MTORC1 Signaling Pathway Involved In The Occurrence And Development Of IBD

Background and aimsInflammatory bowel diseasea(IBD) is a chronic non-specific gastrointestinal inflammatory diseases,consists of Crohn’s disease(CD) and ulcerative colitis(UC),mainly leads to abdominal pain,diarrhea,grossly bloody stools and body weight loss.In western countries,IBD has a prevalence of 150-300 cases per 100,000 inhabitants.IBD is rare in the past China,but as the lifestyle and food habits westernization,the incidence of IBD increased by years.Althought genetic,environmental,microbial and immue factors is thought to be contributed to IBD,the detailed pathophysiological mechanisms remain largely unknown.Chronic inflammation in the intestine ranks among the top three high-risk conditions for colorectal cancer.Patients with inflammatory bowel disease (IBD) have an increased risk of developing colorectal cancer (CRC).Moreover, the duration and anatomic extent of the disease have been known to affect the development of IBD-related CRC.Mammalian target of rapamycin (mTOR) is a highly conserved Ser/Thr protein kinasewhich elicits its pleiotropic functions in the context of two functionally distinct signaling complexes, termed mTOR complex 1 (mTORC1) andmTOR complex 2 (mTORC2). mTORC1, which contains mTOR, mLST8/GβL, Raptor and PRAS40,integrates diverse signals including nutrients, growth factors, energy and stresses to control cell growth, proliferation, survival, and metabolism.Tuberous sclerosis complex (TSC) is the most significant upstream regulator of mTORC1.It could activate GTP enzyme (GTP activating protein, GAP),enabling GTP decomposing into GDP in the small GDP enzyme Rheb, thereby in the inactiviated state. As immediate upstream of the mTORCl, the activation of Rheb could activate mTORC1, resulting in activation of its downstream target of phosphorylation of ribosomal S6 kinase (S6K) and the transcription initiation factor 4E binding protein 1 (4E-BP1).mTORClis sensitive to rapamycin and can be used as a potential immunosuppressant.Different from mTORCl,the function of mTORC2 is still unknown. In yeast and mammalian cells, it has been reported that mTORC2 plays key roles in various biological processes, including cell survival, proliferation,metabolismand cytoskeleton organization.mTORC2 was able to phosphorylate hydrophobic motif (HM) of AKT (Ser473 site), but its indeed function needs to be clarified.More and more researches now have found that mTOR signaling pathway plays an important role in inflammatory response.Inflammatory cytokines such as TNF-a、 VEGF、IL-1β、IL-5、IL-6、IL-12p40 and IFN-y are regulated by mTOR.IL-6 is an important proinflammatory factor,and its functional form is soluble IL-6 receptor,rather than IL-6 membrane receptor.The activation of mTOR increases the expression of soluble IL-6 receptor.Additionally,in the model of inflammatory related gastric cancer(GP130),IL-6 activates STAT3,meanwhile sensitizes PI3K/mTORCl by GP130/JAK signaling pathway.And mTORCl inhibiton RAD001 inhibits inflammation,therefore prevent the occurrence of gastric cancer.Epidemioligoical evidence shows that high fat diet contributes to IBD and persistent activates mTOR signaling pathway. Some scholars found that the level of p-mTOR and the expression of IL-1α、IL-6 andTNF-a in high fat diet feed rat arised at the same time.And the expression level of p-mTOR and TNF-a even positively correlated.In the model of inflammatory bowel disease and colorectal cancan,mTOR-STAT phosphorylation was enhanced in both inflammatory lesions and tumor lesions.And that the proliferation of intestinal epithelial cells are depend on mTORC1.Clinical data showed that mTORC1 signaling pathway activation can be observed up to 57% of IBD patients. So that we can speculate mTOR is closely related to the occurrence of IBD.However,therelationship between mTOR and IBD has not been reported yet.Methods1、Using Cre-loxp system,we have successfully created mice with colonic epithelial cell specific deletion of Tscl.The effect of deletion was identified by PCR and agarose gel electrophoresis for mice genotype,while western blot and IF for protein expression in colonic epithelial cells.2、The impact on mouse of colonicepithelial cell specific deletion of Tscl was assessed by physical development,colon tissue section histological and morphololgical analysis.3、Application of DSS to imitate inflammatory bowel disease model on C57 mice.The severity of IBD is estimated according by stool properties,OB test,grossly bloody stools,body weight loss,HE tissue sections and histological score.4、Oral administration of rapamycin on DSS-induced colitis C57 mice,to figure out whether colitis could be reverse by mTORC1 specific inhibitor rapamycin.5、DSS-induced colitis on colonic epithelial cell specific deletion