Title

Transcription factor NF-kappaB in the colonic epithelium

Date of Completion

January 2000

Keywords

Biology, Molecular|Biology, Cell

Degree

Ph.D.

Abstract

NF-κB is a transcription factor regulating genes that influence cell proliferation, apoptosis and inflammatory responses. We analyzed the influence of NF-κB on turnover of colonic epithelial cells, and studied its regulation by short chain fatty acids generated from dietary fiber metabolism. ^ Since the short chain fatty acid butyrate effects colonic epithelial cell proliferation and intestinal inflammation, we investigated the effect of butyrate on NF-κB activity in human colonic HT-29 cell line. First, we found that NFκB-p50 is constituvely active in these cells. Exposure of human HT-29 cells to butyrate eliminated this constitutive NF-κB activity, and reduced NF-kB activation by of TNF-α over 10-fold. Changes in NF-κB activation did not correlate with NF-κB subunit expression nor in the levels inhibitory molecule, IκBα. These findings suggest that butyrate's influences on colonic inflammatory responses may result, in part, from its influence on NF-κB activation. ^ We also investigated expression and activity of NF-κB in mouse colonic epithelium. NF-kB p65 subunit expression and activity occurred at the bottom of the crypts as a p65-p50 dimer. In contrast, p50 subunit expression was found throughout the crypt, with the p50-p50 dimer active in mature cells. Interestingly, NF-κB1 (p50) deletion largely eliminates NF-κB activity and causes more epithelial cell proliferation and possibly cell death in colonic epithelium. Gene profiling indicates that two growth regulatory molecules are up-regulated in the NF-κB1 knockout mice. Taking together these data demonstrate that NF-kB participates in regulation of cell turnover in the colon. ^ Given the potential growth regulatory role of NF-kB, we examined expression of NF-κB-regulating IκB proteins in normal and transformed colon epithelium. Immunohistochemical analysis of the mouse colon reveals that IκBβ is expressed at high levels in epithelial cells, whereas IκBα is found primarily within lamina propria. Interestingly, immunoblot analysis reveals two larger IκBβ proteins (p106 and p112) in transformed epithelial cells. These p106 and p112 proteins are stably ubiquitinated forms of IκBβ, which may interfere with NF-kB regulation in transformed colonocytes. ^ Taken together, these findings suggest that NF-κB activity and expression are highly regulated in the colon, and that it is involved in the regulation of cell proliferation. ^