Cytokeratin 8 Negatively Regulates TLR/NF-κB Signal Pathway And Protects Mice From Colitis And Colitis-associated Cancer

Chronic inflammation is thought to be the leading cause of many human cancers including colorectal cancer (CRC). CRC is the third common malignancy and one of the major causes of cancer-related death. The gut microbiota is altered in the CRC. NF-κB signal pathway play an important role in inflammatory-to-cancer process. Keratin 8 is a member of intermediate filaments family, which are expressed mainly in epithelial cells and skin appendages, such as colon, liver and lung etc. Previous studies suggest that CK8 is involved in cell migration and adhesion. And CK8 knockout mice spontaneously developed colitis. Our previous study also found that CK8 is a physiological partner of NEMO and inhibits TNFa-induced NF-κB pathway activation. Furthermore, CK8 is down-regulated in colitis and colon cancer patients. These data suggest that CK8 is a novel regulator of NF-κB signal pathway and most likely play an important role in the process of colitis-associated colon cancer.In the present study, we used CK8+/- mice to evaluate the function of CK8 in colitis-associated colon cancer. Genetic ablation of CK8 leads to increased susceptibility to DSS-induced colitis with shortened colon length, increased inflammation index in colon and elevated inflammatory factors such as TNF-α, IL-1α, KC. We further investigated the susceptibility of CK8+/- mice to AOM/DSS-induced colon cancer. he CK8+/- mice showed remarkable weight loss, significantly higher stool index, and more serious fecal occult blood compared with WT mice. Furthermore, the peripheral leukocytes were increased, and red blood cells and hemoglobin were decreased after AOM/DSS treatment in CK8+/- mice. Dissection of the gut at the end of treatment confirmed the presence of macroscopic polyps in the distal colon and rectum of CK8+/-mice and WT mice, and the CK8+/- mice developed significantly more polyps compared with WT mice. Moreover, the polyps were more numerous and significantly larger in the CK8+/- mice. Elevated levels of proinflammatory cytokines and tumor related genes were detected in CK8+/- mice. These data suggest that genetic ablation of CK8 leads to increased susceptibility to AOM/DSS-induced colon cancer.We further investigated the colonic permeability of WT mice and CK+/- mice using the FITC-labelled dextran method. CK8+/- mice showed greater permeability with more seriously damaged crypt in comparison with WT mice. Metagenomic analysis of fecal microbiota suggested that Firmicutes was detected in the CK8+/- mice. Then we infected mice with E.coli by intraperitoneal injection and the CK8+/- mice showed increased susceptibility to E.coli infection. We furthermore examined the effect of CK8 on TLR-mediated NF-κB signal pathway. CK8 overexpression inhibited various TLR ligands-induced activation of NF-κB signaling pathway including LPS, CpG、PolyI:C and Pam3csk4. Further, CK8 inhibited NEMO ubiquitination. Taken together, this study confirmed that CK8 negatively regulates TLR/NF-κB signaling pathway and protects intestinal epithelial cell structure integrity and regulates intestinal homeostasis.