Role And Mechanism Of Death Receptor 5(DR5) In The Regulation Of Colon Stem Cell Stemness

ObjectivesIntestinal epithelium is one of the fastest renewal tissues in the body,serves as an important line of defense for the body to resist the invasion of external microorganisms.The regeneration of intestinal epithelium accounts on intestinal stem cell,generally involves three stages,includes that self-renewal,proliferation,and differentiation of intestinal stem cells(ISC).Under pathological conditions,loss of function of stem cells can cause intestinal epithelial repair disorders,and excessive activation may induce colorectal tumors.Therefore,a comprehensive understanding of working mechanisms to the regulation of intestinal stem cell homeostasis is vital in regulating intestinal epithelial homeostasis,has attracted interest from researchers in basic and clinical fields.Many researchers are continuously seeking to clarify the regulation mechanism of physiological intestinal epithelial homeostasis due to its theoretical and clinical significance and aim for providing new evidence for the treatment of intestinal diseases,such as inflammatory bowel disease and colon tumors.Death receptor 5(DR5),also known as TRAILR2(Tumour necrosis factor-related apoptosis-inducing ligand receptor 2),is a member of the apoptosis-inducing tumor necrosis factor receptor superfamily.It has been raised attention due to its ability in inducing certain tumor cell apoptosis.In recent years,studies have shown that the DR5 mediates other effects besides death signals.Intestinal epithelial cells express DR5,but under physiological conditions,intestinal epithelial DR5 exhibits significant resistance to TRAIL-induced apoptosis.This suggests that DR5 which is expressed by intestinal epithelium may have other effects besides apoptosis under physiological conditions.Our preliminary experiments found that DR5 is predominantly expressed in the stem cell area where locates at the bottom of colonic crypts.It is can be speculated that DR5 may play an important role in the physiological regulation of intestinal stem cell sternness.This project is designed to use DR5 gene-deficient(DR5-/-)mice to study the role and working mechanisms of the colon crypt DR5 in the regulation of stemness.The obtained research results can provide experimental evidence for understanding the regulation of intestinal stem cell homeostasis.Methods1.Experimental animals and subgroups:Experimental animals:Healthy male adult(6-8 weeks)C57BL/6N mice(WT)and healthy male adult(6-8 weeks)DR5 gene-deficient(DR5-/-)mice.Prepare the paraffin sections with colon,then perform HE staining and immunohistochemical staining on the prepared paraffin.Or take the colon tissue and freeze it in the-80? refrigerator for Western blot or QRT-PCR detection.2.BMP signaling pathway inhibitor LDN193189 treatment:male C57BL/6N mice and DR5 gene-deficient mice were randomly divided into WT control groups,DR5 gene-deficient mice control group,and DR5 gene-deficient mice+LDN193189(inhibition of BMP pathway target protein Smad1/5/8 phosphorylation)group treatment group.The control group was intraperitoneally injected with the BMP inhibitor LDN193189(3mg/kg)12 hours before the sample was injected with the same dose of normal saline.3.Ulcerative colitis(UC)model:to prepare a colitis model,C57BL/6N male mice and DR5-/-male mice(6-8 weeks)were given water containing 1.5%DSS(Dextran Sulfate sodium)or 2.5%DSS for 7 days,or 2.5%DSS(Dextran Sulfate sodium)or 2.5%DSS for 6 ays.Rcord the weight of mice,stool shape,occult blood or blood in the stool every day.After 6 or 7 days,start to prepare the paraffin sections with colon,then perform HE staining and immunohistochemical staining on the prepared paraffin.Or take the colon tissue and freeze it in the-80 refrigerator for Western blot or QRT-PCR detection.4.In vitro cell culture:Human normal colonic epithelial cell line CCD841 cells was cultured in DMEM medium containing 10%fetal bovine serum,1%penicillin(100?g/ml),placed at 37?,5%CO2 cell culture cultivate in a box.To study the effect of DR5,selective agonist Bioymifi treatment(10-8M?10-7M?10-6M?10-5M?2×10-5M?5×10-5M)was implemented on cell apoptosis and proliferation;interfere with the effect of endogenous DR5 expression on cell proliferation.Research content:1.In-vivo Experiments:HE staining was used to detect(examine)the depth of colonic crypts;PAS(Periodic Acid-schiff stain)glycogen staining was used to detect(examine)the number of goblet cells.QRT-PCR:QRT-PCR was utilized to detect the following expression:Intestinal stem cell marker genes,include that Lgr5,OLFM4,and Ascl2.Goblet cell marker genes MUC2,TFF3(Trefoil Factor 3).Goblet cell differentiation-related genes KLF4 and ELF3 intestinal epithelial cell marker gene ALPI.BMP signaling pathway ligands BMP2,BMP4,BMP7.BMP signaling pathway downstream target genes ID1,ID3.Wnt pathway ligand wnt3,downstream target protein ?