The loss of circadian rhythms in the intestine leads to aberrant regulation of stem cell signaling pathways and increased tumor initiation.

Many physiological functions exhibit circadian rhythms: oscillations in biological processes that occur in a 24-hour period. These daily rhythms are maintained through a highly conserved molecular pacemaker known as the circadian clock. Circadian disruption has been proposed to cause increased risk of Inflammatory Bowel Disease (IBD) but the underlying mechanisms remain unclear. Patients with IBD experience chronic inflammation and impaired regeneration of intestinal epithelial cells. Previous animal-based studies have revealed that colitis models of IBD are more severe in mice without a circadian clock but the timing of colitis, and whether its inflammatory and regenerative processes have daily rhythms, remains poorly characterized. We tested circadian disruption using Bmal1-/- mutant mice that have a non-functional circadian clock and thus no circadian rhythms. Dextran Sulfate Sodium (DSS) was used to induce colitis. The disease activity of colitis was found to exhibit time-dependent variation in Bmal1+/+ control mice but is constant and elevated in Bmal1-/- mutants, who exhibit poor recovery. Histological analyses indicate worsened colitis severity in Bmal1-/- mutant colon, and colon infiltration of immune system cells shows a daily rhythm that is lost in the Bmal1-/- mutant. Similarly, epithelial proliferation in the colon has a daily rhythm in Bmal1+/+ controls but not in Bmal1-/- mutants. Our results support a critical role of a functional circadian clock in the colon which drives 24-hour rhythms in inflammation and healing, and whose disruption impairs colitis recovery. This indicates that weakening circadian rhythms not only worsens colitis, but delays healing and should be taken into account in the management of IBD. Recognition of this is important in the management of IBD patients required to do shift work.

The circadian clock is a self-sustained molecular timekeeper that drives 24-h (circadian) rhythms in animals. The clock governs important aspects of behavior and physiology including wake/sleep activity cycles that regulate the activity of metabolic and digestive systems. Light/dark cycles (photoperiod) and cycles in the time of feeding synchronize the circadian clock to the surrounding environment, providing an anticipatory benefit that promotes digestive health. The availability of animal models targeting the genetic components of the circadian clock has made it possible to investigate the circadian clock’s role in cellular functions. Circadian clock genes have been shown to regulate the physiological function of hepatocytes, gastrointestinal cells, and adipocytes; disruption of the circadian clock leads to the exacerbation of liver diseases and liver cancer, inflammatory bowel disease and colorectal cancer, and obesity. Previous findings provide strong evidence that the circadian clock plays an integral role in digestive/metabolic disease pathogenesis, hence, the circadian clock is a necessary component in metabolic and digestive health and homeostasis. Circadian rhythms and circadian clock function provide an opportunity to improve the prevention and treatment of digestive and metabolic diseases by aligning digestive system tissue with the 24-h day.

