Gastroenterology

Gastroenterology

Volume 118, Issue 4, April 2000, Pages 724-734
Gastroenterology

Alimentary Tract
The luminal short-chain fatty acid butyrate modulates NF-κB activity in a human colonic epithelial cell line,☆☆,

https://doi.org/10.1016/S0016-5085(00)70142-9Get rights and content

Abstract

Background & Aims: The transcription factor nuclear factor–κB (NF-κB) plays a central role in regulating immune and inflammatory responses. Because butyrate deficiency has been associated with inflammatory bowel disease, we examined the effect of butyrate on NF-κB activity in the human HT-29 colonic cell line. Methods: The influence of butyrate (4 mmol/L) on NF-κB activity was determined using the gel mobility shift assay. The effect of butyrate on the expression of NF-κB subunits and inhibitory proteins was determined by immunoblotting. NF-κB–regulated gene expression was assayed by primer extension of intercellular adhesion molecule 1 and Mn superoxide dismutase messenger RNA, and by analysis of a transfected luciferase reporter. Results: Exposure of HT-29 cells to butyrate eliminated their constitutive NF-κB, p50 dimer activity. This inhibition corresponded with a reduction in p50 nuclear localization, without a reduction in expression. Butyrate also selectively modulated activation of NF-κB, suppressing its activation by tumor necrosis factor α and phorbol ester more than 10-fold, without affecting the activity induced by interleukin (IL)-1β. Butyrate did, however, enhance formation of the stronger p65-p50 transcriptional activator in IL-1β–stimulated cells. The changes in NF-κB activation did not correlate with changes in IκBα levels. Gene expression reflected DNA binding. The influence of butyrate on NF-κB may result in part from its ability to inhibit deacetylases because the specific deacetylase inhibitor trichostatin A has a similar effect. Conclusions: These findings suggest that the influences of butyrate on colonic inflammatory responses may result in part from its influence on NF-κB activation. This activity of butyrate apparently involves its ability to inhibit deacetylases.

GASTROENTEROLOGY 2000;118:724-734

Section snippets

Cell culture and treatments

HT-29 cells were purchased from American Type Culture Collection (Rockville, MD) and were propagated on tissue culture plastic with McCoy's 5A medium supplemented 10% fetal bovine serum, nonessential amino acids, streptomycin (50 μg/mL), and penicillin (50 U/mL).46 All medium components were purchased from Life Technologies (Gaithersburg, MD). TNF-α was purchased from R&D Systems and used at a final concentration 100 μg/mL. IL-1β was purchased from Promega (Madison, WI) and used at a

Influence of butyrate on NF-κB p50 dimer activity

Because evidence shows that butyrate may suppress mucosal inflammation,41, 42, 43 and many anti-inflammatory agents function by suppressing NF-κB activity (e.g., glucocorticoids and salicylates),44, 45 we determined the influence of butyrate on NF-κB in HT-29 cells. HT-29 cells possess a constitutive NF-κB DNA binding activity. Supershift analyses using antibodies to the NF-κB subunits indicate that this constitutive complex is a p50 dimer. As shown in Figure 1A, an antibody to the p50 subunit

Discussion

Butyrate plays a major role in many aspects of intestinal health and function. It is an important energy source for epithelial cells, plays a central role in regulating differentiation and apoptosis, and can suppress the development of colorectal cancer in laboratory animals.27, 35, 36, 37, 38, 39, 53, 56, 57 Evidence also indicates that butyrate may suppress intestinal inflammation.41, 42, 43 The mechanism of this potential anti-inflammatory effect is not clear. We have found that butyrate has

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    Address requests for reprints to: Charles Giardina, Ph.D., Department of Molecular and Cell Biology, University of Connecticut, U-125, 75 North Eagleville Road, Storrs, Connecticut 06269-3125. e-mail: [email protected]; fax: (860) 486-4331.

    ☆☆

    Supported by the National Institutes of Health (R29 CA 79656-01) and by a Research Starter Grant from the Pharmaceutical Research and Manufacturers of America Foundation (to C.G.).

    Drs. Inan and Rasoulpour contributed equally to this manuscript.

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