REV-ERBα and the clock gene machinery in mouse peripheral tissues: a possible role as a synchronizing hinge.

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.