Background The circadian clock is a highly conserved molecular pacemaker found in nearly every cell of the body. It consists of the genes BMAL1 and CLOCK that positively regulate CRY and PER, their negative regulators, resulting in a transcription/translation feedback loop that has a 24 hour cycle. This core clock mechanism drives the rhythmic expression of over 40% of the genome in a tissue-specific manner and therefore imposes 24 hour rhythms on many physiological processes. Shift work, which causes disruptions to the natural 24 hour physiological rhythms, has been shown to lead to an increased incidence of inflammatory bowel disease (IBD). Aims This study aims to characterize daily rhythms in inflammation and regeneration of the colon upon induction of acute colitis. We also aim to investigate the intestinal epithelial-specific effects of circadian clock disruption on overall disease progression. We hypothesize that the absence of a functional circadian clock eliminates proliferation rhythms of intestinal epithelial cells and disrupts the rhythms of inflammatory cytokines, thereby increasing the pathogenesis of IBD. Methods We tested the role of the clock in IBD using BMAL1+/+ (wild type) and BMAL1-/- (null mutant) mice. We also investigated the effects of the circadian clock specific to intestinal epithelial tissue using Vil+/+;BMAL1flox/flox (control) and VilCre/+;BMAL1flox/flox (conditional intestinal epithelial mutant) mice. Dextran Sulfate Sodium (DSS) was applied to induce acute colitis. Results We observed significantly decreased survival of BMAL1 circadian clock mutant mice when given colitis. A histology analysis indicates increased lesioning and overall inflammation in BMAL1-/- colon tissue. Disease activity and cytokine analyses reveal time-dependent severity in inflammatory response that is worse in BMAL1-/- mice. To test the circadian rhythms in intestinal regeneration of mice with IBD, we performed a 24 hour analysis comparing epithelial cell proliferation and cell death in colon tissue. We observed rhythmic expression of phosphor-histone H3 (a mitosis marker) in wild type mice which is eliminated in the BMAL1-/- lacking a circadian clock. Cell death which was measured by caspase 3 did not exhibit any differences between genotypes. Based on these results, we conclude that the loss of clock function leads to impaired regeneration during IBD, in part due to decreased and arrhythmic cell proliferation. Preliminary results in our VilCre/+;BMAL1flox/flox conditional intestinal epithelial mutant mice indicate that some of these effects may be epithelial-specific. Conclusions Our results support a critical role of the circadian clock in inflammatory bowel disease development. These data highlight that the circadian clock affects the regenerative abilities of intestinal epithelial cells. Funding Agencies CIHRChron’s and Colitis Canada, Ontario, University of Windsor

Background Circadian rhythms are autonomously running 24h cycles in bodily processes. In animals these rhythms are driven by a molecular time keeper known as the circadian clock. The clock is a transcription-translation feedback loop composed of the transcription factors Bmal1 and Clock as well as their repressors Per and Cry. The circadian clock regulates over 40% of the genome rhythmically. Chronic circadian disruption, in the case of shift work, can lead to pathologies including cancer. Colorectal cancer is most frequently initiated through a mutation in the Wnt pathway regulator, Apc. Several studies have attempted to provide a mechanistic link between cancer and circadian clock disruption but the use of mice on mixed genetic backgrounds and poor circadian models have made this link unclear. Aims We aim to determine if the circadian clock plays a role in intestinal tumourigenesis. Methods We crossed the Apcmin mouse strain, a common intestinal tumour model, with Bmal1 mutant mice, which lack a functioning circadian clock. After creating an isogenic strain, we examined the number of tumours in control (Bmal1+/+) and clock dead (Bmal1-/-) animals. We derived organoids, a 3D cell culture method, from Apc+/+; Bmal1+/+ (healthy, clock-live), Apc+/+, Bmal1-/-(healthy, clock-dead), Apcmin; Bmal1+/+(adenoma, clock-live), Apcmin; Bmal1-/- (adenoma, clock-live) mouse ileum and collected every 2h from 24-48h after synchronizing their circadian clock. Collected samples were sent for RNA sequencing and assessed for circadian regulated transcripts. This experiment was followed up by in vitro organoid assays. Results The circadian clock controls 41 genes in the intestinal epithelium, including genes like Tead4 which are known to be important in intestinal biology. There are twofold more tumours in Bmal1-/- mice than their Bmal1+/+ littermates, and Bmal1-/- tumours upregulate Tead4 and Hippo pathway targets and downregulate Wnt pathway targets. Bmal1-/- adenoma organoids show increased self-renewal when compared to Bmal1+/+ adenoma organoids. However, this increase in self-renewal is lost when organoids are treated with inhibitors of the hippo pathway. Conclusions The circadian clock is important in maintaining the health of an organism, and disruption of the clock can lead to many health consequences including cancer. We show for the first time that the circadian clock controls the hippo signaling mediator Tead4. Additionally, we show that the loss of the clock leads to an increase in the number of tumours present in the epithelium which are characterized by an increase in hippo signaling. This research shows the important of considering time of day when studying stem cells during homeostasis and in cancer. Funding Agencies CIHRNSERC