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Dysfunction of the circadian clock has been implicated in the pathogenesis of cardiovascular disease. The CLOCK protein is a core molecular component of the circadian oscillator, so that mice with a mutated Clock gene (Clk/Clk) exhibit abnormal rhythms in numerous physiological processes. However, here we report that chronic kidney disease (CKD)-induced cardiac inflammation and fibrosis are attenuated in Clk/Clk mice even though they have high blood pressure and increased serum angiotensin II levels. A search for the underlying cause of the attenuation of heart disorder in Clk/Clk mice with 5/6 nephrectomy (5/6Nx) led to identification of the monocytic expression of G protein-coupled receptor 68 (GPR68) as a risk factor of CKD-induced inflammation and fibrosis of heart. 5/6Nx induces the expression of GPR68 in circulating monocytes via altered CLOCK activation by increasing serum levels of retinol and its binding protein (RBP4). The high-GPR68-expressing monocytes have increased potential for producing inflammatory cytokines, and their cardiac infiltration under CKD conditions exacerbates inflammation and fibrosis of heart. Serum retinol and RBP4 levels in CKD patients are also sufficient to induce the expression of GPR68 in human monocytes. Our present study reveals an uncovered role of monocytic clock genes in CKD-induced heart failure.

Daily rhythmic variations in biological functions affect the efficacy and/or toxicity of drugs: a large number of drugs cannot be expected to exhibit the same potency at different administration times. The "circadian clock" is an endogenous timing system that broadly regulates metabolism, physiology and behavior. In mammals, this clock governs the oscillatory expression of the majority of genes with a period length of approximately 24 h. Genetic studies have revealed that molecular components of the circadian clock regulate the expression of genes responsible for the sensitivity to drugs and their disposition. The circadian control of pharmacodynamics and pharmacokinetics enables 'chrono-pharmaceutical' applications, namely drug administration at appropriate times of day to optimize the therapeutic index (efficacy vs. toxicity). On the other hand, a variety of pathological conditions also exhibit marked day-night changes in symptom intensity. Currently, novel therapeutic approaches are facilitated by the development of chemical compound targeted to key proteins that cause circadian exacerbation of disease events. This review presents an overview of the current understanding of the role of the circadian biological clock in regulating drug efficacy and disease conditions, and also describes the importance of identifying the difference in the circadian machinery between diurnal and nocturnal animals to select the most appropriate times of day to administer drugs in humans.

