Roles And Mechanisms Of Numb In Regulating The Intestinal Mucosal Barrier Function

Background and ObjectiveIntestinal mucosal barrier constitutes of mechanical barrier and immune barrier,chemical barrier, and biological barrier. Intestinal mucus barrier is the covering of epithelialcells surface by the gel-like mucus formed by hydration and glycosylation of mucin. Andintestinal epithelial cell barrier contains epithelial cell itself and tight junctions formedbetween cells,providing a mechanical protection from external injuries from all of sources.Numb molecule because of its able to asymmetrical distribution to the two daughter cells ofstem cells,causing them to have different "Destiny" by inhibiting the Notch signalingpathway,have attracted widespread attention. Previous studies have shown that Numb isexpressed in most tissues, including breast, lung, testis, salivary gland, but expression andfunction of Numb in intestinal epithelial cells has not yet been reported. In Preliminaryexperiments, we have found that Numb is not expressed on stem cells at the bottom of thecrypt, but is common expressed on all epithelial cells along the crypt-villus axis. Previousstudies have found that Numb regulates cell differentiation by inhibiting the Notch pathway,besides, Numb can regulate cell adhesion, cell migration and cell polarity. Inintestinalepithelial cells, whether Numb to promote the formation of mucous barrierthrough increasing Mucin secretion or maintain the barrier integrity of the intestinalepithelial cell through regulation the assembly of apical junctional complex (AJC) is stillunclear.To answer the above mentioned questions, this study revealed the expression andlocalization of Numb molecular in intestine mucosa and its relationship with Notchpathway related molecules Hes1and Hath1. W revealed its inhibition of the Notch pathwayand promotion of goblet cell phenotype in LS174T cell. Using Caco-2monolayer cellmodel, we revealed the role of Numb played in the intestinal epithelial monolayer cellbarrier formation, and revealed the mechanism of Numb maitains epithelial cellspermeability by inhibiting the phosphorylation of the myosin light chain and preventing the reorganization of cell actin cytoskeletonMaterial and Methods1. Detected the expression of Numb-PRRL and Numb-PRRS isoforms in the intestinalepithelial cell lines and intestinal mucosa cells by RT-PCR and Western blot method;Immunohistochemical Staining was performed to detect the expression of Numb, Hes1,Hath1and Muc2in mouse intestinal epithelial cells, and to reveal their distributionrelationship in the intestinal epithelial cells; goblet cells position was localized by AB stainin intestine mucosa.2. Construced interference plasmid pRNAT-U6.1-shRNA-Numb, transfected andscreened stably transfected LS174T cell clones; Notch activity was detected by luciferaseplasmid pGa981-6, and its downstream target genes,such as Hes1and Hath1were detectedby real-time quantitative PCR technique upon Numb expression was interfered.3. MTT assay was performed to detect the effect of Numb expression on cellproliferation, Expression of Muc2was detected by RT-PCR, Western blot andImmunofluorescence staining upon Numb expression supressed, PAS staining to detectLS174T cells mucus secretion.4. RT-PCR and Western blot method was used to detect the expression of Numb in theintestinal epithelial cell line Caco-2; Immunofluorescence staining was used to detect thelocalization relationship of Numb with junctional molecules of ZO-1, E-cadherin and Par3.Immunoprecipitation method was used to detect the interaction between Numb andE-cadherin or Par3.5. Interfered with the expression of Numb molecular; the role of Numb in epithelialcell barrier permeability was assessed by measurement of TEER and FITC-Dextranpenetration assay in Caco-2monolayer. Numb's role in the assembly and maintenance ofcell apical junctional complexes was verified by calcium switch experiment andTNF-a/INF-γ of stimulation experiments.6. Immunofluorescence staining was performed to observe the effect of Numb on thealteration of F-actin of Caco-2cells before and after the calcium switch assay, and theexpression changes of ZO-1, of Occludin, the E-cadhern, beta-catenin, of Claudin-1, ofClaudin-2in physiological and pathological conditions was detected by Western blot; theimpact of Numb on muscle myosin light chain phosphorylation in Caco-2cells was also assessed by western blot.7. Mouse animal model of ulcerative colitis was induced by DSS, colon tissuemorphological changes was evaluated by HE staining, change of Numb expression in colonicepithelial cells under inflammatory conditions was observed by immunofluorescence staining.Results1.Numb-PRRL and Numb-PRRS isoforms are expressed in intestinal epithelial celllines and intestinal mucosa epithelial cells. There is co-localization of Numb with Notchpathway related molecules of Hes1Hath1. Numb also co-localized with the Muc2and ABstaining positive goblet cells.2. Sequencing confirmed that the pRNAT-U6-shRNA-of Numb plasmid wassuccessfully constructed, stable transfected LS174T cell clones were obtained, luciferaseplasmid pGa981-6detection found Numb inhibited Notch pathway activity, inhibited ofHes1expression and promoted Hath1expression.3. Numb inhibited intestinal epithelial cell proliferation; promoted LS174T expressionof goblet cell molecular markers Muc2, PAS staining found Numb promoted LS174T cellsmucus secretion.4. There was Numb expression in Caco-2cell line. Numb co-localized with ZO-1,E-cadherin and Par3in Caco-2cell line. Numb interacted with E-cadherin and Par3.5Inhibition of Numb expression increased the cell barrier permeability in Caco-2. Thecalcium switch assay and TNF-a/INF-γ of stimulation experiment confirmed that Numbplayed a roles in AJC assembly and maintenance of cell junction integrity.6. Inhibition of Numb expression in Caco-2cells altered F-actin structure before andafter the calcium conversion assay; Numb had no effect on expression of ZO-1, of Occludin,E-cadhern, β-catenin and Claudin-1protein in Caco-2cell under normal and inflammatoryconditions, but the raised Claudin-2protein level; Numb inhibited Caco-2cell myosin lightchain phosphorylation.7. HE staining showed DSS successfully induced inflammatory reaction in mice,Numb expression in the colonic epithelial cells was reduced and subcellular distributionwas aberrant under inflammatory conditions.ConclutionsNumb promotes intestinal epithelial cell mucin2expression and mucus secretion by inhibiting the Notch pathway, thus involved in the formation of the intestinal mucus barrier;Numb regulate intestinal epithelial cells AJC assembly and maintain the integrity of theintestinal epithelial cell barrier through inhibition the phosphorylation of myosin light chain,to participate in the formation of intestinal mechanical barrier.