Volume 132, Issue 1, Pages 236-248 (January 2007) Butyrate Induces Intestinal Sodium Absorption via Sp3-Mediated Transcriptional Up- Regulation of Epithelial Sodium Channels  Sebastian Zeissig, Anja Fromm, Joachim Mankertz, Jörg Weiske, Martin Zeitz, Michael Fromm, Jörg–Dieter Schulzke  Gastroenterology  Volume 132, Issue 1, Pages 236-248 (January 2007) DOI: 10.1053/j.gastro.2006.10.033 Copyright © 2007 AGA Institute Terms and Conditions

Figure 1 Butyrate induces transcription of β- and γ-ENaC mRNA. HT-29/B6 cells were incubated with or without sodium butyrate (5 mmol/L, 24 hours). RT-PCR was performed with total RNA using α-, β-, and γ-ENaC specific primers for 32 cycles. Although β- and γ-ENaC mRNA were not detectable in untreated HT-29/B6 cells, butyrate induced transcription of both mRNA. NTC, no template control. One representative experiment is shown; 3 additional experiments gave comparable results. Gastroenterology 2007 132, 236-248DOI: (10.1053/j.gastro.2006.10.033) Copyright © 2007 AGA Institute Terms and Conditions

Figure 2 Butyrate and trichostatin A (TSA) induce transcription from α-, β-, and γ-ENaC promoters. (A) Butyrate induces transcription from α-, β-, and γ-ENaC promoters. HT-29/B6 cells were transiently transfected with luciferase constructs of the human α-ENaC promoter (pahENaC-A, −761/+706 according to the α-ENaC-1 promoter26), the human β-ENaC-1 promoter (pbhENaC-A, −1137/+200), the human γ-ENaC promoter (pghENaC-A, −2926/+35), or the empty vector (pGL3Basic), respectively. Four hours after transfection, cells were incubated with or without sodium butyrate (5 mmol/L, 24 hours). (B) Butyrate dose dependently induces transcription from the γ-ENaC promoter. HT-29/B6 cells were transiently transfected with human γ-ENaC promoter (pghENaC-A, −2926/+35). Four hours after transfection, cells were incubated with sodium butyrate in the indicated concentrations for 24 hours. (C) Butyrate and propionate, but not acetate, induce transcription from the γ-ENaC promoter. HT-29/B6 cells were transfected with pghENaC-A or pGL3Basic. Four hours after transfection, cells were incubated with sodium butyrate (5 mmol/L), propionate (5 mmol/L), and acetate (10 mmol/L) as indicated for 24 hours. (D) Trichostatin A induces transcription from α-, β-, and γ-ENaC promoters. HT-29/B6 cells were transiently transfected with luciferase constructs as described in A. Four hours after transfection, cells were incubated with or without trichostatin A (100 ng/mL, 24 hours). **P < .01, ***P < .001 for TSA or butyrate-treated cells compared with untreated cells. The results are the mean ± SEM of 3 independent experiments done in duplicate. Gastroenterology 2007 132, 236-248DOI: (10.1053/j.gastro.2006.10.033) Copyright © 2007 AGA Institute Terms and Conditions

Figure 3 Transcriptional activation of γ-ENaC by butyrate depends on 2 Sp1 sites in the γ-ENaC promoter. (A) Transcriptional activation of the γ-ENaC promoter by butyrate is not affected by several 5′ truncations. HT-29/B6 cells were transiently transfected with plasmids containing the luciferase gene under control of the human full-length γ-ENaC promoter (pghENaC-A, −2926/+35) or the indicated 5′ truncated constructs. Four hours after transfection, cells were incubated with or without sodium butyrate (5 mmol/L, 24 hours). Normalized luciferase activity did not differ between constructs. Putative Sp1 sites are indicated by boxes. (B) Transcriptional activation of the γ-ENaC promoter by butyrate depends on 2 Sp1 sites. HT-29/B6 cells were transiently transfected with the pghENaC-E luciferase construct or the same plasmid containing mutated nucleotides in the Sp1-5 and/or Sp1-6 site. Mutated sites are shown in gray boxes, and mutated nucleotides are underlined. Four hours after transfection, cells were incubated with or without sodium butyrate (5 mmol/L, 24 hours).***P < .001 for butyrate-treated cells transfected with mutated constructs vs the original pghENaC-E construct. The results are the mean ± SEM of 3 independent experiments done in duplicate. Gastroenterology 2007 132, 236-248DOI: (10.1053/j.gastro.2006.10.033) Copyright © 2007 AGA Institute Terms and Conditions

