The Role Of Death Receptor 5 In The Small Intestinal Epithelial Homeostasis And Its Mechanism

Background and aim:Death receptor 5(DR5),a member of the tumor necrosis factor receptor(TNFR)superfamily,can be activated by TRAIL to trigger apoptosis of tumor cells,but it has no toxicity to normal cells.Recently,it has been found that the activation of DR5 in some cells did not mediate death signals,but regulated cell proliferation and differentiation.DR5 was expressed in human colonic stem cells,and colonic crypt cells were insensitive to TRAILinduced apoptosis.Our previous study found that DR5 did mediate non-death signaling in colonic crypt cells,which could promote colonic stem cell proliferation.Although intestinal epithelial homeostasis depends on the proliferation and differentiation of intestinal stem cells,the small intestine has different functions and composition of intestinal epithelial cells compared with the colon.To date,the physiological role of DR5 in the small intestinal epithelium has not been reported.Considering the important effect of intestinal stem cells on intestinal epithelial homeostasis,we suspected whether DR5 affected the intestinal epithelial homeostasis by participating in the regulation of small intestinal stem cell proliferation and differentiation?To clarify this hypothesis,we used DR5 knockout(DR5-/-)mice to explore the effect of DR5 on intestinal epithelial homeostasis and its mechanism via in vivo and in vitro intestinal organoid experiments.Results:1.DR5 was expressed in small intestinal epithelial cells.DR5 gene knockout inhibited the absorptive function of the small intestinal epithelium and disrupted the integrity of the intestinal epithelial barrier.2.The number of small intestinal epithelial cells was significantly reduced by DR5 gene knockout.RNA sequencing showed that the expression of genes related to the proliferation and differentiation of intestinal stem cells in small intestinal crypt cells was down-regulated after DR5 gene knockout.Compared with wild type(WT)mice,the expressions of intestinal stem cell marker genes such as Lgr5,Ascl2,Axin2,Olfm4,Bmill,Hopx were downregulated in DR5-/-mice,and the number of Olfm4+intestinal stem cells,Ki67+and BrdU+cells in crypt was decreased.For differentiation of small intestinal stem cells,the expression of Hmgcs2 and CDX2 gene,which promoted intestinal stem cell differentiation,was downregulated in DR5-/-mice.In addition,the expressions of intestinal stem cell lineage differentiation genes Elf3,Klf4,Ngn3,Mmp7 and Sox9 were down-regulated,and the gene and protein expressions marking enterocytes,goblet cells,enteroendocrine cells and Paneth cells were down-regulated,and the number of all the type of intestinal epithelial cells was decreased.All these results showed that DR5 gene knockout could inhibit the proliferation and differentiation of small intestinal stem cells,and reduced the number of small intestinal epithelial cells.3.Small intestinal organoids derived from small intestinal crypt cells of DR5-/-mice showed significantly less area growth and bud formation than those of wild-type mice.DR5 selective agonist bioymifi significantly promoted the growth of small intestinal organoids,increased organoid area and budding,and upregulated the expression of genes and proteins indicating proliferation and differentiation of intestinal stem cells.These results suggested that DR5 regulated the proliferation and differentiation of small intestinal stem cells by acting on small intestinal crypt cells.4.DR5 gene knockout prompted Paneth cell apoptosis,inhibited the formation of Paneth cells secretory granules,and then reduced intestinal stem cell niche factors produced by Paneth cells including Wnt ligands Wnt3,Notch ligands Dll1 and D114,and BMP ligands BMP4 and BMP7.In line with these,DR5 gene knockout inhibited the Wnt,Notch,BMP signaling pathways of intestinal stem cell.Bioymifi treatment upregulated the expressions of Wnt3,D114,BMP4 in small intestinal organoids,and then activated Wnt,Notch and BMP signaling pathways to promote proliferation and differentiation of small intestinal stem cells.The results suggested that DR5 regulated the proliferation and differentiation of small intestinal stem cells by promoting the production of intestinal stem cell niche factors from Paneth cells.5.DR5 gene knockout caused down-regulation of ERK1/2 activity in transit amplifying cells(TACs)of small intestinal crypts,thus prompted the DNA damage and apoptosis in TA cells.Bioymifi treatment upregulated ERK1/2 activity in small intestinal organoids.Importantly,the ERK1/2 inhibitor PD0325901 blocked the effect of bioymifi on promoting small intestinal organoid formation and small intestinal stem cell proliferation,differentiation.These suggested that DR5 participated in the stemness regulation of small intestinal stem cells by maintaining the activity of ERK1/2 in small intestinal TA cells.Conclusions:DR5 activation promotes small intestinal stem cell proliferation and differentiation via promoting the production of intestinal stem cell niche factors from Paneth cells and activating TA cells ERK1/2 activity.By means of promoting the proliferation and differentiation of small intestinal stem cells,DR5 maintains the homeostasis of the small intestinal stem cell pool and epithelial cells,which plays an important role in the absorption and barrier function of epithelial cells.DR5 is essential for the small intestinal epithelial homeostasis.