Differential Responses of NF kappa B Activation and Cell Apoptosis in Constitutional Growth and upon LPS Challenge in Two Intestinal Epithelial Cell Lines

• Main Authors: Yin-Chung Au, 區曣中
• Other Authors: 余佳慧
• Format: Others
• Language: zh-TW
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/96838897116629593077

碩士 === 臺灣大學 === 生理學研究所 === 95 === The intestinal epithelial monolayer not only serves for digestive, absorptive and secretive functions, but also acts as the first line of defense against luminal noxious stimuli. Excessive epithelial apoptosis may compromise this barrier function, whereas delayed apoptosis is in part involved in intestinal carcinogenesis. Previous studies have documented that luminal exposure to bacterial lipopolysaccharide (LPS) augments enterocytic cell death in dose- and time-dependent manner, whereas others reported the activation of a pro-inflammatory transcription factor, i.e. nuclear factor kappa B (NFκB) upon LPS challenge, in intestinal epithelial cell lines. The relationship between the two phenomena is still not understood. It remains elusive whether NFκB is pro- or anti-apoptotic in enterocytes. The first aim of the study was to examine the role of NFκB in modulating apoptosis in two intestinal epithelial cell lines, i.e. T84 and Caco-2, in constitutional growth. The second aim was to characterize the phenomena of enhanced cell apoptosis and NFκB activation induced by LPS in these two cell lines. In the first part of the study, colonic carcinoma T84 and Caco-2 cell lines were treated with an NFκB inhibitor, pyrrolidinedithiocarbamate (PDTC), at 0-50μM for various time points to examine its effect on cell apoptosis. The effect of PDTC in modulating nuclear translocation and DNA binding activity of NFκB (heterodimer of p50 and p65 subunits) was first verified. Western blotting results revealed constitutive nuclear translocation of p65 in the two cell lines, which is reduced by PDTC in a time-dependent fashion. The DNA binding activity of p50 was also decreased in cells treated with PDTC. Our results showed that PDTC dose-dependently induced apoptosis, i.e. nuclear condensation and DNA fragmentation, in both T84 and Caco-2 cell as evidenced by Hoechst and TUNEL staining, as well as Cell death ELISA. These results indicated that constitutive NFκB activation plays a pro-survival role in intestinal epithelial carcinoma cell lines. In the second part of the study, confluent T84 and Caco-2 cells were luminally exposed to bacterial LPS (from E. coli, O26:B6 serotype) at 0-50 ug/ml. The level of NFκB activation was examined at 15 and 60 mins post challenge and cell death was examined at 24 hrs after LPS exposure. LPS dose-dependently triggered cell apoptosis, but failed to enhance nuclear translocation of NFκB in Caco-2 cells. In contrast, LPS exposure increased NFκB activation in T84 cells, whereas the level of apoptosis did not change compared to untreated controls. Moreover, immunofluorescent staining showed differential expression of LPS receptor components on the two cell lines. In line with previous reports, the expression of Toll-like receptor (TLR)-4 was detected in T84, but none was seen in Caco-2 cells. On the other hand, expression of CD14 was observed on Caco-2, but not T84 cells. Previous studies have demonstrated that TLR-4 is the main receptor component responsible for inducing downstream NFκB activation. Our current study using functional blocking antibodies revealed that CD14 is involved in the induction of apoptosis by LPS challenge in Caco-2 cells. Taken together, the distinct response in T84 and Caco-2 cells upon LPS challenge may be attributed to the differential expression of LPS receptor components.