Circadian Clock Regulates Intestinal Epithelial Cell Differentiation

Background and Objective:The small intestine is an important digestive and absorptive organ in the human body.Intestinal epithelium is composed of a single-layer columnar epithelial cells connected by the tight junction.The human body can effectively prevent pathogens and toxins from crossing the mucosal barrier to invade inner organs with epithelial mechanical barrier,immune barrier and microbial barrier.Maintenance of intestinal barrier function is critical to health.The mucous layer formed by the secretion of goblet cells reduces the contact between epithelial cells and pathogens,while the antimicrobial peptides secreted by paneth cells also help to remove pathogens.Intestinal epithelium consists of crypt-villus units.The intestinal stem cells,with self-renewal and differentiation potential,produce transit amplifying cells which rapidly split and migrate along the "crypt-villus" axis,differentiate into adult cells.However,mature paneth cells remain at the bottom of the crypt.Absorptive and secretory intestinal epithelial cells share the absorption and defense function of the intestinal epithelium.The fate of intestinal stem cells to be secretory cell or absorptive cells is determined by the Notch signaling pathway.It has been shown that the circadian clock affects cell cycle of stem cells and precursor cells through regulation of the expression of WNT signaling genes in paneth cells.Thus,we assumed that the differentiation of intestinal stem cells and precursor cells may be regulated by circadian clock.Our study will investigate how circadian clock regulates the differentiation of secretory epithelial cells.This will help to determine the relationship between circadian clock dysrhythmia or circadian clock gene mutation and the intestinal homeostasis and intestinal barrier function.Methods:1.Establish Bmall total knockout(Bmall-/-)and Bmall intestinal epithelial specific knockout(Bmall?IEC)mice model.Establish dysrhythmic mice model by the light entrainment.2.Entrain mice under 12L:12D conditions for a week.The Start time of light on was recorded as ZTO.Collected the jejunum,ileum,proximal and distal colon every 4 hours,a total of 7 point from ZTO to ZT24.Tissues were fixed in 4%formalin,embedded,and secretory epithelial cells were stained by immunohistochemistry.The number of each cell types were counted.The whole segment of small intestine and colon was frozen in liquid nitrogen and the mRNA was extracted.The mRNA expression of cell type markers was determined by qRT-PCR.Intestinal epithelial cells were collected and protein was extracted for western blot.3.Analyze binding sites of transcription factors by prediction software,ChIP assay,and dual luciferas reporter assay.4.Feed Bmall?IEC mice and wildtype mice with Salmonella typhimurium to establish oral Salmonella infection model.The body weight and clinical score were recorded.Jejunum and proximal colon were collected,fixed with 4%formalin,embedded,and stained with H&E to investigate pathological changes of tissues.Results:We found that circadian clock genes did not show circadian rhythms in Bmall-/-mice.In Bmall-/-and Bmall?IEC mice intestinal epithelium,the number of secretory cells including goblet cells,paneth cells and enteroendocrine cells decreased significantly.Consistent phenotypes were also observed in light entrainment dysrhythmic mouse model.The expression of the transcription factor Atoh1,which determined the differentiation of intestinal epithelial secretory cells,decreased significantly,while Notch signal was inhibited.Thus,the transcription inhibition of Atoh1 caused by the absence of circadian clock gene did not depend on the Notch signaling pathway.Therefore,we speculate that Bmall may play an important role in the differentiation of intestinal epithelial stem cells into secretory cells by regulating the expression of Atoh1.The enhancement of BMAL1/CLOCK complex on Atoh1 transcription was confirmed by ChIP and dual luciferas reporter assay.Oral salmonella infection model showed that Bmal1?IEC mice had faster weight loss,higher mortality and clinical scores compared with wildtype mice.Reduced number of secretory cells caused by the absence of circadian clock genes reduced the intestinal resistance to pathogens.Conclusions:The circadian clock gene Bmall gates the differentiation direction and differentiation time of intestinal epithelial stem cell/secretory progenitor by regulating the key transcription activator Atoh1.Bmall knockout or dysrhythmia lead to a decreasing number of intestinal epithelial secretory cells,thus affect the intestinal barrier function and reduce the ability of intestinal epithelium to resist pathogenic invasion.