STAT1 promotes megakaryopoiesis downstream of GATA-1 in mice

Thrombocytosis is associated with inflammation, and certain inflammatory cytokines, including IFN-gamma, stimulate megakaryocyte and platelet production. However, the roles of IFN-gamma and its downstream effector STAT1 in megakaryocyte development are poorly understood. We previously reported that STAT1 expression was significantly downregulated in Gata1-knockdown murine megakaryocytes, which also have impaired terminal maturation. Here, we show that ectopic expression of STAT1, or its target effector IRF-1, rescued multiple defects in Gata1-deficient megakaryopoiesis in mice, inducing polyploidization and expression of a subset of platelet-expressing genes. Enforced expression of STAT1, IRF-1, or GATA-1 enhanced phosphorylation of STAT1, STAT3, and STAT5 in cultured Gata1-deficient murine megakaryocytes, with concomitant megakaryocyte maturation. In contrast, enhanced thrombopoietin signaling, conferred by enforced expression of constitutively active JAK2 or c-MPL, induced phosphorylation of STAT3 and STAT5, but not STAT1, and failed to rescue megakaryocyte maturation. Finally, megakaryocytes from Stat1(-/-) mice were defective in polyploidization. Together, these findings reveal a unique role for STAT1 in megakaryopoiesis and provide new insights into how GATA-1 regulates this process. Our studies elucidate potential mechanisms by which various inflammatory disorders can cause elevated platelet counts.     

Zinc finger protein, Hzf, is required for megakaryocyte development and hemostasis

Using an expression gene trapping strategy, we recently identified a novel gene, hematopoietic zinc finger (Hzf), which encodes a protein containing three C(2)H(2)-type zinc fingers that is predominantly expressed in megakaryocytes. Here, we have examined the in vivo function of Hzf by gene targeting and demonstrated that Hzf is essential for megakaryopoiesis and hemostasis in vivo. Hzf-deficient mice exhibited a pronounced tendency to rebleed and had reduced alpha-granule substances in both megakaryocytes and platelets. These mice also had large, faintly stained platelets, whereas the numbers of both megakaryocytes and platelets were normal. These results indicate that Hzf plays important roles in regulating the synthesis of alpha-granule substances and/or their packing into alpha-granules during the process of megakaryopoiesis.     

生物节律在恶性肿瘤中的改变及作用

生物节律对机体的生理、代谢和行为具有广泛而重要的调控作用。生物节律的形成是生命体在进化过程中与地球环境相适应的结果，由此形成以地球自转为周期的自主振荡模式。目前已在多种正常细胞中发现由生物节律基因调控的关键基因转录和表达，而这种调控也在肿瘤细胞的形成和进展中起到关键作用。本文总结了关于生物节律与恶性肿瘤发生、发展进程之间相互联系和相关分子机制的研究进展，为基于生物节律的个性化、精准化肿瘤治疗策略的研究提供参考。     

Continuous expression of Bcl-xL protein during megakaryopoiesis is post-translationally regulated by thrombopoietin-mediated Akt activation, which prevents the cleavage of Bcl-xL

BACKGROUND: One of the important biological activities of thrombopoietin (TPO) is to prevent the apoptosis of megakaryocytes. As the antiapoptotic protein Bcl-xL, which has been proven to be indispensable for erythroid differentiation, is also abundantly expressed in megakaryocytes, it is assumed that Bcl-xL plays an important role in megakaryopoiesis. OBJECTIVES: We investigated the expression of Bcl-xL during megakaryopoiesis and the underlying regulatory mechanism. METHODS AND RESULTS: In stem cell-derived megakaryocytes, expression of Bcl-xL increased in the early and mid-stages of the differentiation. Both in vitro in cell culture and in vivo in an animal model, expression of Bcl-xL protein was maintained until the platelet-producing stage. TPO depletion caused significant decrease in Bcl-xL protein level without affecting its mRNA in both megakaryocytes and TPO-dependent megakaryocytic UT-7/TPO cells, suggesting that Bcl-xL protein level in TPO-dependent cells is post-translationally regulated. In agreement with this finding, we recognized the appearance of a 12-kD fragment of Bcl-xL upon TPO depletion. This cleavage of Bcl-xL was inhibited by a caspase-3-specific inhibitor. Furthermore, pretreatment of UT-7/TPO with a phosphatidylinositol 3-kinase (PI3 K) inhibitor resulted in the cleavage of Bcl-xL even in the presence of TPO. We thus hypothesized that PI3 K inhibits the activation of caspase-3 and consequent cleavage of Bcl-xL. To prove this, we prepared UT-7/TPO cells transfected with constitutively active Akt-1. When TPO was depleted, the transfectant was significantly less liable to caspase-3 activation and Bcl-xL cleavage. CONCLUSIONS: Bcl-xL protein is expressed throughout megakaryopoiesis until platelets are produced, and its expression level is at least in part post-translationally regulated through TPO-mediated Akt activation.     

