Su1870 Differential Effects of Acute, Chronic and Combined Stress Paradigms on Small Intestinal and Colonic Permeability in Rats

Abstract

Background: Kruppel-like factor 5 (KLF5) is a zinc-finger transcription factor abundantly expressed in the crypts of Lieberkuhn, the proliferative compartment of the intestinal epithelium. KLF5 has been shown to regulate intestinal epithelial cell proliferation and embryonic enterocyte differentiation (Bell SM et al., Developmental Biology 2013; 375:128-39). Our previous study shows that KLF5 regulates maintenance of intestinal crypt architecture and epithelial barrier integrity (McConnell BB et al., Gastroenterology 2011; 141:1302-13). Aim: To assess the role of KLF5 in regulating barrier function of intestinal epithelial cells via tight junction complexes. Methods: We established stable KLF5 knockdown in Caco-2 BBe cells using lentiviral delivery system. We used transepithelial electrical resistance (TEER) measurement and FITC-dextran assay to assess the formation and function of tight junctions in KLF5 knockdown and control Caco-2 BBe cells grown on transwell plates. We examined expression levels and distributions of apical junctional complex components using Western blot and immunofluorescent staining. Additionally, we estimated the effects of short-term downregulation of KLF5 in Caco-2 BBe using siRNA technique. Furthermore, using luciferase assay approach, we tested the effects of KLF5 levels on occludin (OCLN) promoter activity in DLD-1 and HCT116 colorectal cancer cell lines. Results: Caco-2 BBe cell line with stable knockdown of KLF5 (Caco-2 BBE ΔKLF5) showed reduced levels of TEER and increased permeability as measured with FITC-dextran assay in comparison to control cell line (Caco2 BBE Ctrl.) over three weeks period. Caco-2 BBE ΔKLF5 cell line was characterized by decreased levels and altered distribution of tight junction proteins, such as ZO-1 and occludin, and adherens junction proteins, such as E-cadherin and β-catenin in comparison to Caco2 BBE Ctrl. However, Caco-2 BBE ΔKLF5 unexpectedly exhibited higher expression level of KLF5 compared to Caco-2 BBE Ctrl. by the end of the three weeks period. This phenomenon can be due to compensatory mechanism or due to selective advantage. Short-term KLF5 knockdown by siRNA in Caco-2 BBe cell line led to an increase in occludin protein level, but no significant changes in other apical junctional complex components were observed. Consistent with this result, OCLN promoter activity was increased upon KLF5 knockdown as tested in both DLD-1 and HCT116 cell lines. Conclusion: Our preliminary data suggest that altered expression of KLF5 results in impaired barrier function of Caco-2 BBe monolayer and modification to the levels of expression and/or distribution of tight junction components.