Abstract
BackgroundCircadian rhythms are endogenous, self-sustained oscillations with approximately 24-hr rhythmicity that are manifested in various physiological and metabolic processes. The circadian organization of these processes in mammals is governed by the master oscillator within the suprachiasmatic nuclei (SCN) of the hypothalamus. Recent findings revealed that circadian oscillators exist in most organs, tissues, and even in immortalized cells, and that the oscillators in peripheral tissues are likely to be coordinated by SCN, the master oscillator. Some candidates for endogenous entrainment factors have sporadically been reported, however, their details remain mainly obscure.ResultsWe developed the in vitro real-time oscillation monitoring system (IV-ROMS) by measuring the activity of luciferase coupled to the oscillatory gene promoter using photomultiplier tubes and applied this system to screen and identify factors able to influence circadian rhythmicity. Using this IV-ROMS as the primary screening of entrainment factors for circadian clocks, we identified 12 candidates as the potential entrainment factor in a total of 299 peptides and bioactive lipids. Among them, four candidates (endothelin-1, all-trans retinoic acid, 9-cis retinoic acid, and 13-cis retinoic acid) have already been reported as the entrainment factors in vivo and in vitro. We demonstrated that one of the novel candidates, 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), a natural ligand of the peroxisome proliferator-activated receptor-γ (PPAR-γ), triggers the rhythmic expression of endogenous clock genes in NIH3T3 cells. Furthermore, we showed that 15d-PGJ2 transiently induces Cry1, Cry2, and Rorα mRNA expressions and that 15d-PGJ2-induced entrainment signaling pathway is PPAR-γ – and MAPKs (ERK, JNK, p38MAPK)-independent.ConclusionHere, we identified 15d-PGJ2 as an entrainment factor in vitro. Using our developed IV-ROMS to screen 299 compounds, we found eight novel and four known molecules to be potential entrainment factors for circadian clocks, indicating that this assay system is a powerful and useful tool in initial screenings.
Highlights
IntroductionSelf-sustained oscillations with approximately 24-hr rhythmicity that are manifested in various physiological and metabolic processes
Circadian rhythms are endogenous, self-sustained oscillations with approximately 24-hr rhythmicity that are manifested in various physiological and metabolic processes
Per2 is considered to be one of the core molecule for molecular clocks since gene-knockout analysis revealed that mouse Per2 (mPer2) mutants display a shorted circadian period followed by a loss of circadian rhythmicity in constant darkness [33]
Summary
Self-sustained oscillations with approximately 24-hr rhythmicity that are manifested in various physiological and metabolic processes. Circadian rhythms are endogenous self-sustained oscillations with approximately 24-hr rhythmicity that are manifested in various physiological and metabolic processes [1,2] In mammals the circadian orchestration of these processes is governed by pacemaker cells located within the suprachiasmatic nuclei (SCN) of the hypothalamus. Principally two basic helix-loop-helix-PAS transcriptional factors, CLOCK and BMAL1, regulate gene expression by interacting with a promoter element termed E-box [12,13] Target genes of these transcriptional factors include several repressor proteins, including PER1, PER2, PER3, CRY1, and CRY2, which function to inhibit the activity of CLOCK/BMAL1 complex by entering into the nucleus [14,15], thereby generating a circadian oscillation of their own transcription
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