In mammals, the circadian pacemaker resides in the paired suprachiasmatic nuclei (SCN) and influences a multitude of biological processes, including the sleep-wake rhythm. Circadian rhythms regulate diverse physiologic processes, including homeostatic functions of steroid hormones and their receptors. Perturbation of these rhythms is associated with pathogenic conditions such as cancer, metabolic syndrome, cardiovascular disease, sleep disorder and depression. Clock genes ultimately control a vast array of circadian rhythms involved in physiology and behavior. They regulate several diseases described above. Chronotherapy is especially relevant when the risk and/or intensity of symptoms of a disease vary predictably over time. The effectiveness and toxicity of several drugs vary depending on the dosing time. Such chronopharmacological phenomena are influenced by not only the pharmacodynamics but also the pharmacokinetics of a medication. The underlying mechanisms are associated with the 24-h rhythms of biochemical, physiological, and behavioral processes under the control of the circadian clock. Identifying a rhythmic marker based on the molecular clock for choosing dosing time can lead to the progress and diffusion of chronopharmacotherapy. To monitor the rhythmic markers such as clock genes, it might be useful to choose the most appropriate time of a day for the administration of a drug, to increase its therapeutic effects and/or reduce its side effects. On the contrary, several drugs affect the molecular clock and alter the 24-h rhythms of various processes. Alterations in rhythmicity are sometimes associated with therapeutic effects, or it might lead to illness and altered homeostatic regulation. Furthermore, to produce new rhythmicity by manipulating the molecular clock of organs by rhythmic administration of drugs at altered feeding schedules appears to lead to a new concept of chronopharmacotherapy. An approach to increase the efficiency of pharmacotherapy is administering drugs at times when they are best tolerated. From the perspective of pharmaceutics, the application of biological rhythm to pharmacotherapy can be accomplished by the appropriate timing of administration of conventionally formulated tablets and capsules, and also by the use of special drug-delivery system to synchronize drug concentrations to the rhythms in disease activity. New drugs targeting the molecular clock are being developed to manage diseases in human. For instance, novel molecular mechanisms that mediate renal dysfunction in mice with chronic kidney disease (CKD) have been identified by examining the relationship between the circadian clock and CKD aggravation. The inhibition of cell cycle regulatory factor ameliorated renal inflammation in a mouse model of CKD. A novel inhibitor of cell cycle regulatory factor has been identified, supporting the potential utility of cell cycle regulatory factor inhibition in the treatment of CKD. Although malignant phenotypes of triple-negative breast cancer are subject to circadian alterations, the role of cancer stem cells (CSCs) in defining this circadian change remains unclear. The effectiveness of anticancer drugs varies with the circadian dynamics of CSCs, which are regulated by the tumor microenvironmental factors. The finding reveals that the circadian dynamics of CSCs are regulated by the tumor microenvironment and provides a proof of principle of its implication for chronotherapy against triple-negative breast cancer. Therefore, we present an overview of the dosing-time-dependent alterations in therapeutic outcome and safety of a drug and the regulatory system of biological rhythm from the perspective of clock genes and the possibility of pharmacotherapy based on the molecular clock., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Although malignant phenotypes of triple-negative breast cancer (TNBC) are subject to circadian alterations, the role of cancer stem cells (CSC) in defining this circadian change remains unclear. CSC are often characterized by high aldehyde dehydrogenase (ALDH) activity, which is associated with the malignancy of cancer cells and is used for identification and isolation of CSC. Here, we show that the population of ALDH-positive cells in a mouse 4T1 breast tumor model exhibits pronounced circadian alterations. Alterations in the number of ALDH-positive cells were generated by time-dependent increases and decreases in the expression of Aldh3a1 Importantly, circadian clock genes were rhythmically expressed in ALDH-negative cells, but not in ALDH-positive cells. Circadian expression of Aldh3a1 in ALDH-positive cells was dependent on the time-dependent release of Wingless-type mmtv integration site family 10a (WNT10a) from ALDH-negative cells. Furthermore, antitumor and antimetastatic effects of ALDH inhibitor N,N-diethylaminobenzaldehyde were enhanced by administration at the time of day when ALDH activity was increased in 4T1 tumor cells. Our findings reveal a new role for the circadian clock within the tumor microenvironment in regulating the circadian dynamics of CSC. These results should enable the development of novel therapeutic strategies for treatment of TNBC with ALDH inhibitors. Significance: This seminal report reveals that circadian dynamics of CSC are regulated by the tumor microenvironment and provides a proof of principle of its implication for chronotherapy in TNBC. Cancer Res; 78(13); 3698-708. ©2018 AACR ., (©2018 American Association for Cancer Research.)

A number of diverse cell-surface proteins are anchored to the cytoskeleton via scaffold proteins. Na + /H + exchanger regulatory factor-1 (NHERF1), encoded by the Slc9a3r1 gene, functions as a scaffold protein, which is implicated in the regulation of membrane expression of various cell-surface proteins. Here, we demonstrate that the circadian clock component PERIOD2 (PER2) modulates transcription of the mouse Slc9a3r1 gene, generating diurnal accumulation of NHERF1 in the mouse liver. Basal expression of Slc9a3r1 was dependent on transcriptional activation by p65/p50. PER2 bound to p65 protein and prevented p65/p50-mediated transactivation of Slc9a3r1. The time-dependent interaction between PER2 and p65 underlay diurnal oscillation in the hepatic expression of Slc9a3r1/NHERF1. The results of immunoprecipitation experiments and liquid chromatography-mass spectrometry analysis of mouse liver revealed that NHERF1 time-dependently interacted with fatty acid transport protein-5 (FATP5). Temporary accumulation of NHERF1 protein stabilized plasmalemmal localization of FATP5, thereby enhancing hepatic uptake of fatty acids at certain times of the day. Our results suggest an unacknowledged role for PER2 in regulating the diurnal expression of NHERF1 in mouse liver. This machinery also contributed to diurnal changes in the ability of hepatic cells to uptake fatty acids.