Genetic and Molecular Analysis of Wild-Derived Arrhythmic Mice

A new circadian variant was isolated by screening the intercross offspring of wild-caught mice (Mus musculus castaneus). This variant was characterized by an initial maintenance of damped oscillations and subsequent loss of rhythmicity after being transferred from light-dark (LD) cycles to constant darkness (DD). To map the genes responsible for the persistence of rhythmicity (circadian ratio) and the length of free-running period (τ), quantitative trait locus (QTL) analysis was performed using F₂ mice obtained from an F₁ cross between the circadian variant and C57BL/6J mice. As a result, a significant QTL with a main effect for circadian ratio (Arrhythmicity; Arrh-1) was mapped on Chromosome (Chr) 8. For τ, four significant QTLs, Short free-running period (Sfp-1) (Chr 1), Sfp-2 (Chr 6), Sfp-3 (Chr 8), Sfp-4 (Chr 11) were determined. An epistatic interaction was detected between Chr 3 (Arrh-2) and Chr 5 (Arrh-3). An in situ hybridization study of clock genes and mouse Period1::luciferase (mPer1::luc) real-time monitoring analysis in the suprachiasmatic nucleus (SCN) suggested that arrhythmicity in this variant might not be attributed to core circadian mechanisms in the SCN neurons. Our strategy using wild-derived variant mice may provide a novel opportunity to evaluate circadian and its related disorders in human that arise from the interaction between multiple variant genes.     

Transgenic,inducible RNAi in megakaryocytes and platelets in mice

Summary Background: RNA interference (RNAi) is a powerful tool for suppressing gene function. The tetracycline (tet)‐regulated expression system has recently been adapted to allow inducible RNAi in mice, however its efficiency in a particular cell type in vivo depends on a transgenic tet transactivator expression pattern and is often highly variable. Objective: We aimed to establish a transgenic strategy that allows efficient and inducible gene knockdown in particular hematopoietic lineages in mice. Methods and results: Using a tet‐regulated reporter gene strategy, we found that transgenic mice expressing the rtTA (tet‐on) transactivator under control of the cytomegalovirus (CMV) promoter (CMV‐rtTA mice) display inducible reporter gene expression with unusual and near‐complete efficiency in megakaryocytes and platelets. To test whether the CMV‐rtTA transgene can drive inducible and efficient gene knockdown within this lineage, we generated a novel mouse strain harboring a tet‐regulated short hairpin RNA (shRNA) targeting Bcl‐x_L, a pro‐survival Bcl‐2 family member known to be essential for maintaining platelet survival. Doxycycline treatment of adult mice carrying both transgenes induces shRNA expression, depletes Bcl‐x_L in megakaryocytes and triggers severe thrombocytopenia, whereas doxycycline withdrawal shuts off shRNA expression, normalizes Bcl‐x_L levels and restores platelet numbers. These effects are akin to those observed with drugs that target Bcl‐x_L, clearly demonstrating that this transgenic system allows efficient and inducible inhibition of genes in megakaryocytes and platelets. Conclusions: We have established a novel transgenic strategy for inducible gene knockdown in megakaryocytes and platelets that will be useful for characterizing genes involved in platelet production and function in adult mice.     

Fluoxetine normalizes disrupted light-induced entrainment,fragmented ultradian rhythms and altered hippocampal clock gene expression in an animal model of high trait anxiety- and depression-related behavior

Introduction Disturbances of circadian rhythms are a key symptom of mood and anxiety disorders. Selective serotonin reuptake inhibitors (SSRIs) - commonly used antidepressant drugs – also modulate aspects of circadian rhythmicity. However, their potential to restore circadian disturbances in depression remains to be investigated.

Materials and methods The effects of the SSRI fluoxetine on genetically based, depression-related circadian disruptions at the behavioral and molecular level were examined using mice selectively bred for high anxiety-related and co-segregating depression-like behavior (HAB) and normal anxiety/depression behavior mice (NAB).

Results The length of the circadian period was increased in fluoxetine-treated HAB as compared to NAB mice while the number of activity bouts and light-induced entrainment were comparable. No difference in hippocampal Cry2 expression, previously reported to be dysbalanced in untreated HAB mice, was observed, while Per2 and Per3 mRNA levels were higher in HAB mice under fluoxetine treatment.

Discussion The present findings provide evidence that fluoxetine treatment normalizes disrupted circadian locomotor activity and clock gene expression in a genetic mouse model of high trait anxiety and depression. An interaction between the molecular mechanisms mediating the antidepressant response to fluoxetine and the endogenous regulation of circadian rhythms in genetically based mood and anxiety disorders is proposed.     

