Volume 118, Issue 4, Pages 724-734 (April 2000) The luminal short-chain fatty acid butyrate modulates NF-κB activity in a human colonic epithelial cell line  Mehmet Sait Inan*, Reza J. Rasoulpour*, Lei Yin*, Andrea K. Hubbard‡, Daniel W. Rosenberg‡, Charles Giardina*  Gastroenterology  Volume 118, Issue 4, Pages 724-734 (April 2000) DOI: 10.1016/S0016-5085(00)70142-9 Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 1 (A) HT-29 cells and mouse colonic cells possess a constitutive NF-κB activity in the form of a p50 dimer. Nuclear extracts were prepared from HT-29 cells or mouse colonic epithelial cells and analyzed by EMSA in the absence or presence of antibodies to p50 or p65. (B) Butyrate inhibits p50 dimer activity in HT-29 cells. HT-29 cells were treated with 4 mmol/L butyrate for the indicated times; nuclear extracts were prepared and analyzed by EMSA. (C) ALP assay. Butyrate activation of ALP expression in HT-29 cells occurs after NF-κB inactivation. HT-29 cells were treated with 4 mmol/L butyrate under conditions inducing the acquisition of many differentiated phenotypes, including increased ALP expression. Cellular lysates were prepared at different times after butyrate treatment and assayed for ALP activity as described in Materials and Methods. Gastroenterology 2000 118, 724-734DOI: (10.1016/S0016-5085(00)70142-9) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 1 (A) HT-29 cells and mouse colonic cells possess a constitutive NF-κB activity in the form of a p50 dimer. Nuclear extracts were prepared from HT-29 cells or mouse colonic epithelial cells and analyzed by EMSA in the absence or presence of antibodies to p50 or p65. (B) Butyrate inhibits p50 dimer activity in HT-29 cells. HT-29 cells were treated with 4 mmol/L butyrate for the indicated times; nuclear extracts were prepared and analyzed by EMSA. (C) ALP assay. Butyrate activation of ALP expression in HT-29 cells occurs after NF-κB inactivation. HT-29 cells were treated with 4 mmol/L butyrate under conditions inducing the acquisition of many differentiated phenotypes, including increased ALP expression. Cellular lysates were prepared at different times after butyrate treatment and assayed for ALP activity as described in Materials and Methods. Gastroenterology 2000 118, 724-734DOI: (10.1016/S0016-5085(00)70142-9) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 2 (A) Immunoblot analysis indicates that butyrate inhibits p50 nuclear localization. HT-29 cells were incubated with 4 mmol/L butyrate for the indicated times and then fractionated into nuclear and cytoplasmic fractions. Proteins (25 μg) were then analyzed by immunoblotting with an antibody that recognizes both p50 and p105. (B) Butyrate does not affect p50/p105 mRNA levels. HT-29 cells were incubated with 0 or 4 mmol/L butyrate for 24 hours. RNA was then prepared and assayed by primer extension. Actin mRNA was used as a control. The 2 lanes on the left are results obtained with RNA isolated from untreated cells (performed in duplicate); the 2 lanes on the right are results obtained with RNA isolated from butyrate-treated cells (also performed in duplicate). Gastroenterology 2000 118, 724-734DOI: (10.1016/S0016-5085(00)70142-9) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 3 (A) Butyrate treatment of HT-29 cells influences NF-κB activation by TNF-α and IL-1β, favoring the formation of the p65-p50 heterodimer and suppressing the overall level of activation by TNF-α. Cells were pretreated with 4 mmol/L butyrate (BA) for 1 hour or 18 hours as indicated. After this pretreatment, cells were exposed to TNF-α or IL-1β for an additional 6 hours (as indicated). Nuclear extracts were then prepared and analyzed by EMSA. (B) Butyrate also inhibits NF-κB activation by PMA. Cells were pretreated with 4 mmol/L butyrate for 18 hours and then treated with the indicated concentration of PMA. Nuclear extracts were then prepared and analyzed by EMSA. (C) Effect of butyrate on cytokine responses is observed after 30 minutes or 6 hours of cytokine exposure. Cells were treated with 4 mmol/L butyrate (BA) for 18 hours where indicated. Cells were then treated with TNF-α or IL-1β for 30 minutes or 6 hours. Nuclear extracts were tested for NF-κB DNA binding activity by EMSA. Gastroenterology 2000 118, 724-734DOI: (10.1016/S0016-5085(00)70142-9) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 3 (A) Butyrate treatment of HT-29 cells influences NF-κB activation by TNF-α and IL-1β, favoring the formation of the p65-p50 heterodimer and suppressing the overall level of activation by