The in vitro real-time oscillation monitoring system identifies potential entrainment factors for circadian clocks
The in vitro real-time oscillation monitoring system identifies potential entrainment factors for circadian clocks
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DOI：
10.1186/1471-2199-7-5
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发表时间：
2006-02-16
影响因子：
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通讯作者：
Takumi, T
中科院分区：
文献类型：
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作者：
Nakahata, Y;Akashi, M;Takumi, T
背景资料：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节律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，昼夜节律振荡器存在于大多数器官，组织，甚至在永生化细胞，和周围组织中的振荡器很可能是由SCN，主振荡器协调。一些候选人的内源性夹带因素已经零星报道，但是，他们的细节仍然主要是objectives.Results：我们开发了体外实时振荡监测系统（IV-ROMS）通过测量荧光素酶的活性耦合到振荡基因启动子使用光电倍增管和应用该系统筛选和识别能够影响昼夜节律的因素。使用该IV-ROMS作为生物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确定了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、全反式视黄酸、9-顺式视黄酸和13-顺式视黄酸）已经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-Delta（12，14）-前列腺素J（2）（15 d-PGJ（2）），过氧化物酶体增殖物激活受体-γ（PPAR-gamma）的天然配体，触发了NIH 3 T3细胞中内源性时钟基因的节律性表达。此外，我们还发现15 d-PGJ（2）瞬时诱导Cry 1、Cry 2和Ror α mRNA的表达，并且15 d-PGJ（2）诱导的夹带信号通路不依赖于PPAR-gamma和MAPK（ERK、JNK、p38 MAPK）。使用我们开发的IV-ROMS筛选299种化合物，我们发现8种新的和4种已知的分子是生物钟的潜在夹带因子，这表明该测定系统在初始筛选中是一个强大而有用的工具。
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-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), 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-PGJ(2) transiently induces Cry1, Cry2, and Ror alpha mRNA expressions and that 15d-PGJ(2)-induced entrainment signaling pathway is PPAR-gamma - and MAPKs (ERK, JNK, p38MAPK)-independent.Conclusion: Here, we identified 15d-PGJ(2) 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.