Circad 5 Full Version
Julieann Rohde
The circadian oscillator in eukaryotes consists of several interlocking feedback loops through which the expression of clock genes is controlled. It is generally assumed that all plant cells contain essentially identical and cell-autonomous multiloop clocks. Here, we show that the circadian clock in the roots of mature Arabidopsis plants differs markedly from that in the shoots and that the root clock is synchronized by a photosynthesis-related signal from the shoot. Two of the feedback loops of the plant circadian clock are disengaged in roots, because two key clock components, the transcription factors CCA1 and LHY, are able to inhibit gene expression in shoots but not in roots. Thus, the plant clock is organ-specific but not organ-autonomous.
Day-night changes in the storage capacity of the urinary bladder are indispensable for sound sleep. Connexin 43 (Cx43), a major gap junction protein, forms hemichannels as a pathway of ATP in other cell types, and the urinary bladder utilizes ATP as a mechanotransduction signals to modulate its capacity. Here, we demonstrate that the circadian clock of the urothelium regulates diurnal ATP release through Cx43 hemichannels. Cx43 was expressed in human and mouse urothelium, and clock genes oscillated in the mouse urothelium accompanied by daily cycles in the expression of Cx43 and extracellular ATP release into the bladder lumen. Equivalent chronological changes in Cx43 and ATP were observed in immortalized human urothelial cells, but these diurnal changes were lost in both arrhythmic Bmal1-knockout mice and in BMAL1-knockdown urothelial cells. ATP release was increased by Cx43 overexpression and was decreased in Cx43 knockdown or in the presence of a selective Cx43 hemichannel blocker, which indicated that Cx43 hemichannels are considered part of the components regulating ATP release in the urothelium. Thus, a functional circadian rhythm exists in the urothelium, and coordinates Cx43 expression and function as hemichannels that provide a direct pathway of ATP release for mechanotransduction and signalling in the urothelium.
Based on the above, we hypothesized that the increased functional bladder capacity during sleep-time at night was promoted by the decrease of mechanotransduction signalling by ATP through C43 hemichannels in the urothelium via the functional circadian clock. Here, we investigated the circadian role of the urothelium for ATP release through regulating Cx43 expression and the function of hemichannels in urothelial cells in mice and humans.
We found circadian profiles of the clock genes, as well as Cx43 and ATP release in the urothelium. To investigate the causal-relationship among these findings, we used hTERT-immortalized human urothelial cells (TRT-HU1)27,35,36,37.
To demonstrate that the Cx43 expression and ATP release are under the control of the circadian clock, we investigated the influence of BMAL1-knockdown on the circadian rhythm in urothelial cells. After the transfection of five different BMAL1 shRNA plasmids (sh1-5) to TRT-HU1, BMAL1 shRNA plasmid (sh2) transfected cells (sh2 TRT-HU1) had the highest efficacy in BMAL1 knockdown by real-time quantitative RT-PCR and immunoblotting (Fig. 3d). After serum shock, the oscillation of PER2 and REV-ERBα as well as BMAL1 mRNA expression became unclear in sh2 TRT-HU1 compared with that in parent TRT-HU1 (Fig. 3e, Supplementary Fig. S3). Accordingly, the oscillation of Cx43 protein expression was lost in sh2 TRT-HU1 (Fig. 3f). In addition, the circadian rhythm of mechanically induced ATP release disappeared in sh2 TRT-HU1 (Fig. 3g). Furthermore, to confirm whether Cx43 mRNA expression was regulated by Rev-erbα and Sp-1 in urothelial cells as in smooth muscle cells11, a Cx43 promoter-reporter assay was performed using TRT-HU1. As a result, Cx43 transcription was upregulated in coexistence with Rev-erbα and Sp-1 in TRT-HU1 (Supplementary Fig. S4). These results suggest that the circadian clock system may regulate the oscillation of Cx43 expression and ATP release in urothelial cells.
To investigate whether Cx43 hemichannels function in vivo, GAP19 peptide was used for the examination of ATP release by bladder distention in mice at ZT19. The concentration of ATP release was significantly lower in the presence of GAP19 peptide than that in the presence of GAP19 scramble peptide (Fig. 4f). Based on these results, Cx43 hemichannels are considered part of the components regulating ATP release in the urothelium. Therefore, we propose that circadian rhythm in the urothelium coordinates the diurnal change in ATP release via Cx43 hemichannels, which modulate functional bladder capacity (Supplementary Fig. S10).
