivo. Furthermore, phase mapping of the molecular clockwork in vitro showed that PF-670462 treatment lengthened the period in a phase-specific manner, selectively extending the duration of PER2-mediated transcriptional feedback. These findings suggested that CK1δ inhibition might be effective in increasing the amplitude and synchronization of disrupted circadian oscillators. This was tested using arrhythmic SCN slices derived from Vipr2−/− mice, in which PF-670462 treatment transiently restored robust circadian rhythms of PER2::Luc bioluminescence. Moreover, in mice rendered behaviorally arrhythmic by the Vipr2−/− mutation or by constant light, daily treatment with PF-670462 elicited robust 24-h activity cycles that persisted throughout treatment. Accordingly, selective pharmacological targeting of the endogenous circadian regulator CK1δ offers an avenue for therapeutic modulation of perturbed circadian behavior.

Selective inhibition of casein kinase 1 epsilon minimally alters circadian clock period.

The circadian clock links our daily cycles of sleep and activity to the external environment. Deregulation of the clock is implicated in a number of human disorders, including depression, seasonal affective disorder, and metabolic disorders. Casein kinase 1 epsilon (CK1ϵ) and casein kinase 1 delta (CK1δ) are closely related Ser-Thr protein kinases that serve as key clock regulators as demonstrated by mammalian mutations in each that dramatically alter the circadian period. Therefore, inhibitors of CK1δ/ϵ may have utility in treating circadian disorders. Although we previously demonstrated that a pan-CK1δ/ϵ inhibitor, 4-[3-cyclohexyl-5-(4-fluoro-phenyl)-3H-imidazol-4-yl]-pyrimidin-2-ylamine (PF-670462), causes a significant phase delay in animal models of circadian rhythm, it remains unclear whether one of the kinases has a predominant role in regulating the circadian clock. To test this, we have characterized 3-(3-chloro-phenoxymethyl)-1-(tetrahydro-pyran-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-ylamine (PF-4800567), a novel and potent inhibitor of CK1ϵ (IC50 = 32 nM) with greater than 20-fold selectivity over CK1δ. PF-4800567 completely blocks CK1ϵ-mediated PER3 nuclear localization and PER2 degradation. In cycling Rat1 fibroblasts and a mouse model of circadian rhythm, however, PF-4800567 has only a minimal effect on the circadian clock at concentrations substantially over its CK1ϵ IC50. This is in contrast to the pan-CK1δ/ϵ inhibitor PF-670462 that robustly alters the circadian clock under similar conditions. These data indicate that CK1ϵ is not the predominant mediator of circadian timing relative to CK1δ. PF-4800567 should prove useful in probing unique roles between these two kinases in multiple signaling pathways.

Ligand modulation of REV-ERBα function resets the peripheral circadian clock in a phasic manner

The nuclear receptor REV-ERBα is a key negative-feedback regulator of the biological clock. REV-ERBα binds to ROR elements of the Bmal1 (Arntl) promoter and represses Bmal1 transcription. This stabilizing negative loop is important for precise control of the circadian pacemaker. In the present study, we identified a novel synthetic REV-ERBα ligand, which enhances the recruitment of nuclear receptor co-repressor (NCoR) to REV-ERBα. In order to explore REV-ERBα action on resetting responses of the molecular clock, we first established the rhythmic transcription profile and expression level of REV-ERBα in Rat-1 fibroblasts. When applied at different phases of the circadian oscillation to cell models containing stably transfected Bmal1::Luc or Per2::Luc, the REV-ERBα ligand induced phase-dependent bi-directional phase shifts. When the phase changes were plotted against time, a clear phase response curve was revealed, with a significant peak-to-trough amplitude of ca. 5 hours. The phase-resetting effect was also observed when the compound was applied to primary lung fibroblasts and ectopic lung slices from transgenic PER2::Luc mice. Therefore, similar regulation of REV-ERBα function by endogenous ligands, such as heme, is likely to be an important mechanism for clock resetting. In addition, we identify a new means to generate phasic shifts in the clock.