TNF-α. Cells were pretreated with 4 mmol/L butyrate (BA) for 1 hour or 18 hours as indicated. After this pretreatment, cells were exposed to TNF-α or IL-1β for an additional 6 hours (as indicated). Nuclear extracts were then prepared and analyzed by EMSA. (B) Butyrate also inhibits NF-κB activation by PMA. Cells were pretreated with 4 mmol/L butyrate for 18 hours and then treated with the indicated concentration of PMA. Nuclear extracts were then prepared and analyzed by EMSA. (C) Effect of butyrate on cytokine responses is observed after 30 minutes or 6 hours of cytokine exposure. Cells were treated with 4 mmol/L butyrate (BA) for 18 hours where indicated. Cells were then treated with TNF-α or IL-1β for 30 minutes or 6 hours. Nuclear extracts were tested for NF-κB DNA binding activity by EMSA. Gastroenterology 2000 118, 724-734DOI: (10.1016/S0016-5085(00)70142-9) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 3 (A) Butyrate treatment of HT-29 cells influences NF-κB activation by TNF-α and IL-1β, favoring the formation of the p65-p50 heterodimer and suppressing the overall level of activation by TNF-α. Cells were pretreated with 4 mmol/L butyrate (BA) for 1 hour or 18 hours as indicated. After this pretreatment, cells were exposed to TNF-α or IL-1β for an additional 6 hours (as indicated). Nuclear extracts were then prepared and analyzed by EMSA. (B) Butyrate also inhibits NF-κB activation by PMA. Cells were pretreated with 4 mmol/L butyrate for 18 hours and then treated with the indicated concentration of PMA. Nuclear extracts were then prepared and analyzed by EMSA. (C) Effect of butyrate on cytokine responses is observed after 30 minutes or 6 hours of cytokine exposure. Cells were treated with 4 mmol/L butyrate (BA) for 18 hours where indicated. Cells were then treated with TNF-α or IL-1β for 30 minutes or 6 hours. Nuclear extracts were tested for NF-κB DNA binding activity by EMSA. Gastroenterology 2000 118, 724-734DOI: (10.1016/S0016-5085(00)70142-9) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 4 Supershift analysis of NF-κB DNA binding activity formed in HT-29 cells under different treatment conditions. Nuclear extracts were prepared from untreated HT-29 cells and from HT-29 cells treated with TNF-α or IL-1β for 6 hours, with or without a prior 18-hour exposure to 4 mmol/L butyrate (BA). (A) Results obtained by inclusion of an anti-p50 antibody in the DNA binding reaction; (B) results when an anti-p65 antibody is used. Labels B and A with arrows denote protein-DNA complexes with different mobilities. Gastroenterology 2000 118, 724-734DOI: (10.1016/S0016-5085(00)70142-9) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 5 (A) Effects of butyrate on p65, p50, and p105 levels in TNF-α– and IL-1β–treated cells. Cells were pretreated with 4 mmol/L butyrate for 18 hours where indicated (B), and then exposed to TNF-α (T) or IL-1β (IL) for 6 hours. Nuclear and cytoplasmic extracts were then prepared, with 30 μg of each analyzed by immunoblotting. Blots were first probed with anti-p50/p105 antibody, and then with an anti-p65 antibody. (B) Butyrate does not influence TNF-α–induced IκBα degradation immediately after cytokine exposure. Cells were treated for the indicated times with TNF-α and then processed to determine IκBα levels by immunoblotting. Where indicated, cells were pretreated for 18 hours with 4 mmol/L butyrate. (C) Butyrate treatment leads to lower levels of IκBα in cells treated with TNF-α and IL-1β for a prolonged 6-hour exposure. Cells were treated as in A, except that blots were probed for IκBα. Gastroenterology 2000 118, 724-734DOI: (10.1016/S0016-5085(00)70142-9) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 6 Butyrate influences the expression level of 2 endogenous NF-κB–regulated genes: ICAM-1 and MnSOD. Cells were pretreated with 4 mmol/L butyrate (BA) for 18 hours, as indicated, and then exposed to TNF-α or IL-1β for 6 hours. Control cells were incubated without cytokine for 6 hours. RNA was isolated, and 25 μg was analyzed by primer extension using primers specific for MnSOD, ICAM-1, and β-actin. The 2 bands detected by the primer extension reaction for MnSOD are the result of 2 transcriptional start sites. Gastroenterology 2000 118, 724-734DOI: (10.1016/S0016-5085(00)70142-9) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 7 (A) NF-κB transcriptional activation in HT-29 cells is enhanced by the p65 subunit. HT-29 cells were cotransfected with an NF-κB–luciferase reporter construct (shown schematically in C) and increasing amounts of a CMV-regulated p50 expression