-catenin,downstream target gene c-myc.Notch signaling pathway ligand the expression of DLL1,DLL4,downstream gene Hes1.Western blot was utilized to detect:The colon tissue DR5,stem cell marker Lgr5,CDX2,a key factor used in regulating intestinal epithelial differentiation,Hesl,and a key downstream protein of the Notch signaling pathway.The expression of BMP signaling pathway downstream target protein p-Smad1/5,and expression of BMP signaling pathway ligand BMP4;And the expression of inflammatory mediator iNOS.2.Ulcerative colitis(UC)model:Daily record the weight of mice,stool shape,occult blood or blood in the stool;record the length of the colon while collecting materials,the disease activity index,and inflammation score.3.In vitro cell experiment:Cell cycle detection(DNA content detection)and CCK8(Cell Counting Kit-8)methods are used to detect the effect of Bioymifi on the proliferation of CCD841 cells;Western blot was used to detect the effect of Bioymifi treatment on the expression of cell stem cell markers Lgr5 and the apoptotic proteins Cleaved caspase3 and caspase3.Results1.Both Colonic epithelial cells DR5,and DR5 show a positive expression in the nucleus and cytoplasm of the stem cell area where locates at the bottom of the crypt according to experimental results.Moreover,it has been found that the nuclear expression gradually weakens as it migrates to the intestinal lumen.2.We found that DR5 plays an important role in the self-renewal and proliferation of intestinal stem cells:In particular,first,it has not found any difference between wild-type mice and DR5 gene-deficient mice with respect to the colonic crypt depth;Comparing with wild-type mice,DR5 gene-deficient mice have significantly down-regulated expression of stem cell across the marker genes Lgr5,Olfm4,Ascl2,and Lgr5;The number of Ki67-positive cells of DR5 gene-deficient mice in crypts was found significantly reduced.The results of the study suggest that DR5 gene defects suppress the self-renewal and proliferation of the stem cells.3.The role of DR5 in the differentiation of stem cell:Compared with the control group of wild mice,the number of PAS-positive and MUC-2-positive cells in colonic crypts of DR5 gene-deficient mice was significantly increased.Goblet cell marker genes MUC2,TFF3,stem cell differentiation markers KLF4,and ELF3 were up-regulated;Moreover,the number,and protein expression of differentiation marker CDX2 positive cells of intestinal epithelium also increased significantly,and the expression of intestinal alkaline phosphatase(ALPI)was significantly up-regulated.4.The potential mechanisms of DR5 in regulating stem cell stemness4.1 siRNA down-regulated the expression of DR5 gene of CCD841 cells was leading to a decrease in the number of S-phase cells,and this suggests that endogenous DR5 was involved in regulating the cell proliferation,which is consistent with observations in vivo.After treating the CCD841 cells with different concentrations of selective DR5 small molecule agonist Bioymifi.It indicates that the high concentrations(above 2×10-5M)of Bioymifi induced apoptosis of CCD841 cells,while concentrations lower than this(2×10-5M)did not cause apoptosis.Correspondingly,after treating cells with low concentrations of Bioymifi(10-7M,10-6M),indicates that it failed to induce apoptosis and S-phase cells increased,and the expression of stem cell marker Lgr5 was up-regulated.The result suggests that low concentration can promote cell proliferation.With simulating the cells using the Bioymifi,the DR5 trend to translate to the nucleus.Comparing with wild mice,DR5 gene-deficient mice do not show any changes in colonic crypt cell apoptosis.Looking