Circadian clock disruption by selective removal of endogenous carbon monoxide

Saika Minegishi,Koji Kano,Ikuko Sagami,Hiroaki Kitagishi,Shigeru Negi

doi:10.1038/s41598-018-30425-6

Abstract

Circadian rhythms are regulated by transcription-translation feedback loops (TTFL) of clock genes. Previous studies have demonstrated that core transcriptional factors, NPAS2 and CLOCK, in the TTFL can reversibly bind carbon monoxide (CO) in vitro. However, little is known about whether endogenous CO, which is continuously produced during a heme metabolic process, is involved in the circadian system. Here we show that selective removal of endogenous CO in mice considerably disrupts rhythmic expression of the clock genes. A highly selective CO scavenger, hemoCD1, which is a supramolecular complex of an iron(II)porphyrin with a per-O-methyl-β-cyclodextrin dimer, was used to remove endogenous CO in mice. Intraperitoneal administration of hemoCD1 to mice immediately reduced the amount of internal CO. The removal of CO promoted the bindings of NPAS2 and CLOCK to DNA (E-box) in the murine liver, resulting in up-regulation of the E-box-controlled clock genes (Per1, Per2, Cry1, Cry2, and Rev-erbα). Within 3 h after the administration, most hemoCD1 in mice was excreted in the urine, and heme oxygenase-1 (HO-1) was gradually induced in the liver. Increased endogenous CO production due to the overexpression of HO-1 caused dissociation of NPAS2 and CLOCK from E-box, which in turn induced down-regulation of the clock genes. The down-regulation continued over 12 h even after the internal CO level recovered to normal. The late down-regulation was ascribed to an inflammatory response caused by the endogenous CO reduction. The CO pseudo-knockdown experiments provided the clear evidence that endogenous CO contributes to regulation in the mammalian circadian clock.

Highlights

The circadian rhythm is a naturally occurring day-and-night oscillation system that controls physiological and behavioral cycles and is regulated in almost all cells[1,2,3,4]
After the i.p. administration of hemoCD1, endogenous Carbon monoxide (CO) in the mice was bound to hemoCD1 via ligand exchange with O2 and excreted in the urine in a form complexed with hemoCD1 (CO-hemoCD1) as previously demonstrated[34]
The amount of hemoCD1 administered was sufficient to deplete the endogenous CO in mice; the excreted amount of endogenous CO as CO-hemoCD1 was saturated when hemoCD1 was administered at concentrations higher than 1.0 mM (0.15 mL)[34]

Summary

Introduction

The circadian rhythm is a naturally occurring day-and-night oscillation system that controls physiological and behavioral cycles and is regulated in almost all cells[1,2,3,4]. CLOCK and NPAS2 are the transcriptional factors that play central and overlapping roles in the TTFL1–7 Both proteins form heterodimers with BMAL1 and bind to a specific DNA sequence called E-box. The role of endogenous CO in the circadian clock system has been recently studied by using HO-knockout/knockdown systems[24], suggesting that rhythmic heme degradation generating endogenous CO is required for keeping the E-box-controlled circadian rhythms. We show that selective removal of endogenous CO in mice significantly affects the expression levels of the E-box-controlled clock genes in the murine liver. The pseudo-knockdown study for CO in vivo provides the clear experimental evidence that endogenous CO contributes to regulation of the mammalian circadian clock through acting on NPAS2 and CLOCK and modulating the clock genes related to inflammatory responses

Methods

Results

Conclusion