Functions Of STAT3 Arginine Mono-methylation In The Regulation Of Inflammatory Bowel Disease

Background and purposeInflammatory bowel disease(IBD)is a complex genetic disease caused by dysfunction of mucosal immune system and intestinal barrier.The pathogenesis of IBD is complicated and the etiology remains unclear.STAT3 is an important transcription factor with diverse functions,which plays critical roles in the pathogenesis of IBD by inducing Th17 cell differentiation and pro-inflammatory cytokine secretion.Post-translational modifications of STAT3,such as phosphorylation,acetylation and palmitoylation,have been thoroughly studied in the pathogenesis of IBD,but the role of arginine(R)methylation of STAT3 in the regulation of IBD has not been reported so far.Accordingly,we proposed whether STAT3 arginine methylation involves in the regulation of IBD.MethodsIn view of the above scientific issues,we put forward a scientific hypothesis:STAT3 arginine residue is methylated by arginine methyltransferase,and methylated STAT3 plays an important role in regulating immune cells activity,and thus participates in the regulation of IBD.Based on the scientific hypothesis,we designed experiment program and used a series of advanced techniques and methods such as high-resolution mass spectrometry,RNA-Seq,flow cytometry,immunofluorescence,Western blot,immunohistochemistry,real-time fluorescence quantitative PCR,and we also constructed the classic colitis mouse model to verify the scientific hypothesis.ResultsThe mass spectrometry(MS)analysis revealed STAT3-R31 site was mono-methylated.To test this new finding,we have done the following experiments.(1)Mono-meR31-STAT3 antibody was synthesized to verify the modification.(2)Co-transfection of STAT3 and arginine methyltransferase 1(PRMT1)further confirmed that PRMT1 could catalyze STAT3-R31 methylation.(3)The R31 residue of STAT3 was mutated by point mutation technique,and found that the stability of STAT3 protein was decreased and the ubiquitination level was increased,suggesting that R31 residue of STAT3 might play a significant role in stabilizing STAT3 protein.These results demonstrated that PRMT1 catalyzed STAT3-R31 mono-methylation,which was the basis of STAT3 protein function.On the other hand,we used STAT3-R31K knock-in mice to further explore the function of R31 residue.The results were presented below.(1)The expression levels of STAT3 protein were decreased in almost all the organs including the intestinal epithelium in the STAT3-R31+/-mice compared to the STAT3-WT mice,confirming R31 residue of STAT3 maintained the stability of STAT3 protein.(2)STAT3-R31K mutation impaired T cells activation,mitochondrial function and induced T cells apoptosis in vitro,and RNA-seq analysis revealed the DEGs were mainly enriched in the regulation of cytokines gene sets and IBD pathway.(3)Th17 differentiation was inhibited in STAT3-R31+/-mice or by PRMT1 inhibitor,indicating PRMT1 might be involved in Th17 cell differentiation through STAT3-R31 methylation.(4)To our surprise,the symptoms of DSS-induced colitis in STAT3-R31K+/-mice were more severe than the STAT3-WT mice with more severe damage in intestinal barrier.(5)The protein expression level of STAT3,mono-meR31-STAT3 and PRMT1 were significantly increased in DSS-induced mice,which further confirmed PRMT1 catalyzed STAT3-R31 mono-methylation played important role in the regulation of IBD.Although there were some conflicts between in vitro and in vivo experiments,our study still gave an important hint that STAT3-R31 methylation played a critical role in the regulation of IBD.ConclusionOur study firstly revealed PRMT1 catalyzed STAT3 protein mono-methylation at arginine(R)31 residue.STAT3-R31 mutation impaired the protein stability,and accelerated the protein degradation through ubiquitin-proteasome pathway.We also confirmed STAT3-R31 participated in the regulation of na(?)ve CD4+T cells activation,Th17 cell differentiation and DSS-induced colitis.Therefore,we proposed a new mechanism that mono-meR31-STAT3 played important role in the regulation of IBD pathogenesis.This study was original and innovative,providing a new research direction for studying the mechanism of STAT3 and giving an important reference for the clinical treatment of IBD.