In mammals, circadian rhythms in physiological function are generated by a molecular oscillator driven by transcriptional-translational feedback loop consisting of negative and positive regulators. Disruption of this circadian clock machinery is thought to increase the risk of cancer development, but the potential contributions of each component of circadian clock to oncogenesis have been little explored. Here we reported that negative and positive transcriptional regulators of circadian feedback loop had different roles in oncogene-induced neoplastic transformation. Mouse embryonic fibroblasts prepared from animals deficient in negative circadian clock regulators, Period2 (Per2) or Cryptochrome1/2 (Cry1/2), were prone to transformation induced by co-expression of H-ras(V12) and SV40 large T antigen (SV40LT). In contrast, mouse embryonic fibroblasts prepared from mice deficient in positive circadian clock regulators, Bmal1 or Clock, showed resistance to oncogene-induced transformation. In Per2 mutant and Cry1/2-null cells, the introduction of oncogenes induced expression of ATF4, a potent repressor of cell senescence-associated proteins p16INK4a and p19ARF. Elevated levels of ATF4 were sufficient to suppress expression of these proteins and drive oncogenic transformation. Conversely, in Bmal1-null and Clock mutant cells, the expression of ATF4 was not induced by oncogene introduction, which allowed constitutive expression of p16INK4a and p19ARF triggering cellular senescence. Although genetic ablation of either negative or positive transcriptional regulators of the circadian clock leads to disrupted rhythms in physiological functions, our findings define their different contributions to neoplastic cellular transformation., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Iron is an important biological catalyst and is critical for DNA synthesis during cell proliferation. Cellular iron uptake is enhanced in tumor cells to support increased DNA synthesis. Circadian variations in DNA synthesis and proliferation have been identified in tumor cells, but their relationship with intracellular iron levels is unclear. In this study, we identified a 24-h rhythm in iron regulatory protein 2 (IRP2) levels in colon-26 tumors implanted in mice. Our findings suggest that IRP2 regulates the 24-h rhythm of transferrin receptor 1 (Tfr1) mRNA expression post-transcriptionally, by binding to RNA stem-loop structures known as iron-response elements. We also found thatIrp2mRNA transcription is promoted by circadian clock genes, including brain and muscle Arnt-like 1 (BMAL1) and the circadian locomotor output cycles kaput (CLOCK) heterodimer. Moreover, growth in colon-26(Δ19) tumors expressing the clock-mutant protein (CLOCK(Δ19)) was low compared with that in wild-type colon-26 tumor. The time-dependent variation of cellular iron levels, and the proliferation rate in wild-type colon-26 tumor was decreased by CLOCK(Δ19)expression. Our findings suggest that circadian organization contributes to tumor cell proliferation by regulating iron metabolism in the tumor., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Cisplatin, cis-diamminedichloro-platinum (CDDP), is a widely used anticancer agent, the clinical applications of which have been limited by severe nephrotoxicity. Although dosing time-dependent differences in CDDP-induced nephrotoxicity have been reported in both humans and laboratory animals, the underlying mechanism remains unknown. In the present study, we investigated the molecular mechanism for the dosing-time dependency of the nephrotoxic effect of CDDP in mice. CDDP-induced nephrotoxicity was significantly attenuated by injecting CDDP at times of the day when its renal clearance was enhanced. The dosing-time dependency of the nephrotoxic effect was parallel to that of CDDP incorporation into renal DNA. Two types of transporters, organic cation transporter 2 (OCT2, encoded by Slc22a2) and multidrug and toxin extrusion 1 (MATE1, encoded by Slc47a1), are responsible for the renal excretion of CDDP. The expression of OCT2, but not MATE1, exhibited a significant time-dependent oscillation in the kidneys of mice. The circadian expression of OCT2 was closely related to the dosing-time dependency of CDDP incorporation into renal DNA. Molecular components of the circadian clock regulated the renal expression of Slc22a2 mRNA by mediating peroxisome proliferator-activated receptor-α, which resulted in rhythmic oscillations in OCT2 protein levels. These findings indicate a clock-regulated mechanism of dosing time-dependent changes in CDDP-induced nephrotoxicity and also suggest a molecular link between the circadian clock and renal xenobiotic excretion.

