The in vitro real-time oscillation monitoring system identifies potential entrainment factors for circadian clocks.
BACKGROUND
Circadian 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.


RESULTS
We 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-Delta12,14-prostaglandin J2 (15d-PGJ2), a natural ligand of the peroxisome proliferator-activated receptor-gamma (PPAR-gamma), triggers the rhythmic expression of endogenous clock genes in NIH3T3 cells. Furthermore, we showed that 15d-PGJ2 transiently induces Cry1, Cry2, and Roralpha mRNA expressions and that 15d-PGJ2-induced entrainment signaling pathway is PPAR-gamma--and MAPKs (ERK, JNK, p38MAPK)-independent.


CONCLUSIONS
Here, 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.
Nakahata
Yasukazu
Y
Osaka Bioscience Institute, Suita, Osaka 565-0874, Japan. yasu_nakahata@yahoo.co.jp
Akashi
Makoto
M
Trcka
Daniel
D
Yasuda
Akio
A
Takumi
Toru
T
D000818:Animals; D030801:Animals, Genetically Modified; D001695:Biology / Q000379:methods; D018797:Cell Cycle Proteins; D002460:Cell Line; D002940:Circadian Rhythm; D003199:Computer Systems; D005347:Fibroblasts / Q000378:metabolism; D005786:Gene Expression Regulation; D005821:Genetic Techniques; D007031:Hypothalamus / Q000502:physiology; D017403:In Situ Hybridization; D066298:In Vitro Techniques; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D041681:NIH 3T3 Cells; D009687:Nuclear Proteins / Q000235:genetics; D009991:Oscillometry; D047495:PPAR gamma / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D056950:Period Circadian Proteins; D017440:Photoperiod; D011401:Promoter Regions, Genetic; D015230:Prostaglandin D2 / Q000031:analogs & derivatives / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D015398:Signal Transduction; D013493:Suprachiasmatic Nucleus / Q000502:physiology; D013997:Time Factors; D014157:Transcription Factors / Q000235:genetics; D015854:Up-Regulation
C477819:15-deoxyprostaglandin J2; D018797:Cell Cycle Proteins; D009687:Nuclear Proteins; D047495:PPAR gamma; D010455:Peptides; C513312:Per2 protein, mouse; C513311:Per2 protein, rat; D056950:Period Circadian Proteins; D012333:RNA, Messenger; D014157:Transcription Factors; D008156:Luciferases; D015230:Prostaglandin D2