Lithium Impacts on the Amplitude and Period of the Molecular Circadian Clockwork

Lithium salt has been widely used in treatment of Bipolar Disorder, a mental disturbance associated with circadian rhythm disruptions. Lithium mildly but consistently lengthens circadian period of behavioural rhythms in multiple organisms. To systematically address the impacts of lithium on circadian pacemaking and the underlying mechanisms, we measured locomotor activity in mice in vivo following chronic lithium treatment, and also tracked clock protein dynamics (PER2::Luciferase) in vitro in lithium-treated tissue slices/cells. Lithium lengthens period of both the locomotor activity rhythms, as well as the molecular oscillations in the suprachiasmatic nucleus, lung tissues and fibroblast cells. In addition, we also identified significantly elevated PER2::LUC expression and oscillation amplitude in both central and peripheral pacemakers. Elevation of PER2::LUC by lithium was not associated with changes in protein stabilities of PER2, but instead with increased transcription of Per2 gene. Although lithium and GSK3 inhibition showed opposing effects on clock period, they acted in a similar fashion to up-regulate PER2 expression and oscillation amplitude. Collectively, our data have identified a novel amplitude-enhancing effect of lithium on the PER2 protein rhythms in the central and peripheral circadian clockwork, which may involve a GSK3-mediated signalling pathway. These findings may advance our understanding of the therapeutic actions of lithium in Bipolar Disorder or other psychiatric diseases that involve circadian rhythm disruptions.

A Circadian Clock Is Not Required in an Arctic Mammal

Seasonally breeding mammals use the annual change in the photoperiod cycle to drive rhythmic nocturnal melatonin signals from the pineal gland, providing a critical cue to time seasonal reproduction. Paradoxically, species resident at high latitudes achieve tight regulation of the temporal pattern of growth and reproduction despite the absence of photoperiodic information for most of the year. In this study, we show that the melatonin rhythm of reindeer (Rangifer tarandus) is acutely responsive to the light/dark cycle but not to circadian phase, and also that two key clock genes monitored in reindeer fibroblast cells display little, if any, circadian rhythmicity. The molecular clockwork that normally drives cellular circadian rhythms is evidently weak or even absent in this species, and instead, melatonin-mediated seasonal timing may be driven directly by photic information received at a limited time of year specific to the equinoxes.

The Circadian Clock in Murine Chondrocytes Regulates Genes Controlling Key Aspects of Cartilage Homeostasis

ObjectiveTo characterize the circadian clock in murine cartilage tissue and identify tissue-specific clock target genes, and to investigate whether the circadian clock changes during aging or during cartilage degeneration using an experimental mouse model of osteoarthritis (OA). MethodsCartilage explants were obtained from aged and young adult mice after transduction with the circadian clock fusion protein reporter PER2::luc, and real-time bioluminescence recordings were used to characterize the properties of the clock. Time-series microarrays were performed on mouse cartilage tissue to identify genes expressed in a circadian manner. Rhythmic genes were confirmed by quantitative reverse transcription–polymerase chain reaction using mouse tissue, primary chondrocytes, and a human chondrocyte cell line. Experimental OA was induced in mice by destabilization of the medial meniscus (DMM), and articular cartilage samples were microdissected and subjected to microarray analysis. ResultsMouse cartilage tissue and a human chondrocyte cell line were found to contain intrinsic molecular circadian clocks. The cartilage clock could be reset by temperature signals, while the circadian period was temperature compensated. PER2::luc bioluminescence demonstrated that circadian oscillations were significantly lower in amplitude in cartilage from aged mice. Time-series microarray analyses of the mouse tissue identified the first circadian transcriptome in cartilage, revealing that 615 genes (∼3.9% of the expressed genes) displayed a circadian pattern of expression. This included genes involved in cartilage homeostasis and survival, as well as genes with potential importance in the pathogenesis of OA. Several clock genes were disrupted in the early stages of cartilage degeneration in the DMM mouse model of OA. ConclusionThese results reveal an autonomous circadian clock in chondrocytes that can be implicated in key aspects of cartilage biology and pathology. Consequently, circadian disruption (e.g., during aging) may compromise tissue homeostasis and increase susceptibility to joint damage or disease.