ABCG2, encoding breast cancer resistance protein (BCRP), is a member of the ATP-binding cassette transporter family and is often associated with cancer chemotherapeutic resistance. BCRP is also expressed in a variety of normal cells and acts as a xenobiotic efflux transporter. Because intestinal BCRP limits systemic exposure to xenobiotics, alterations in the function and expression of this transporter could account for part of the variation in oral drug absorption. In this study, we show that ATF4, a molecular component of the circadian clock, induces circadian expression of the Abcg2 gene in mouse small intestine. Three types of leader exons (termed exons 1A, 1B, and 1C) are identified in the 5'-untranslated region of mouse Abcg2 transcripts. The exon 1B-containing Abcg2 transcript was the only isoform detected in mouse small intestine, and its mRNA levels oscillated in a circadian time-dependent manner. ATF4 bound time-dependently to the cAMP response element within the exon 1B promoter region of the Abcg2 gene, thereby causing the oscillation of BCRP protein abundance and its efflux pump function. The circadian clock-ATF4 pathway appears to enhance the function of BCRP during a specific time window and to modulate intestinal drug absorption. Our findings suggest a mechanism underlying circadian change in xenobiotic detoxification.

Activating transcription factor (ATF)/cAMP-response element (CRE)-binding (CREB) proteins induce the CRE-mediated gene transcription depending on the cAMP stimulation. cAMP-dependent signaling oscillates in a circadian manner, which in turn also sustains core oscillation machinery of the circadian clock. Here, we show that among the ATF/CREB family proteins, ATF4 is essential for the circadian expression of the Period2 (Per2) gene, a key component of the circadian clock. Transcription of the Atf4 gene was regulated by core components of the circadian clock, and its expression exhibited circadian oscillation in mouse tissues as well as embryonic fibroblasts. ATF4 bound to the CRE of the Per2 promoter in a circadian time-dependent manner and periodically activated the transcription of the Per2 gene. Consequently, the oscillation of the Per2 expression was attenuated in embryonic cells prepared from Atf4-null mice. Furthermore, the loss of ATF4 also disrupted the rhythms in the expression of other clock genes. These results suggest that ATF4 is a component responsible for sustaining circadian oscillation of CRE-mediated gene expression and also constitute a molecular link connecting cAMP-dependent signaling to the circadian clock.

In all living organisms, one of the most indispensable biological functions is the circadian clock (suprachiasmatic nuclei; SCN), which acts like a multifunction timer to regulate homeostatic systems such as sleep and activity, hormone levels, appetite, and other bodily functions with 24h cycles. Circadian rhythms regulate diverse physiologic processes, including homeostatic functions of steroid hormones and their receptors. Perturbations of these rhythms are associated with pathogenic conditions such as depression, diabetes and cancer. Clock genes are identified as the genes that ultimately control a vast array of circadian rhythms in physiology and behavior. Clock gene regulates several diseases such as cancer, metabolic syndrome and sleep etc. CLOCK mutation affects the expression of rhythmic genes in wild-type (WT) tissue, but also affects that of non-rhythmic genes. On the other hand, the change of the drug pharmacodynamic and pharmacokinetic (PK/PD) parameters are influenced by not only inter-individual variability but also intra-individual variabilities of medications. Identification of a rhythmic marker for selecting dosing time will lead to improved progress and diffusion of chronopharmacotherapy. The mechanisms underlying chronopharmacological findings should be clarified from viewpoint of clock genes. On the other hand, several drugs have an effect on molecular clock. Thus, the knowledge of intra- and inter-individual variability of molecular clock should be applied for the clinical practice. Therefore, we introduce the regulatory system of biological rhythm from viewpoints of clock genes and the possibility of pharmacotherapy based on the intra- and inter-individual variability of clock genes., (2010 Elsevier B.V. All rights reserved.)

Bronchial asthma is characterized by chronic airways inflammation and reversible airflow limitation. In patients with asthma, symptoms generally worsen during the early hours of the morning, and pulmonary function often deteriorates at the same time, suggesting a role for chronopharmacotherapy. Several drugs for asthma have been developed based on chronopharmacology. Most medications employed for the chronotherapy of asthma are administered once at night with the goal of preventing chronic airway inflammation or development of airflow limitation. In addition to bronchodilators, the inhaled glucocorticosteroid ciclesonide is now available with once-daily dosing, which also improves patients' compliance. Numerous investigations have demonstrated the usefulness of chronotherapy for asthma, especially for patients with nocturnal asthma. This review focuses on chronotherapy of asthma, and also provides a molecular biological explanation for the influence of asthma medications on the clock genes., (2010 Elsevier B.V. All rights reserved.)