(C and D) Immunoblots for -catenin levels in CCD 841 CoN cells under the same treatment conditions utilized for the assays for which the results are shown in panels A and B are shown beneath each time point. -catenin expression neither altered the phosphorylated IB kinase / complex nor activated activator protein 1 signals. Furthermore, inhibition of glycogen synthase kinase 3 was associated with increased -catenin expression and attenuated NF-B activity and IL-8 expression in BFT-exposed cells. These findings suggest the unfavorable regulation of NF-B-mediated inflammatory responses by -catenin in intestinal epithelial cells stimulated with BFT, resulting in attenuation of acute inflammation in ETBF infection. (ETBF) is associated with intestinal diseases, such as colitis, inflammatory bowel disease, and colorectal cancer (1,C3). The important cause of these diseases is known to be the enterotoxin produced by ETBF strains (2, 4). Exposure of intestinal epithelial cells to enterotoxin (BFT) rapidly activates nuclear transcriptional factors, such as nuclear factor kappa B (NF-B) and activator protein 1 (AP-1), leading to the release of proinflammatory mediators, such Rabbit Polyclonal to OR1L8 as interleukin-8 (IL-8) (5,C8). We previously found that the activated signals of NF-B and AP-1 in BFT-exposed intestinal epithelial cells gradually decline after exposure (5,C8). Therefore, it is possible that some factors may modulate the activities of transcriptional factors in BFT-exposed cells and contribute to the regulation of enteric inflammation. Although ETBF strains are considered enteric pathogens, clinical studies have revealed that in many cases of infection, bacteria alone are present without symptoms of enteritis (4, 8, 9). Therefore, it is believed that some negative regulatory signals for enteric inflammation might be induced after intestinal epithelial cells are exposed Drostanolone Propionate to BFT derived from ETBF. In the present study, we propose that altered expression of -catenin is one of these regulatory signals. -Catenin is a member of the Wnt/-catenin pathway, which regulates various cellular processes, such as cellular proliferation, differentiation, and development, as well as intercellular adhesion (10,C12). In the absence of extracellular Wnt ligands, the canonical Wnt/-catenin pathway is inactive (Wnt-off state) and -catenin is maintained at low levels in the cytoplasm due to its degradation through the ubiquitin-proteasome pathway. The -catenin destruction complex is formed by the scaffold protein axin, adenomatous polyposis coli protein (APC), glycogen synthase kinase 3 (GSK-3), and casein kinase I isoform (CK1). In this complex, -catenin is phosphorylated at the N-terminal domain (first at Ser45 by CK1 and then at Ser33, Ser37, and Thr41 Drostanolone Propionate by GSK-3), followed by polyubiquitination and subsequent degradation by the ubiquitin-proteasome-mediated pathway (13, 14). In intercellular adhesion, -catenin localizes to the plasma membrane, acting as a bridge between E-cadherin and cytoskeleton-associated actin to form adherent junctions between cells (13). BFT is a metalloprotease and can destroy the tight junctions in the intestinal epithelium by cleaving E-cadherin, resulting in the release of -catenin and the loss of tight junctions (2, 15, 16). From the perspective of clinical findings associated with ETBF infection, these results may lead to the leakage of the intestinal barrier and the diarrhea that are characteristically observed in ETBF infection (15, 16). However, the role of -catenin as a cellular signaling intermediate in the induction of proinflammatory responses by BFT has not been clarified. NF-B is a dimeric transcription factor composed of homodimers or heterodimers of Rel proteins, of which there are five family members in mammalian cells (i.e., RelA [p65], c-Rel, Rel B, NF-B1 [p50], and NF-B2 [p52]) (6, 17). We previously demonstrated that BFT primarily induces p65 and p50 heterodimers in intestinal epithelial cells (6, 18). These NF-B dimers are held in the cytoplasm in an inactive state by physical interaction with IB proteins. Therefore, IB is a negative regulator of NF-B signaling. In the context of enteric inflammation, the regulation of -catenin and IB seems to be similar. Thus, -catenin and IB are associated with phosphorylation of the same N-terminal serine sequence sites, ubiquitination by the same E3 ligase complex, and subsequent proteasomal degradation (19). As evidence to support this, the degradation of -catenin has been related to the activation of NF-B signals in intestinal epithelial cells infected with (20, 21). However, the opposite results have also been reported. For example, inflammatory cytokine expression and NF-B activity are all significantly reduced Drostanolone Propionate when -catenin is knocked down in human monocytic THP-1 cells stimulated with the Der.