Department of Medical Sciences, Scientific Institute and Regional General Hospital, Casa Sollievo della Sofferenza, Italy.
J Biol Regul Homeost Agents. 2012 Apr-Jun;26(2):265-76.
Rhythmic oscillations of cellular biological processes are driven by translational-transcriptional feedback loops that realize molecular clocks ticking in every single cell, driven by neural and humoral outputs from the suprachiasmatic nuclei of the hypothalamus that are entrained by environmental photon inputs. The nuclear receptor REV-ERBα has the capability to reset the molecular oscillators of peripheral tissues. The aim of our study was to evaluate the clock gene machinery function in light/dark cycles (LD) and in constant darkness (DD) exploiting in particular the REV-ERBα pattern of expression by using data from two independent experimental settings to reduce procedure related influences. In the LD study C57BL/6 male mice housed on a 12L:12D cycle were sacrificed at 4 h intervals. Liver, kidney, spleen, thymus and testis were harvested and blood was collected. Expression levels of PER1, PER2, CRY1, CRY2, BMAL1, REV-ERBα, CLOCK were evaluated by qRT-PCR. In the DD study Balb/c male mice in the third DD cycle as a continuation of the dark phase of the last LD cycle were sacrificed at 4 h intervals. Lung, heart, liver, stomach, kidney, spleen, and testis were harvested and mRNA expression of PER1, PER2, CRY1, CRY2, BMAL1, REV-ERBα, CLOCK, was evaluated by qRT-PCR. A statistically significant difference was found for the size of the semi-interquartile range of acrophases of clock gene expression in different organs evaluated in LD and DD conditions (4:38∓1:12h versus 1:16∓0:10h, p=0.026). A statistically significant difference was found for the acrophases of clock gene expression in different organs evaluated in LD (p=0.01) and in DD (p<0.0001). In LD study only REV-ERBα showed concomitant expression in the different peripheral tissues with the phase peaking around 07:03∓0.8h. In the DD study all the core clock genes showed concomitant phases in different peripheral mouse tissues and REV-ERB alpha expression peaked around 07:09∓0.9h. In conclusion, REV-ERBα is the only clock gene that maintains its timing of oscillation in the LD study and in the DD study and its phase of expression remains concomitant in the different mouse peripheral tissues in the presence of LD alternance, or in constant darkness. Oscillation in REV-ERBα ligands (heme, carbon monoxide) may affect not only the phase and amplitude of circadian rhythms, but also physiological outputs of the circadian system and REV-ERBalpha may participate in the entrainment of central and peripheral clocks, functioning as a synchronizing hinge of the clock gene machinery.
细胞生物过程的节律性振荡由翻译-转录反馈环驱动，这些反馈环在每个细胞中实现分子钟的计时，由下丘脑视交叉上核的神经和体液输出驱动，并通过环境光子输入进行调整。核受体 REV-ERBα 具有重置外周组织分子振荡器的能力。我们的研究目的是通过利用来自两个独立实验设置的数据来评估光/暗循环 (LD) 和持续黑暗 (DD) 中的时钟基因机制功能，以减少与程序相关的影响。在 LD 研究中，将 C57BL/6 雄性小鼠饲养在 12L:12D 周期上，并在 4 小时间隔处处死。采集肝脏、肾脏、脾脏、胸腺和睾丸，并采集血液。通过 qRT-PCR 评估 PER1、PER2、CRY1、CRY2、BMAL1、REV-ERBα、CLOCK 的表达水平。在 DD 研究中，作为最后一个 LD 循环的暗期的延续，在第三个 DD 循环中处死 Balb/c 雄性小鼠，并在 4 小时间隔处处死。采集肺、心、肝、胃、肾、脾和睾丸，并通过 qRT-PCR 评估 PER1、PER2、CRY1、CRY2、BMAL1、REV-ERBα、CLOCK 的 mRNA 表达。在 LD 和 DD 条件下，不同器官时钟基因表达的峰相位半四分位距的大小存在统计学显著差异（4:38∓1:12h 与 1:16∓0:10h，p=0.026）。在 LD（p=0.01）和 DD（p<0.0001）中，不同器官时钟基因表达的峰相位存在统计学显著差异。在 LD 研究中，只有 REV-ERBα 在不同的外周组织中表现出伴随表达，相位峰值约为 07:03∓0.8h。在 DD 研究中，所有核心时钟基因在不同的小鼠外周组织中表现出伴随的相位，REV-ERBα 的表达峰值约为 07:09∓0.9h。总之，REV-ERBα 是唯一一种在 LD 研究和 DD 研究中保持其振荡时间的时钟基因，并且在 LD 交替或持续黑暗的情况下，其表达相位在不同的小鼠外周组织中保持伴随。REV-ERBα 配体（血红素、一氧化碳）的振荡可能不仅影响昼夜节律的相位和幅度，还影响昼夜节律系统的生理输出，REV-ERBalpha 可能参与中央和外周时钟的同步，作为时钟基因机制的同步铰链。