The chondrocyte clock gene Bmal1 controls cartilage homeostasis and integrity

Osteoarthritis (OA) is the most prevalent and debilitating joint disease, and there are currently no effective disease-modifying treatments available. Multiple risk factors for OA, such as aging, result in progressive damage and loss of articular cartilage. Autonomous circadian clocks have been identified in mouse cartilage, and environmental disruption of circadian rhythms in mice predisposes animals to OA-like damage. However, the contribution of the cartilage clock mechanisms to the maintenance of tissue homeostasis is still unclear. Here, we have shown that expression of the core clock transcription factor BMAL1 is disrupted in human OA cartilage and in aged mouse cartilage. Furthermore, targeted Bmal1 ablation in mouse chondrocytes abolished their circadian rhythm and caused progressive degeneration of articular cartilage. We determined that BMAL1 directs the circadian expression of many genes implicated in cartilage homeostasis, including those involved in catabolic, anabolic, and apoptotic pathways. Loss of BMAL1 reduced the levels of phosphorylated SMAD2/3 (p-SMAD2/3) and NFATC2 and decreased expression of the major matrix-related genes Sox9, Acan, and Col2a1, but increased p-SMAD1/5 levels. Together, these results define a regulatory mechanism that links chondrocyte BMAL1 to the maintenance and repair of cartilage and suggest that circadian rhythm disruption is a risk factor for joint diseases such as OA.

The E3 ubiquitin ligase UBE3A is an integral component of the molecular circadian clock through regulating the BMAL1 transcription factor

Post-translational modifications (such as ubiquitination) of clock proteins are critical in maintaining the precision and robustness of the evolutionarily conserved circadian clock. Ubiquitination of the core clock transcription factor BMAL1 (brain and muscle Arnt-like 1) has recently been reported. However, it remains unknown whether BMAL1 ubiquitination affects circadian pacemaking and what ubiquitin ligase(s) is involved. Here, we show that activating UBE3A (by expressing viral oncogenes E6/E7) disrupts circadian oscillations in mouse embryonic fibroblasts, measured using PER2::Luc dynamics, and rhythms in endogenous messenger ribonucleic acid and protein levels of BMAL1. Over-expression of E6/E7 reduced the level of BMAL1, increasing its ubiquitination and proteasomal degradation. UBE3A could bind to and degrade BMAL1 in a ubiquitin ligase-dependent manner. This occurred both in the presence and absence of E6/E7. We provide in vitro (knockdown/over-expression in mammalian cells) and in vivo (genetic manipulation in Drosophila) evidence for an endogenous role of UBE3A in regulating circadian dynamics and rhythmic locomotor behaviour. Together, our data reveal an essential and conserved role of UBE3A in the regulation of the circadian system in mammals and flies and identify a novel mechanistic link between oncogene E6/E7-mediated cell transformation and circadian (BMAL1) disruption.

Identification of Tctex2β, a Novel Dynein Light Chain Family Member That Interacts with Different Transforming Growth Factor-β Receptors

Endoglin is a membrane-inserted protein that is preferentially synthesized in angiogenic vascular endothelial and smooth muscle cells. Endoglin associates with members of the transforming growth factor-β (TGF-β) receptor family and has been identified as the gene involved in hereditary hemorrhagic telangiectasia. Although endoglin is known to affect cell responses to TGF-β, its mode of action is largely unknown. We performed yeast two-hybrid screening of a human placental cDNA library and isolated a new endoglin-binding partner, a novel 221-amino acid member of the Tctex1/2 family of cytoplasmic dynein light chains named Tctex2β, as the founder of a new Tctex1/2 subfamily. The interaction was localized exclusively to the cytoplasmic domain of endoglin. Reverse transcription-PCR showed expression of Tctex2β in a wide range of tissues, including vascular endothelial and smooth muscle cells, placenta, and testis, as well as in several tumor cell lines. High expression levels were found in human umbilical vein endothelial cells and the large cell lung cancer cell line. Forced expression of Tctex2β had a profound inhibitory effect on TGF-β signaling. Additional Tctex2β-interacting receptors were identified to be the TGF-β type II receptor and most likely beta-glycan, but not ALK5, ALK1, or the bone morphogenetic protein type II receptor. Upon fluorescence tagging, co-localization of Tctex2β and endoglin, as well as Tctex2β, endoglin, and the TGF-β type II receptor, was observed by different microscopy techniques. Our findings link endoglin for the first time to microtubule-based minus end-directed transport machinery, suggesting that some endoglin functions might be regulated and directed by its interaction with the cytoplasmic dynein light chain Tctex2β.