vector (as indicated). Transfections were performed with p50 alone (□) or in the presence of 0.05 μg of a p65 expression vector (▩). The total amount of CMV expression vector in the transfection mixture was kept constant by inclusion of an empty expression vector. Twenty-four hours after transfection, luciferase assays were performed. Control experiments indicate that p65 activation is specific for the NF-κB–regulated promoter and is not observed on the control plasmid. (B) Butyrate influences expression of a transfected NF-κB reporter plasmid. HT-29 cells were transfected with an NF-κB–regulated luciferase reporter construct or a control luciferase construct lacking NF-κB binding sites. Cells were treated with 4 mmol/L butyrate for 18 hours. Cell extracts were prepared and assayed for luciferase activity. (C) Schematic drawing of the luciferase reporters used in this study. Gastroenterology 2000 118, 724-734DOI: (10.1016/S0016-5085(00)70142-9) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 7 (A) NF-κB transcriptional activation in HT-29 cells is enhanced by the p65 subunit. HT-29 cells were cotransfected with an NF-κB–luciferase reporter construct (shown schematically in C) and increasing amounts of a CMV-regulated p50 expression vector (as indicated). Transfections were performed with p50 alone (□) or in the presence of 0.05 μg of a p65 expression vector (▩). The total amount of CMV expression vector in the transfection mixture was kept constant by inclusion of an empty expression vector. Twenty-four hours after transfection, luciferase assays were performed. Control experiments indicate that p65 activation is specific for the NF-κB–regulated promoter and is not observed on the control plasmid. (B) Butyrate influences expression of a transfected NF-κB reporter plasmid. HT-29 cells were transfected with an NF-κB–regulated luciferase reporter construct or a control luciferase construct lacking NF-κB binding sites. Cells were treated with 4 mmol/L butyrate for 18 hours. Cell extracts were prepared and assayed for luciferase activity. (C) Schematic drawing of the luciferase reporters used in this study. Gastroenterology 2000 118, 724-734DOI: (10.1016/S0016-5085(00)70142-9) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 8 (A) The histone deacetylase inhibitor TSA reproduces butyrate's effect on p50 dimer activity, whereas the glutathione synthesis inhibitor BSO does not. Cells were incubated with increasing concentrations of TSA (0, 2, and 4 μmol/L) or BSO (0, 2, and 4 mmol/L) for 18 hours, after which nuclear extracts were prepared and analyzed by EMSA. (B) TSA reproduces butyrate's effect on cytokine activation of NF-κB. Cells were preincubated with 2 μmol/L TSA for 18 hours and then treated with TNF-α or IL-1β, as indicated. Nuclear extracts were then prepared and assayed for NF-κB activity by EMSA. (C) TSA does not prevent IκBα degradation by TNF-α. Extracts from cells treated in B were analyzed by immunoblotting using an antibody against IκBα. (D) Propionate (PA) reproduces some of butyrate's effect on NF-κB, but is less potent. Cells were preincubated with 4 mmol/L propionate for 18 hours and then treated with TNF-α or IL-1β, as indicated. Nuclear extracts were then prepared and assayed for NF-κB activity by EMSA. Gastroenterology 2000 118, 724-734DOI: (10.1016/S0016-5085(00)70142-9) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 8 (A) The histone deacetylase inhibitor TSA reproduces butyrate's effect on p50 dimer activity, whereas the glutathione synthesis inhibitor BSO does not. Cells were incubated with increasing concentrations of TSA (0, 2, and 4 μmol/L) or BSO (0, 2, and 4 mmol/L) for 18 hours, after which nuclear extracts were prepared and analyzed by EMSA. (B) TSA reproduces butyrate's effect on cytokine activation of NF-κB. Cells were preincubated with 2 μmol/L TSA for 18 hours and then treated with TNF-α or IL-1β, as indicated. Nuclear extracts were then prepared and assayed for NF-κB activity by EMSA. (C) TSA does not prevent IκBα degradation by TNF-α. Extracts from cells treated in B were analyzed by immunoblotting using an antibody against IκBα. (D) Propionate (PA) reproduces some of butyrate's effect on NF-κB, but is less potent. Cells were preincubated with 4 mmol/L propionate for 18 hours and then treated with TNF-α or IL-1β, as indicated. Nuclear extracts were then prepared and assayed for NF-κB activity by EMSA. Gastroenterology 2000 118, 724-734DOI: (10.1016/S0016-5085(00)70142-9) Copyright © 2000 American Gastroenterological Association Terms and Conditions