Nakahata Yasukazu, Akashi Makoto, Trcka Daniel, Yasuda Akio, Takumi Toru
Osaka Bioscience Institute, Suita, Osaka 565-0874, Japan.
BMC Mol Biol. 2006 Feb 16;7:5. doi: 10.1186/1471-2199-7-5.
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.
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.
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.
昼夜节律是内源性的、自我维持的振荡，具有约24小时的节律性，表现在各种生理和代谢过程中。哺乳动物这些过程的昼夜组织由下丘脑视交叉上核（SCN）内的主振荡器控制。最近的研究发现，大多数器官、组织甚至永生化细胞中都存在昼夜振荡器，并且外周组织中的振荡器可能由主振荡器SCN协调。然而，一些内源性夹带因子的候选物已被零星报道，但其细节仍主要不清楚。
我们通过使用光电倍增管测量与振荡基因启动子偶联的荧光素酶的活性，开发了体外实时振荡监测系统（IV-ROMS），并应用该系统筛选和鉴定能够影响昼夜节律性的因子。使用该IV-ROMS作为昼夜节律时钟夹带因子的初步筛选，我们在总共299种肽和生物活性脂质中鉴定出12种候选物作为潜在的夹带因子。其中，四种候选物（内皮素-1、全反式维甲酸、9-顺式维甲酸和13-顺式维甲酸）已被报道为体内和体外的夹带因子。我们证明了一种新的候选物，15-脱氧-Δ12,14-前列腺素J2（15d-PGJ2），过氧化物酶体增殖物激活受体-γ（PPAR-γ）的天然配体，触发NIH3T3细胞中内源性时钟基因的节律性表达。此外，我们表明15d-PGJ2瞬时诱导Cry1、Cry2和Roralpha mRNA表达，并且15d-PGJ2诱导的夹带信号通路独立于PPAR-γ和丝裂原活化蛋白激酶（ERK、JNK、p38MAPK）。
在这里，我们在体外鉴定出15d-PGJ2作为夹带因子。使用我们开发的IV-ROMS筛选299种化合物，我们发现八种新的和四种已知的分子是昼夜节律时钟的潜在夹带因子，表明该检测系统是初始筛选中一种强大而有用的工具。
