Agez L, Laurent V, Pévet P, Masson-Pévet M, Gauer F
Institut des Neurosciences Cellulaires et Intégratives, Département de Neurobiologie des Rythmes, CNRS UMR 7168-LC2, IFR 37 Neurosciences, Université Louis Pasteur, Strasbourg, France.
Neuroscience. 2007 Jan 19;144(2):522-30. doi: 10.1016/j.neuroscience.2006.09.030. Epub 2006 Oct 24.
The pineal hormone melatonin nocturnal synthesis feeds back on the suprachiasmatic nuclei (SCN), the central circadian clock. Indeed, daily melatonin injections in free-running rats resynchronize their locomotor activity to 24 h. However, the molecular mechanisms underlying this chronobiotic effect of the hormone are poorly understood. The endogenous circadian machinery involves positive and negative transcriptional feedback loops implicating different genes (particularly period (Per) 1-3, Clock, Bmal1, cryptochrome (Cry) 1-2). While CLOCK:BMAL1 heterodimer activates the rhythmic transcription of per and cry genes, the PER and CRY proteins inhibit the CLOCK:BMAL1 complex. In previous studies, we observed that the immediate resetting effect of a melatonin injection at the end of the subjective day on the SCN circadian activity did not directly involve the above-mentioned clock genes. Recently, nuclear orphan receptors (NORs) have been presented as functional links between the regulatory loops of the molecular clock. These NORs bind to a retinoic acid receptor-related orphan receptor response element (RORE) domain and activate (RORalpha) or repress (REV-ERBalpha) bmal1 expression. In this study, we investigated whether melatonin exerts its chronobiotic effects through transcriptional regulation of these transcription factors. We monitored roralpha, rorbeta and rev-erbalpha messenger RNA (mRNA) expression levels by quantitative in situ hybridization, up to 36 h following a melatonin injection at circadian time (CT) 11.5. Results clearly showed that, while roralpha was not affected by melatonin, the hormone partially prevented the decrease of the rorbeta mRNA expression observed in control animals during the first hours following the injection. The major result is that the rev-erbalpha mRNA expression rhythm was 1.3+/-0.8-h phase-advanced in melatonin-treated animals during the first subjective night following the melatonin administration. Moreover, the bmal1 mRNA expression was 1.9+/-0.9-h phase-shifted in the second subjective night following the melatonin injection. These results clearly suggest that the NOR genes could be the link between the chronobiotic action of melatonin and the core of the molecular circadian clock.
松果体激素褪黑素的夜间合成会反馈作用于视交叉上核（SCN），即中枢生物钟。实际上，对自由活动的大鼠每日注射褪黑素可使其运动活动重新同步至24小时节律。然而，该激素这种时间生物学效应背后的分子机制仍知之甚少。内源性生物钟机制涉及正、负转录反馈环，涉及不同基因（特别是周期基因（Per）1 - 3、Clock、Bmal1、隐花色素基因（Cry）1 - 2）。虽然CLOCK:BMAL1异二聚体激活per和cry基因的节律性转录，但PER和CRY蛋白会抑制CLOCK:BMAL1复合物。在先前的研究中，我们观察到在主观日结束时注射褪黑素对视交叉上核昼夜活动的即时重置效应并不直接涉及上述生物钟基因。最近，核孤儿受体（NORs）被认为是分子生物钟调节环之间的功能联系。这些NORs与视黄酸受体相关孤儿受体反应元件（RORE）结构域结合，激活（RORα）或抑制（REV - ERBα）bmal1表达。在本研究中，我们调查了褪黑素是否通过对这些转录因子的转录调控发挥其时间生物学效应。我们通过定量原位杂交监测了在昼夜时间（CT）11.5注射褪黑素后长达36小时的rorα、rorβ和rev - erbα信使核糖核酸（mRNA）表达水平。结果清楚地表明，虽然rorα不受褪黑素影响，但该激素部分阻止了在注射后最初几小时内对照动物中观察到的rorβ mRNA表达的下降。主要结果是，在给予褪黑素后的第一个主观夜间，褪黑素处理动物的rev - erbα mRNA表达节律提前了1.3±0.8小时。此外，在注射褪黑素后的第二个主观夜间，bmal1 mRNA表达发生了1.9±0.9小时的相位偏移。这些结果清楚地表明，NOR基因可能是褪黑素的时间生物学作用与分子生物钟核心之间的联系。
