E3 Ubiquitin Ligase RNF128 Inhibits STAT3 Signaling By Mediating Ubiquitin Modification Of IL-6 Receptor

Background:Inflammatory bowel diseases（IBD）are persistent and intractable immune inflammatory diseases of the intestine and are a leading factor in the initiation,formation and progression of colitis-associated colorectal cancer（CAC）.Therefore,exploring the pathogenesis of IBD can provide new ideas for the treatment of IBD and prevention of CAC.E3 ubiquitin ligase RNF128 is an important immunomodulatory molecule widely involved in the development of inflammatory diseases and tumors.We found that RNF128 can inhibit the progression of colitis and intestinal inflammation to CAC by regulating IL-6-mediated IL-6R-STAT3 signaling pathway,but the exact molecular regulatory mechanism remains unknown.Therefore,exploring the pathogenesis of IBD can provide new ideas for the treatment of IBD and prevention of CAC in humans.Objective:To elucidate the role of RNF128 in intestinal inflammation and to reveal the exact molecular mechanism by which RNF128 regulates IL-6-triggered signal transduction and activator of transcription 3（STAT3）activation during the progression of chronic intestinal inflammation to CAC.Methods:1.WT and Rnf128^-/-mice were used to construct a precancerous colitis model by oxidizing azoxymethane（AOM）and dextran sodium sulfate（DSS）administration for14 days.Mouse colon tissue was collected for heat map analysis of gene gntology（GO）biological processes,kyoto encyclopedia of genes and genomes（KEGG）pathways and inflammatory cytokines by whole genome RNA-seq.Then,the inflammatory cytokine expression was detected by Real-time fluorescence quantitative polymerase chain reaction（RT-q PCR）and Enzyme-linked immunosorbent assay（ELISA）to further confirm the protective effect of RNF128 on colitis and its relationship with inflammation.2.Western-blot and immunohistochemical staining（IHC）were used to detect the expression of p-STAT3^Y705in WT and Rnf128^-/-mouse colon tissues and human colorectal cancer cells overexpressing and knocking down RNF128（IL-6 stimulated）to confirm whether RNF128 regulates IL-6 triggered activation of STAT3.3.Coimmunoprecipitation（Co-IP）was used to detect the interaction of RNF128with signaling molecules critical to the IL-6/STAT3 signaling pathway.Then,the interaction between RNF128 and IL-6 receptor（IL-6R）IL-6Rαand glycoprotein 130（gp130）and their colocalization in cells under IL-6 stimulation were examined by Co-IP and immunofluorescence（Immunofluorescence,IF）methods.4.Western-blot was used to detect RNF128-mediated degradation of key signaling molecules of IL-6/STAT3 signaling pathway.Mutants were then constructed to detect the manner in which RNF128 ubiquitinates and degrades IL-6Rαor gp130using Co-IP,thereby clarifying the molecular mechanism by which RNF128 regulates the IL-6/STAT3 signaling pathway.5.Mice were injected intraperitoneally with STAT3 activation inhibitor（Stattic）to observe whether Stattic could reverse the susceptibility of Rnf128^-/-mice to AOM/DSS-induced colitis:survival rate and body weight changes were observed during drug administration and modeling.The length of mouse colon was measured at day 14,and colon histopathology was scored by HE staining,inflammatory cytokine expression was detected by RT-q PCR and p-STAT3^Y705was detected by immunoblotting.Thus,we further verified whether RNF128 inhibited AOM/DSS-induced precancerous colitis in vivo by regulating the IL-6-STAT3pathway.Results:1.In the experiment of precancerous colitis induced by AOM/DSS,colon tissues of mice with precancerous colitis were analyzed by genome-wide RNA-seq,RT-q PCR and ELISA.The results revealed that RNF128 promotes the production of inflammatory factors such as IL-6,revealing that Rnf128^-/-mice suffer from excessive and chronic intestinal inflammation in the early stages of carcinogenesis.2.We found that the expression of p-STAT3^Y705was significantly higher in colonic epithelial cells of Rnf128-deficient mice than in WT mice by western blotting,IF and IHC methods.Then overexpression and knockdown of RNF128 in human colon cancer cells further confirmed that RNF128 inhibited IL-6-mediated STAT3activation.3.We found that RNF128 interacts with IL-6Rαor gp130 in the presence of IL-6using Co-IP and protein immunoblotting methods,and that IL-6 enhances their interactions.Further by IF,RNF128 was found to be significantly colocalized with IL-6Rαor gp130 in the cell membrane and cytoplasm with IL-6 trigger.4.We found by overexpression and knockdown of RNF128 experiments that RNF128 was able to mediate the degradation of RNF128 with IL-6Rαand gp130 but not JAK1、JAK2 and STAT3 via the lysosomal pathway.RNF128 also degraded IL-6Rαand gp130 in a dose-dependent manner as well as reduced the IL-6Rαand gp130 half-life;whereas RNF128 without ligase activity was unable to degrade them.Also,in the early inflammatory stage of CAC carcinogenesis,the expressions of IL-6Rαand gp130 in colon tissue of Rnf128^-/-mice were higher than those of WT mice.5.We first found that RNF128 mediates K48-linked polyubiquitination of IL-6Rαand gp130 by overexpressing and knocking down RNF128.We then found that RNF128 mediated the polyubiquitination and degradation of IL-6Rαat K398/K401 and gp130 at K718/K816/K866.6.The STAT3 activation inhibitors（Stattic）can block the STAT3 activation at the early transformation stage of CAC,thereby inhibiting excessive intestinal inflammation caused by Rnf128 defects after AOM/DSS treatment.Conclusions:1.In the early stage of CAC cancer inflammation,RNF128 inhibits the production of IL-6 and other pro-inflammatory cytokines.2.RNF128 inhibits the activation of STAT3 in colon epithelial cells.3.RNF128 interacts with IL-6Rαand gp130 in the cell membrane and cytoplasm.4.RNF128 mediated the polyubiquitination of IL-6Rαat K398/K401 and gp130at K718/K816/K866 in an E3 ligase-dependent manner,and translocated to lysosome degradation.5.The STAT3 activation inhibitor Stattic inhibits the susceptibility of Rnf128deletion to colitis in the early stage of CAC cancer inflammation.E3 ubiquitin ligase RNF128 exerts a protective effect against colitis by mediating the activation of IL-6-STAT3 signaling pathway by mediating IL-6R polyubiquitination and degradation on the membrane and in the cytoplasm.