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      The Circadian Clock Gene Bmal1 Controls Intestinal Exporter MRP2 and Drug Disposition

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          Abstract

          The intestinal exporter MRP2 plays an important role in disposition and elimination of a wide range of drugs. Here, we aimed to clarify the impact of circadian clock on intestinal MRP2, and to determine the molecular mechanisms for generation of diurnal MRP2 expression.

          Methods: The regulatory effects of Bmal1 on intestinal MRP2 expression were assessed using intestine-specific Bmal1 knockout ( Bmal1 iKO ) mice and colon cancer cells. The relative mRNA and protein levels were determined by qPCR and Western blotting, respectively. Everted gut sac, cell viability and in situ intestinal perfusion experiments were performed to evaluate intestinal efflux of the MRP2 substrate methotrexate (MTX). Toxicity and pharmacokinetic experiments were performed with Bmal1 iKO mice and control littermates ( Bmal1 fl/fl mice) after oral gavage of MTX. Transcriptional gene regulation was investigated using luciferase reporter, mobility shift and chromatin immunoprecipitation (ChIP) assays.

          Results: Bmal1 iKO mice were generated by inter-crossing the mice carrying a Bmal1 exon 8 floxed allele ( Bmal1 fl/fl ) with Villin-Cre mice. Intestinal MRP2 expression exhibited a diurnal oscillation in Bmal1 fl/fl mice with a zenith value at ZT6. Bmal1 ablation caused reductions in Mrp2 mRNA and protein levels [as well as in transport activity (measured by MTX)], and blunted their diurnal rhythms. Intestinal ablation of Bmal1 abrogated circadian time-dependency of MTX pharmacokinetics and toxicity. Bmal1/BMAL1 regulation of rhythmic Mrp2/MRP2 expression was also confirmed in the colon cancer CT26 and Caco-2 cells. Based on a combination of luciferase reporter, mobility shift and ChIP assays, we found that Dbp activated and E4bp4 repressed Mrp2 transcription via specific binding to a same D-box (-100/-89 bp) element in promoter region. Further, Bmal1 directly activated the transcription of Dbp and Rev-erbα through the E-boxes, whereas it negatively regulated E4bp4 via the transcriptional repressor Rev-erbα. Positive regulation of Mrp2 by Rev-erbα was also observed, and attained through modulation of E4bp4.

          Conclusion: Bmal1 coordinates temporal expressions of DBP (a MRP2 activator), REV-ERBα (an E4BP4 repressor) and E4BP4 (a MRP2 repressor), generating diurnal MRP2 expression.

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          Most cited references44

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          The orphan nuclear receptor REV-ERBalpha controls circadian transcription within the positive limb of the mammalian circadian oscillator.

          Mammalian circadian rhythms are generated by a feedback loop in which BMAL1 and CLOCK, players of the positive limb, activate transcription of the cryptochrome and period genes, components of the negative limb. Bmal1 and Per transcription cycles display nearly opposite phases and are thus governed by different mechanisms. Here, we identify the orphan nuclear receptor REV-ERBalpha as the major regulator of cyclic Bmal1 transcription. Circadian Rev-erbalpha expression is controlled by components of the general feedback loop. Thus, REV-ERBalpha constitutes a molecular link through which components of the negative limb drive antiphasic expression of components of the positive limb. While REV-ERBalpha influences the period length and affects the phase-shifting properties of the clock, it is not required for circadian rhythm generation.
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            Molecular architecture of the mammalian circadian clock.

            Circadian clocks coordinate physiology and behavior with the 24h solar day to provide temporal homeostasis with the external environment. The molecular clocks that drive these intrinsic rhythmic changes are based on interlocked transcription/translation feedback loops that integrate with diverse environmental and metabolic stimuli to generate internal 24h timing. In this review we highlight recent advances in our understanding of the core molecular clock and how it utilizes diverse transcriptional and post-transcriptional mechanisms to impart temporal control onto mammalian physiology. Understanding the way in which biological rhythms are generated throughout the body may provide avenues for temporally directed therapeutics to improve health and prevent disease. Copyright © 2013 Elsevier Ltd. All rights reserved.
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              Electrophoretic mobility shift assay (EMSA) for detecting protein-nucleic acid interactions.

              The gel electrophoresis mobility shift assay (EMSA) is used to detect protein complexes with nucleic acids. It is the core technology underlying a wide range of qualitative and quantitative analyses for the characterization of interacting systems. In the classical assay, solutions of protein and nucleic acid are combined and the resulting mixtures are subjected to electrophoresis under native conditions through polyacrylamide or agarose gel. After electrophoresis, the distribution of species containing nucleic acid is determined, usually by autoradiography of 32P-labeled nucleic acid. In general, protein-nucleic acid complexes migrate more slowly than the corresponding free nucleic acid. In this protocol, we identify the most important factors that determine the stabilities and electrophoretic mobilities of complexes under assay conditions. A representative protocol is provided and commonly used variants are discussed. Expected outcomes are briefly described. References to extensions of the method and a troubleshooting guide are provided.
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                Author and article information

                Journal
                Theranostics
                Theranostics
                thno
                Theranostics
                Ivyspring International Publisher (Sydney )
                1838-7640
                2019
                13 April 2019
                : 9
                : 10
                : 2754-2767
                Affiliations
                [1 ]Research Center for Biopharmaceutics and Pharmacokinetics, College of Pharmacy, Jinan University, 601 Huangpu Avenue West, Guangzhou, China.
                [2 ]Integrated Chinese and Western Medicine Postdoctoral research station, Jinan University, 601 Huangpu Avenue West, Guangzhou, China
                Author notes
                ✉ Corresponding author: Baojian Wu, Ph.D., College of Pharmacy, Jinan University. E-mail: bj.wu@ 123456hotmail.com or tbaojianwu@ 123456jnu.edu.cn

                Competing Interests: The authors have declared that no competing interest exists.

                Article
                thnov09p2754
                10.7150/thno.33395
                6568180
                31244920
                1ee36944-360d-448b-b985-17867df31ab7
                © Ivyspring International Publisher

                This is an open access article distributed under the terms of the Creative Commons Attribution (CC BY-NC) license ( https://creativecommons.org/licenses/by-nc/4.0/). See http://ivyspring.com/terms for full terms and conditions.

                History
                : 22 January 2019
                : 31 March 2019
                Categories
                Research Paper

                Molecular medicine
                bmal1,mrp2,dbp,e4bp4,mtx,chronotoxicity
                Molecular medicine
                bmal1, mrp2, dbp, e4bp4, mtx, chronotoxicity

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