The fundamental adaptive advantage of the circadian clock is that it allows for predictive, rather than entirely reactive, homeostatic regulation of physiological functions. Diurnal change in functional bladder capacity provides an adaptive advantage to secure sound sleep as well as to function correctly for daily increases in urine production in the arousal phase5,6,7,8. In the current study, we focused on the circadian clock function of the urothelium and presented evidence for the existence of Cx43 in the human and mouse urothelium, the daily rhythms of clock genes and Cx43 in the mouse urothelium and immortalized human urothelial cells, and the function of Cx43 as a hemichannel for ATP release. Circadian clock-regulated ATP release from Cx43 might contribute in part to the adaptation of functional bladder capacity in daily life.
An important result in this study was the finding that the bladder urothelium has a circadian rhythm generated by the circadian clock and that this indeed modulates urothelial ATP release mechanically stimulated by bladder distension in a circadian manner. Diurnal variations in ATP play a crucial role in various organisms. In rodents, the diurnal change in extracellular ATP levels in astrocytes may provide a mechanism for the clock control of gliotransmission between astrocytes and neurons, which might be associated with sleep-wake changes in the brain by modulating energy metabolism and glial activity43,44. Clinically, the diurnal enhancement of pain hypersensitivity might be mediated by the clock gene controlled glucocorticoid-induced enhancement of extracellular ATP release in the spinal cord45. As above, diurnal variations in ATP levels might be an important mechanism in nature and it is noteworthy that the physiological role of the circadian clock in the urothelium has been demonstrated here.
The mechanism of circadian ATP release from the urothelium was also demonstrated in this study. Cx43 expression had a circadian rhythm in the bladder urothelium and Cx43 functioned as a hemichannel to release ATP in urothelial cells. In addition, the concentration of mechanically induced ATP release had oscillations that correlated with Cx43 expression in the urothelium. It is generally understood that urothelial cells sense the extension stimulus and release ATP, which is essential for the micturition reflex19,46. Stretch-induced urothelial ATP release evokes afferent nerves within the suburothelial tissues via P2X and P2Y receptors and conveys a sense of bladder filling to the central nervous system23,24,25,26,27. Considering the diurnal change of mechanically induced ATP release via Cx43 hemichannels in the present study and the increased functional bladder capacity in Cx43 hetero-knockout mice11,47,48, we infer that the diurnal rhythm of micturition is regulated by diurnal changes in the level of perception for bladder distention.
Another limitation is that roles of the circadian clock and ATP in physiological or pathological conditions in humans have not been investigated yet. Urothelial ATP signalling is involved in pathological conditions such as overactive bladder, but the role of P2X2/3 receptors is still controversial in physiological conditions19,54,55. Interestingly, it was suggested that the circadian clock in the bladder is modulated by endogenous purinergic receptors and that dysregulation of the interaction between the clock and major endogenous receptors may have an effect on maintaining bladder functions56. Further studies are expected to elucidate the roles of the circadian clock and ATP signalling in human micturition.
In conclusion, a functional circadian rhythm exists in the urothelium, and coordinates Cx43 expression and function as hemichannels that provide a direct pathway of ATP release for mechanotransduction and signalling in the urothelium. This understanding warrants further investigation for diseases characterized as disorders of micturition rhythm including nocturnal enuresis and nocturia.
Author Contributions All authors contributed to the design of experiments and analysed data. P.S.F. supervised experiments and wrote the manuscript. S.M.-F. performed circadian measurements of gene expression, circulating progenitors, all Q-PCR, ELISA, in vitro experiments, in vivo effects of adrenergic agonists, immunostainings, western blots and denervation experiments, and prepared figures and wrote the manuscript. D.L. performed circadian progenitor assessments, long-term competitive reconstitutions and 6OHDA experiments. M.B. performed circadian progenitor assessments and G-CSF mobilization experiments.
Haematopoietic stem cells (HSCs) circulate in the blood, where they can home to sites throughout the body. The release of these cells into the blood stream has now been found to be regulated by circadian rhythms. In mice, HSCs undergo pronounced fluctuations corresponding to circadian oscillations induced by continuous light or by a 12-hour time-shift or 'jet lag'. Timing of the expression of the chemokine CXCL12 in the stem cell niche was also in step with the oscillations in response to adrenergic signals delivered locally by nerves in the bone marrow. The rhythmic release of stem cells into the blood during the animal's resting period suggests a possible role in regeneration.
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