557 colectomy rate in hospitalized ulcerative colitis patients undergoing therapy with cyclosporine (csa)

Background and Aims: Although expression of the di/tripeptide transporter PepT1 has been observed in colon under inflammatory conditions, the inducing factors and underlying mechanisms have not yet been investigated. Here, we addressed the role of pathogenic bacteria in the regulation of colonic PepT1 expression/function and the potential role of PepT1 in bacterial-epithelial interaction. Methods: Colonic HT29-Cl.19A cells were infected with enteropathogenic E. coli (EPEC). PepT1 promoter activity and PepT1 expression/activity were analyzed using the luciferase assay, RT-PCR, nuclear run-on assay, immunoblotting, immunofluorescence staining and uptake experiments. Cdx2-PepT1 promoter binding was assessed by gel-shift and chromatin immunoprecipitation assay. In Vitro experiments were validated by ex vivo and In Vivo infection of wild type and PepT1 over-expressing mice with Citrobacter rodentium. Interleukin (IL)-8 and keratinocyte-derived chemokine (KC) expression levels were quantified by real-time RT-PCR and ELISA. Results: EPEC transcriptionally induced PepT1 expression/activity in HT29-Cl.19A cells. Cdx2 over-expression in HT29-Cl.19A cells induced PepT1 expression and Cdx2 silencing markedly reduced EPECinduced PepT1 expression, indicating the importance of Cdx2 in PepT1 expression. Furthermore, PepT1 expression required intimate adherence of EPEC to host cells through lipid rafts (LRs). Importantly, PepT1 expressed upon EPEC infection is functionally localized in LRs, and PepT1 associated with LRs delayed EPEC-LR binding as monitored in real time by an electric cell-substrate impedance-sensing technique. Remarkably, PepT1 over-expression in HT29-Cl.19A cells reduced EPEC-triggered NF-κB and MAP kinase activation and IL-8 production. In agreement with In Vitro data, ex vivo and In Vivo experiments showed that C. rodentium increased PepT1 mRNA and protein expression levels in mouse colon. Furthermore, PepT1 over-expression in mouse colon reduced C. rodentium adherence and C. rodentium-induced KC production. Conclusions: We demonstrate that i) EPEC transcriptionally induces functional PepT1 expression in LRs of colonocytes by intimately attaching to host cell membranes through LRs, ii) the transcription factor Cdx2 is crucial for EPECinduced PepT1 expression, and iii) PepT1 associated with LRs is involved in bacterialepithelial interaction and intestinal inflammation. Our findings not only reveal a novel mechanism underlying the regulation of colonic epithelial PepT1 expression/function under pathological conditions, but also highlight the potential contribution of this transporter to host defense mechanisms in response to pathogenic attack.