at the nuclear expression of DR5 in the stem cell area at the bottom of colonic crypts,we can see that the role of DR5 is related to its subcellular localization and physiological conditions.Moreover,we can see that the nuclear expression of DR5 in the stem cell region of the lower colon crypts always being part of the regulation activity of proliferation and differentiation of stem cell,which has nothing to do with death signals4.2 The effect of DR5 on the pathways related to stem cell stemness regulation:Compared with the group of wild mice,the expression signaling pathway ligand BMP4 in colon tissue was increased in the group of gene-deficient mice with DR5.And BMP4 and BMP7 expression remain the same.The physiological colonic crypt cells have significantly high expression of BMP4;The expression of core protein p-Smad 1/5 where to locate downstream of BMP pathway was increased,the expression of downstream target genes ID1 and ID3 of the BMP pathway increased,which suggest that the defects of DR5 gene caused abnormal activation of colonic BMP4/Smad signaling pathway.On the other hand,the expression of Wnt signaling pathways ligand wnt3,downstream target protein ?-catenin,and target gene c-myc remain the same;The expression of Notch signaling pathway ligands DLL1 and DLL4 did not change as well,and the expression of the downstream key gene Hesl was down-regulated.4.3 The role of BMP signaling pathway DR5 in regulating stem cell sternness:Intraperitoneal injection of BMP/Smad pathway inhibitor LDN193189,the expression of downstream target genes ID1 and ID3 of BMP/Smad pathway in DR5 gene-deficient mice was significantly down-regulated,while the up-regulation of BMP4 expression was not affected,indicating that LDN193189 inhibited BMP/Smad in DR5 gene-deficient mice Excessive activation of the pathway.Compared with the control DR5 gene-deficient mice,the number of Ki67-positive cells in the crypts of the DR5 gene-deficient mice after treatment with LDN193189 was significantly increased,and there was no significant difference in the number of proliferating cells in the crypts of the DR5 gene-deficient mice and wild mice after treatment.After treatment with LDN193189,the expression of the goblet cell marker genes MUC2 and TFF3 of the DR5 gene-deficient mice was reduced,and the number of goblet cells was significantly reduced.There was no statistical difference between the mice and the wild mice.After treatment with LDN193189,the expression of Hesl,the target gene of Notch signaling pathway in DR5 gene-deficient mice,was reversed,which was no different from wild-type mice.Research suggests that blocking the BMP pathway reverses the changes in stem cell sternness caused by DR5 gene defects.5.The role of DR5 in ulcerative colitis(UC):Compared with the group of wild-type mouse DSS-induced colitis model,the DSS model of the group of DR5 gene-deficient mice shows a significantly high death rate.It has lower body weight,an increased disease activity index,colonic epithelial injury score,and crypt injury score,and increased expression of inflammatory mediator iNOS.The expression of stem cell marker Lgr5 decreased,and the number of Ki67-positive cells in crypts decreased.Compared with the wild-type mouse DSS model,the expressions of BMP4 and p-Smad1/5 in the DR5 gene-deficient DSS model mice were significantly up-regulated.Conclusions1.Colonic epithelial cells express DR5,and DR5 is predominantly expressed in the stem cell area at the bottom of the crypt.2.Colonic epithelial DR5 gene defect inhibits the self-renewal and proliferation of colonic stem cells,and promotes the differentiation of stem cells into goblet cells and intestinal epithelial cells.3.The effect of DR5 gene defect on stem cell stemness is related to the activation of the BMP4/Smad signaling pathway in crypt cells.DR5 is essential for maintaining the low BMP4/Smad signal gradient at the bottom of the crypt,and DR5 is essential for the self-renewal of intestinal stem cells at the bottom of the crypt.The maintenance of proliferation and poor differentiation is of great significance.4.DR5 gene-deficient mice are more likely to induce dextran sodium sulfate(DSS)colitis,which may be related to the activation of BMP4/Smad signaling pathway and inhibition of stem cell self-renewal and proliferation.