15-deoxy-Δ12,14 prostaglandin J2-induced heme oxygenase-1 in megakaryocytes regulates thrombopoiesis

Summary.  Background:  Platelet production is an intricate process that is poorly understood. Recently, we demonstrated that the natural peroxisome proliferator-activated receptor gamma (PPARγ) ligand, 15-deoxy-Δ^12,14 prostaglandin J₂ (15d-PGJ₂), augments platelet numbers by increasing platelet release from megakaryocytes through the induction of reactive oxygen species (ROS). 15d-PGJ₂ can exert effects independent of PPARγ, such as increasing oxidative stress. Heme oxygenase-1 (HO-1) is a potent antioxidant and may influence platelet production. Objectives:  To further investigate the influence of 15d-PGJ₂ on megakaryocytes and to understand whether HO-1 plays a role in platelet production. Methods:  Meg-01 cells (a primary megakaryoblastic cell line) and primary human megakaryocytes derived from cord blood were used to examine the effects of 15d-PGJ₂ on HO-1 expression in megakaryocytes and their daughter platelets. The role of HO-1 activity in thrombopoiesis was studied using established in vitro models of platelet production. Results and conclusions:  15d-PGJ₂ potently induced HO-1 protein expression in Meg-01 cells and primary human megakaryocytes. The platelets produced from these megakaryocytes also expressed elevated levels of HO-1. 15d-PGJ₂-induced HO-1 was independent of PPARγ, but could be replicated using other electrophilic prostaglandins, suggesting that the electrophilic properties of 15d-PGJ₂ were important for HO-1 induction. Interestingly, inhibiting HO-1 activity enhanced ROS generation and augmented 15d-PGJ₂-induced platelet production, which could be attenuated by antioxidants. These new data reveal that HO-1 negatively regulates thrombopoiesis by inhibiting ROS.     

Delayed circadian rhythms and substance abuse: dopamine transmission’s time has come

Disrupted sleep and circadian rhythms are linked with substance abuse risk. Human studies that investigate relationships between sleep, circadian rhythm, and substance use reward generally rely on indirect means to infer dopaminergic function, such as functional magnetic resonance imaging. In this issue of the JCI, Zhang and colleagues used positron emission tomography (PET) to image striatal dopamine D1 (D1R) and D2/3 receptor (D/3R) availability in healthy adults. The authors assessed rest-activity rhythms, then conducted PET scans using radioligand antagonists selective for D1 receptors or D2/D3 receptors to measure D1R and D2/3R availability. They also measured the subjective drug effects of oral methylphenidate. Higher D1R availability in caudate and a greater methylphenidate reward sensitivity were associated with delayed rest-activity rhythms. Unexpectedly, lower overall activity was associated with higher D2/3R availability in the nucleus accumbens, which coincided with greater methylphenidate reward score. These findings may inform personalized prevention and/or treatment interventions.     

Circadian rhythms in drug action and drug metabolism in the mouse.

The relationship between circadian rhythms in the pharmacological actions of meperidine and hexobarbital and similar rhythms in the hepatic metabolism of these drugs was examined in mice under a variety of environmental alterations to determine whether such rhythms may be causally related. The rate of metabolism of p-nitroanisole and hexobarbital by hepatic 9000 X g supernatant fractions was found to be higher at 2400 hours (middark phase) compared to 1200 hours (midlight phase). The rhythms in in vitro hexobarbital and in vivo merperidine metabolism were inversely related in time with similar rhythms in duration of hexobarbital sleep time and meperidine analgesia. Exposure of mice to continuous lighting abolished the rhythms in metabolism and response to meperidine and hexobarbital. Reversal of the usual lighting cycle inverted the rhythm in hexobarbital metablism while abolishing the rhythm in pharmacological response to hexobarbital; meperidine was similarly affected. Adrenalectomy abolished the rhythm in hexobarbital metabolism, diminished the amplitude of the circadian variation in meperidine metabolism and abolished the rhythm in hexobarbital hypnosis and meperidine analgesia. These results indicate that circadian rhythms in the action of hexobarbital and meperidine are well correlated with similar rhythms in the disposition of these drugs.     

