RNF186 Affects Ulcerative Colitis By Regulating The EFNB1-EPHB2 Induced Autophagy

Inflammatory bowel diseases(IBD),which include Crohn disease(CD)and ulcerative colitis(UC),are characterized by chronic and recurrent inflammation of the gastrointestinal tract despite exhibiting distinct pathophysiological features.The clinical manifestations of this disease are recurrent abdominal pain and diarrhea,bloody mucopurulent stool.IBD brought great physical and mental pain to the patients due to the persistent symptoms.Moreover,patients with a long history of IBD face an increased risk of developing colorectal cancer or developing small bowel adenocarcinoma.At present,there is no effective treatment for IBD.The molecular and cellular mechanism of IBD has not yet been fully clarified,and increasing evidence showes that genetic,environmental and immune factors are associated with the occurrence and development of this disease.Genome-wide association studies(GWAS)have identified several IBD susceptible genes,and several of these genes encode proteins which are relevant to autophagy,including autophagy related16 like 1(Atg16l1),unc-51 like autophagy activating kinase 1(ULK1)and immunity related GTPase M(IRGM).These findings suggest that autophagy may play an essential role in the host defense against intracellular pathogens and maintenance of intestinal homeostasis.In the previous genetic studies,RING finger protein 186(RNF186),which encodes a RING-finger domain-containing E3 ubiquitin-protein ligase,contains two rare single nucleotide polymorphisms(SNP),which were found to alter the amino acid sequence of RNF186:RNF186^A64T and RNF186^R179X,are related to the UC susceptibility.Interestingly,these RNF186 variants are related to different UC susceptibility:individuals who carried the RNF186^A64T variant were susceptible to UC,whereas carriers of the RNF186^R179Xvariant were protected from UC.While the mechanism of these two variants leading to distinct UC susceptibility was still unclear.By using the molecular biology techniques and experimental animal model in this study,we find that RNF186 regulates EFNB1-EPHB2signaling by ubiquitinating EPHB2,which is essential for EFNB1-EPHB2-induced autophagy in the colonic epithelial cells.Additionally,treatment with ephrin-B1-Fc recombinant protein effectively relieves DSS-induced mouse colitis,which suggests that ephrin-B1-Fc may have the potential to be the drug for the treatment of human ulcerative colitis.RNF186 gene is knocked out in colonic epithelial cells by CRISPR-Cas9,and the protein expression in colonic tissue of patients with ulcerative colitis is analyzed.It is found that RNF186 participates in the maintainence of basal level of autophagy in the intestinal epithelial cells.Identification of interacting proteins by co-immunoprecipitation and mass spectrometry experiments reveal that receptor tyrosine kinases EPHB2 and EPHB3 are interacted and ubiquitinated in vivo and in vitro by RNF186.Furthermore,a human UC susceptible mutant of RNF186,RNF186^A64T loses the interaction with EPHB2 and has decreased E3 catalytic ability for EPHB2,whereas the UC protective mutant,RNF186^R179X,increases the interaction with EPHB2.These results suggest that RNF186 is involved in the regulation of the susceptibility of ulcerative colitis through EPHB2,and RNF186^A64T is a loss-of-function variant,while RNF186^R179X is a gain-of-function variant.Through western blotting and immunofluorescence experiments,it is further found that the ligand EFNB1(ephrin-B1)of EPHB2 induce autophagy in intestinal epithelial cells,which is depended on RNF186 and EPHB2.The ubiquitination site was identified by mass spectrometry,and the lysine 892(K892)of EPHB2 is ubiquitinated by RNF186,and EFNB1-EPHB2-induced autophagy is dependent on the ubiquitination of EPHB2 K892 mediated by RNF186.In addition,this site is crucial for the interaction between EPHB2 and autophagy related protein MAP1LC3B.Gentamycin protection assay shows that autophagy induced by EFNB1-RNF186-EPHB2 axis is essential for the clearance of intracellular bacterial in the intestinal epithelial cells.Inactivation of this pathway leads to defect of eliminating intracellular bacterial in the intestinal epithelial cells,which suggests that this pathway play an important role in the host defense against intracellular pathogens and maintenance of intestinal homeostasis.rnf186^-/-or ephb2^-/-mice shows much more severe phenotype than that of control mice in a dextran sulfate(DSS)-induced colitis model,which verified the important role of these two molecules in the pathogenesis of ulcerative colitis.Additionally,injection of recombinant protein ephrin-B1-Fc into mice activates autophagy in the colonic epithelial cells and alleviates the phenotype of DSS-induced colitis in mice,which process depends on the EPHB2.In conclusion,the functional mechanism of RNF186 in IBD is analyzed at the cellular and mouse individual levels in this study.It is the first report that RNF186 affects the occurrence and development of ulcerative colitis via regulating autophagy induced by EFNB1-EPHB2 pathway.Also,it is proposed that the recombinant protein ephrin-B1-Fc may be a promising medicine for the human ulcerative colitis.