of Tscl/Raptor mice,toexplore mTORC1 activation or inhibition effects on IBD.6、Using high-thoughput sequencing on mRNA chip of colon epithelial cellfrom knockout mice,to clarify how mTORCl signaling pathway involved in the occurrence and development of IBD.Results1、The mice model of colonic epithelial cell specificity knockout Tscl was constructed successfully.Tscl deletion has no obvious effect on physical development,colonic structure and function in 2 months old mice.We generated mice with a conditionally ablated Tscl gene incolonic epithelial cells using a Cre expression cassette under the control of the Carl promote.The effect of deletion was identified by PCR and agarose gel electrophoresis for mice genotype.After we separate the entire colon,thecolonic epithelial cells were purificated and colon tissues were fixed with 10% neutral buffered formalin, embeddedin paraffinand cut into 5um sections.Tissue sections were used to monitor mTORCl activation by assaying the level of Tscl by western blot and phosphor-S6(235/236) by IF.The specific reducementof Tscl and enhancement of phospho-S6 was observed in Tsc1KO mice.In summary,Tsc1KO micehavecolonic epithelial cellsspecific inactivation of the Tscl gene and overactivation of mTORCl signaling.Then we compare the appearance,the color and the body weight of Tscl KO mice.We also measure the entire colonic length and assess the histologicaland morphological structure and function(HE,PAS) though microscope,but found no obvious different bwteen KO and WT mice.So,we can say that Tscl deletion has no important effect on physical development,colonic structure and function in 2 months old mice.2、mTORCl specific inhibitor rapamycin reverse DSS-induced IBD model in C57 mice.Application of DSS to imitate IBD model on C57 mice.The severity of IBD is estimated according by stool properties,OB test,grossly bloody stools,body weight loss,HE tissue sections and histological score.We found that,after drinking DSS,C57 mice showed different degrees of diarrhea,OB test positive or grossly bloody stools,body weight loss gradually.Colon tissue congestion,edema and shorten.Under the microscope,we observed colonic structure damage seriously,small ulcer thoughout the colon especially the the end of the colon,inflammation cells infiltrate the mucosa and submucosa of the colon.While oral giving rapamycin meanwhile,both clinical parameters and pathological score improved.To conclusion,mTORC1 specific inhibitor rapamycin is able to reverse DSS-induced IBD model in C57 mice.3、Overactivation of mTORC1 signaling in colonic epithelial cells in mice exacerbate IBD.On the basis of the above,we futher discuss DSS-induced colitis on colonicepithelial cell specific deletion of Tscl/Raptor mice,explore mTORC1 activation or inhibition effects on IBD.We observe that Tscl knockout mice undergo a more serious colitis than its littermates after drinking DSS.Then we repeat the DSS colitis model on Raptor knockout mice,and the above-mentioned indexes have a various degrees of improvement.Therefore,we believe excessive activation of mTORC1 exacerbate the DSS induced inflammatory bowel disease.4、high-thoughput sequencing on mRNA chip of colonic epithelial cell from knockout mice to figure out mTORCl signaling pathway involved in the occurrence and development of IBDApplication high-thoughput sequencing on mRNA chip of colonic epithelial cell from knockout mice,we found that inflammation and chemotaxis related mRNA expression in Tscl knockout mice is significant higher than that in Tscl WT mice.Screening results showed that the expression of IL-6、IL-17、IL-23、TNFaand COX,which is closely associated to IBD,is at least 3 times higher in Tscl KO mice.Therefore,we hypothesized that deletion of Tscl gene activates mTORCl signaling pathways,upregulates inflammatory or proinflammatory cytokines levels and downregulates anti-inflammatory factor level, so that promote IBD development.Conclusion1、We have successfully constructed mice models Tscl knockout in colon epithelial cell and using PCR and IF to verified the knockout effect.Tscl deletion has no obvious effect on colonic structure and function in 2 months old mice.2、DSS was used to induced colits in C57 mice,mTORCl specific inhibitor rapamycin can reverse DSS-induced IBD model in C57 mice.3、Colonic epithelial cell specific deletion of Tscl undergo a more sevious colitis than its littermates.Overactivation of mTORC1 signaling in colon epithelial cells in mice exacerbate IBD.4、high-thoughput sequencing on mRNA chip of colon epithelial cell from knockout mice clarified mTORCl signaling pathway involved in the occurrence and development of IBD.5、mTOR signaling pathway provide a new explanation mechanism to IBD occurrence and development.mTORCl inhibitor is expected to become promising treatment of IBD.