title: Regulation of molecular clock oscillations and phagocytic activity via muscarinic Ca2+signaling in human retinal pigment epithelial cells.
abstract: Vertebrate eyes are known to contain circadian clocks, however, the intracellular mechanisms regulating the retinal clockwork remain largely unknown. To address this, we generated a cell line (hRPE-YC) from human retinal pigmental epithelium, which stably co-expressed reporters for molecular clock oscillations (Bmal1-luciferase) and intracellular Ca2+concentrations (YC3.6). The hRPE-YC cells demonstrated circadian rhythms in Bmal1 transcription. Also, these cells represented circadian rhythms in Ca2+-spiking frequencies, which were canceled by dominant-negative Bmal1 transfections. The muscarinic agonist carbachol, but not photic stimulation, phase-shifted Bmal1 transcriptional rhythms with a type-1 phase response curve. This is consistent with significant M3 muscarinic receptor expression and little photo-sensor (Cry2 and Opn4) expression in these cells. Moreover, forskolin phase-shifted Bmal1 transcriptional rhythm with a type-0 phase response curve, in accordance with long-lasting CREB phosphorylation levels after forskolin exposure. Interestingly, the hRPE-YC cells demonstrated apparent circadian rhythms in phagocytic activities, which were abolished by carbachol or dominant-negative Bmal1 transfection. Because phagocytosis in RPE cells determines photoreceptor disc shedding, molecular clock oscillations and cytosolic Ca2+signaling may be the driving forces for disc-shedding rhythms known in various vertebrates. In conclusion, the present study provides a cellular model to understand molecular and intracellular signaling mechanisms underlying human retinal circadian clocks.
authors.affiliation: Graduate School of Science and Engineering, University of Toyama, 3190 Gofuku, Toyama city, Toyama 930-8555, Japan.
authors.forename: Rina
authors.initials: R
authors.lastname: Ikarashi
authors.affiliation: Graduate School of Science and Engineering, University of Toyama, 3190 Gofuku, Toyama city, Toyama 930-8555, Japan.
authors.forename: Honami
authors.initials: H
authors.lastname: Akechi
authors.affiliation: Graduate School of Science and Engineering, University of Toyama, 3190 Gofuku, Toyama city, Toyama 930-8555, Japan.
authors.forename: Yuzuki
authors.initials: Y
authors.lastname: Kanda
authors.affiliation: Graduate School of Innovative Life Science, University of Toyama, 3190 Gofuku, Toyama city, Toyama 930-8555, Japan.
authors.forename: Alsawaf
authors.initials: A
authors.lastname: Ahmad
authors.affiliation: Graduate School of Innovative Life Science, University of Toyama, 3190 Gofuku, Toyama city, Toyama 930-8555, Japan.
authors.forename: Kouhei
authors.initials: K
authors.lastname: Takeuchi
authors.affiliation: Graduate School of Science and Engineering, University of Toyama, 3190 Gofuku, Toyama city, Toyama 930-8555, Japan.
authors.forename: Eri
authors.initials: E
authors.lastname: Morioka
authors.affiliation: Advanced Core Technology Department, Research and Development Division, Olympus Co. Ltd., 2-3 Kuboyama, Hachioji, Tokyo 192-8512, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Sugiyama
authors.affiliation: Department of Psychiatry, Tokyo Metropolitan Police Hospital, 4-22-1 Nakano, Nakano-ku, Tokyo 164-8541, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Ebisawa
authors.affiliation: Department of Physiology, Saitama Medical University, 38 Morohongo, Moroyama, Iruma-gun, Saitama 350-0495, Japan.|Molecular Clock Project, Project Research Division, Research Center for Genomic Medicine, Saitama Medical University, 1397-1 Yamane, Hidaka city, Saitama, 350-1241, Japan.
authors.forename: Masaaki
authors.initials: M
authors.lastname: Ikeda
authors.affiliation: Graduate School of Science and Engineering, University of Toyama, 3190 Gofuku, Toyama city, Toyama 930-8555, Japan.|Graduate School of Innovative Life Science, University of Toyama, 3190 Gofuku, Toyama city, Toyama 930-8555, Japan.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Ikeda
mesh_terms: D001683:Biological Clocks / Q000502:physiology*; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000502:physiology*; D002461:Cell Line, Transformed; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D010587:Phagocytosis / Q000502:physiology*; D043587:Receptor, Muscarinic M3 / Q000096:biosynthesis*; D055213:Retinal Pigment Epithelium / Q000166:cytology / Q000378:metabolism*
chemical_list: D043587:Receptor, Muscarinic M3; D002118:Calcium