CLUSTER AND TRIGEMINAL AUTONOMIC CEPHALALGIAS

The suprachiasmatic nucleus (SCN) of the hypothalamus has been termed the master circadian pacemaker of mammals. Recent discoveries of damped circadian oscillators in other tissues have led to the hypothesis that the SCN synchronizes and sustains daily rhythms in these tissues. We studied the effects of constant lighting (LL) and of SCN lesions on behavioral rhythmicity and Period 1 (Per 1) gene activity in the SCN and olfactory bulb (OB). We found that LL had similar effects on cyclic locomotor and feeding behaviors and Per 1 expression in the SCN but had no effect on rhythmic Per 1 expression in the OB. LL lengthened the period of locomotor and SCN rhythms by approximately 1.6 hr. After 2 weeks in LL, nearly 35% of rats lost behavioral rhythmicity. Of these, 90% showed no rhythm in Per 1-driven expression in their SCN. Returning the animals to constant darkness rapidly restored their daily cycles of running wheel activity and gene expression in the SCN. In contrast, the OB remained rhythmic with no significant change in period, even when cultured from animals that had been behaviorally arrhythmic for 1 month. Similarly, we found that lesions of the SCN abolished circadian rhythms in behavior but not in the OB. Together, these results suggest that LL causes the SCN to lose circadian rhythmicity and its ability to coordinate daily locomotor and feeding rhythms. The SCN, however, is not required to sustain all rhythms because the OB continues to oscillate in vivo when the SCN is arrhythmic or ablated.
Comments: As the central generator for cluster appears to be near the hypothalamic circadian nuclei (May A, Bahra A, Buchel C, Frackowiak RS, Goadsby PJ. PET and MRA findings in cluster headache and MRA in experimental pain. Neurology. 2000;55:1328-1335), it behooves us to follow the work on understanding the training of circadian and circannual rhythms in order to better understand cluster. Stewart J. Tepper     

2,2,2-Tribromoethanol phase-shifts the circadian rhythm of the liver clock in Per2::Luciferase knockin mice: lack of dependence on anesthetic activity

Comprehensive gene expression profiling in mice in response to the inhalation of sevoflurane has revealed that circadian clock gene expression is affected strongly in the liver, heart, lung, and kidney, in this order, but moderately in the spleen and slightly in the brain. Therefore, we examined whether the administration of general anesthetics at different times of the day induces phase shifts of the liver clock in Per2::Luciferase knockin mice. One to 4 days of intraperitoneal injection of 2,2,2-tribromoethanol (240 mg/kg, anesthetic time 60 min) or 2,2,2-trichloroethanol (240 mg/kg, 60 min), common anesthetics in veterinary surgery, caused phase delays when injected during the daytime and phase advances when injected during the nighttime. Inhalation administration of isoflurane for 30 or 60 min during the daytime did not induce a phase delay. Injection of propofol (300 mg/kg, 17 min) during the daytime induced an insignificant phase delay of the Per2 bioluminescence rhythm. Injection of 2,2,2-tribromoethanol did not induce a phase shift in the suprachiasmatic nucleus, the main oscillator, or in behavioral locomotor rhythms, suggesting that 2,2,2-tribromoethanol induced phase shifts of the liver clock independent of the main suprachiasmatic clock. The expression of clock genes, such as Bmal1 and Clock, in mouse liver was decreased strongly 1 and 4 h after a single injection of 2,2,2-tribromoethanol. These results demonstrate that 2,2,2-tribromoethanol or 2,2,2-trichloroethanol produce phase shifts of the peripheral clock, independent of anesthetic activity. These anesthetics may cause circadian rhythm disorders in peripheral organs when administered as general anesthetics several times during the day.     

Functional relationship between circadian rhythm and control of food intake

Living things on the earth including bacteria, plants and animals show circadian rhythms in their behaviors and physiological phenomena, and these circadian rhythms are usually synchronized with environmental changes having the period of 24 h on the earth. In mammals including human beings, the hypothalamic suprachiasmatic nucleus (SCN) functions as a master circadian oscillator, and generates a circadian rhythm of food intake. Sometimes the circadian oscillation of the SCN is disturbed with physical and psychological stressors. This review describes the functional relationship in respect to connections between the circadian oscillator in the SCN and food regulatory centers and neurons in the brain focusing on its mechanism in human beings, and a possible involvement of the circadian oscillator of the SCN in the abnormality of the appetite control.     

Cross-talk between the circadian clock and the cell cycle in cancer

Abstract

The circadian clock is an endogenous timekeeper system that controls the daily rhythms of a variety of physiological processes. Accumulating evidence indicates that genetic changes or unhealthy lifestyle can lead to a disruption of circadian homeostasis, which is a risk factor for severe dysfunctions and pathologies including cancer. Cell cycle, proliferation, and cell death are closely intertwined with the circadian clock, and thus disruption of circadian rhythms appears to be linked to cancer development and progression. At the molecular level, the cell cycle machinery and the circadian clocks are controlled by similar mechanisms, including feedback loops of genes and protein products that display periodic activation and repression. Here, we review the circadian rhythmicity of genes associated with the cell cycle, proliferation, and apoptosis, and we highlight the potential connection between these processes, the circadian clock, and neoplastic transformations. Understanding these interconnections might have potential implications for the prevention and therapy of malignant diseases.