CK8Induces The Formation Of Colitis By Negative Regulating NF-κB Signal Pathway

NF-κB signal pathway is widely involved in the regulation of many biologicalprocesses, including cell proliferation, survival, apoptosis, immune response,inflammation etc. CK8is a component of keratin intermediate filament, whoseexpression is closely related to the occurrence of liver tumor. Previously, we foundthat CK8can inhibit the activation of NF-κB signal pathway inducted by TNF,suggesting that CK8is a negative regulator of NF-κB. The present research focuseson identifying the role of CK8in the negative regulation of NF-κB pathway andmolecular mechanism, and exploring its physiologic pathology role of the negativeregulation of NF-κB pathway.We first used CK8knockout mouse to verificate the negative regulation ofNF-κB by CK8in vivo. We use AOM+DSS-induced colon cancer model to evaluatethe function of CK8in colon cancer. The results show that mesenteric lymph nodesCD4+CD69+T cell ratio increased significantly, suggesting CK8knockout may playan important role in the process of colon cancer.We also confirmed the negative regulation of CK8on NF-κB signaling pathwayinduced by TNF. Using alkaline phosphatase reporter gene analysis, we found thatCK8inhibited TNF-induced activation of NF-κB signaling pathway in adose-dependent manner. TNF-induced IκB phosphorylation was also inhibited byCK8overexpression. Gel supershift assay confirmed the CK8reduced DNA bindingactivity of NF-κB. Realtime PCR analyisis demonstrated that CK8inhibited theNF-κB downstream target genes such as IL-1A. In addition, CK8inhibited NF-κBactivation induced by Toll-like receptor5(TLR5) agonist flagellin CLB502, in adose-dependent manner. Further, we found that CK8blocked NF-κB pathway throughthe IKK complex. Furthermore, we found that CK8regulated NEMO ubiquitinationby blocking the interaction between NEMO and TRAF2.To further explore the function of CK8,CK8RNAi lentiviral vectors wereconstructed. With lentiviral packaging, high titers of virus particles were obtained andinfected into human colon cancer cells HCT116. Thus, CK8knockdown stable celllines were generated, which providing a ideal modelfor the subsequent experiments. In order to the specificity of the mouse phenotype, we also prepared mouse CK8overexpression lentivirus.Taken together, this study confirm that cytokeratin CK8negatively regualtesNF-κB signaling pathway through blocking the interaction between TRAF2andNEMO. CK8deletion in mice lead to ecotopic activation of NF-κB signaling pathwaywith the spontaneous colitis accompanied by hyperplasia of the colon.