Figure 4 Sp1 and Sp3 bind to the Sp1-5 site in the γ-ENaC promoter in vitro. (A) EMSAs were performed using nuclear extracts from control and sodium butyrate-treated (5 mmol/L, 24 hours) HT-29/B6 cells. The annealed oligonucleotide containing the sequence between −62 and −32 of pghENaC-E including the Sp1-5 site was labeled using the T4 polynucleotide kinase. Competition experiments were carried out with 50-fold excess of (a) unlabeled Sp1 consensus oligonucleotide (lanes 3, 8); (b) an unlabeled oligonucleotide similar to lanes 2 and 7 but with a mutated Sp1-5 site as described in “Electrophoretic mobility shift assay” in the Materials and Methods section (lanes 4 and 9); (c) an unrelated AP2 consensus oligonucleotide (lanes 5 and 10). (B) EMSAs were performed as described for A, and anti-Sp1 and anti-Sp3 antibodies were used to supershift complexes. The positions for Sp1 and Sp3 binding are indicated by arrows. (C) Cotransfection of Sp1 and Sp3 increases butyrate-dependent induction of the γ-ENaC promoter through Sp1-5 and Sp1-6 sites. HT-29/B6 cells were transiently transfected with the pghENaC-E luciferase construct, the pghENaC-E/M5+M6 construct with mutated Sp1-5 and Sp1-6 sites (as shown in Figure 3B), or pGL3Basic. Expression constructs for full-length Sp1 and Sp3 or the empty vector were cotransfected. Four hours after transfection, cells were incubated with or without sodium butyrate (5 mmol/L) for 24 hours. The results are the mean ± SEM of 3 independent experiments done in duplicate. Gastroenterology 2007 132, 236-248DOI: (10.1053/j.gastro.2006.10.033) Copyright © 2007 AGA Institute Terms and Conditions

Figure 5 Sp3 but not Sp1 binds to the γ-ENaC promoter in vivo, and Sp3 binding is increased upon butyrate stimulation. (A) Schematic representation of the 3’ portion of the γ-ENaC promoter. Arrows indicate the position of the primers for ChIP analysis. Sp1-5 and Sp1-6 sites are indicated as boxes. (B) Quantitative ChIP assay of Sp1 and Sp3 binding to the γ-ENaC promoter. HT-29/B6 cells were incubated with or without sodium butyrate (5 mmol/L, 24 hours). Chromatin proteins and DNA were cross-linked by formaldehyde. The cross-linked chromatin was sheared and then immunoprecipitated using specific antibodies as indicated. Purified immunoprecipitated DNA was quantified using primer/probe sets corresponding to the γ-ENaC promoter. Amplification results are shown as fold increase of the amount of γ-ENaC promoter in Sp1 or Sp3 antibody immunoprecipitated DNA compared with IgG antibody immunoprecipitated DNA. The results are the mean ± SEM of 3 independent immunoprecipitations followed by triplicate real-time PCR. **P < .01 vs Sp3 immunoprecipitation in untreated cells. (C) Western blot analysis of Sp1 and Sp3 in whole cell extracts of HT-29/B6 cells incubated with or without 5 mmol/L sodium butyrate for indicated periods. One representative experiment is shown; 3 additional independent experiments gave comparable results. Sp1 and Sp3 revealed its classical pattern, as described by Sapetschnig et al.59 Sp1*, phosphorylated form of Sp1. Sp3li-1 and -2, long isoforms of Sp3; Sp3si-1 and -2, small isoforms of Sp3. Anti-β-actin was used to demonstrate equal loading. Gastroenterology 2007 132, 236-248DOI: (10.1053/j.gastro.2006.10.033) Copyright © 2007 AGA Institute Terms and Conditions

Figure 6 Butyrate increases dexamethasone-dependent electrogenic sodium absorption in HT-29/B6-GR cells and induces β- and γ-ENaC transcription. (A) Induction of electrogenic sodium absorption in HT-29/B6-GR cells by dexamethasone and/or butyrate. Stable glucocorticoid receptor-transfected HT-29/B6-GR cells were grown on permeable supports for 7 days until confluence, as indicated by transepithelial resistances (Rt) of ∼300 Ω · cm2. Cells were then stimulated for 72 hours with dexamethasone (10-6 mol/L) and/or sodium butyrate (5 mmol/L, 24 hours), and monolayers were mounted into Ussing chambers. Electrogenic sodium absorption via ENaC was determined after 1-hour incubation in the Ussing chamber by addition of 10−4 mol/L amiloride. The results are the mean ± SEM of n = 6 samples per condition. ***P < .001 compared with dexamethasone treatment. (B) Real-time PCR using primer/probe sets specific for α-, β-, and γ-ENaC. HT-29/B6-GR cells were grown on permeable supports as described above and were stimulated for 72 hours with dexamethasone (10−6 mol/L) and/or sodium butyrate (5 mmol/L, 24 hours). PCR was performed in triplicates, and results were normalized to the endogenous control. Fold induction represents the relative expression of α-, β, and γ-ENaC mRNA in dexamethasone- and/or butyrate-treated cells over that of untreated controls. Note second y-axis for γ-ENaC. The results are the mean ± SEM of n = 4 independent samples per condition investigated in triplicates. *P < .05, **P < .01, ***P < .001 compared with untreated controls. (C) Confocal laser scanning microscopy of γ-ENaC. HT-29/B6-GR cells were grown on permeable supports as described above and were stimulated for 72 hours with dexamethasone (10−6 mol/L) and/or sodium butyrate (5 mmol/L, 24 hours). Cell layers were stained with anti-γ-ENaC antibody (red), anti-E-cadherin antibody (green) to visualize the lateral cell membrane, and DAPI (blue) to counterstain nuclei. In cells treated with butyrate, γ-ENaC remained intracellular (arrows) and was not transported to the apical cell membrane (arrowhead). Bar indicates 20 μm. All images were taken with the same instrument settings. Gastroenterology 2007 132, 236-248DOI: (10.1053/j.gastro.2006.10.033) Copyright © 2007 AGA Institute Terms and Conditions

Figure 7 Butyrate increases dexamethasone-dependent electrogenic sodium absorption in rat distal colon. (A) Time course of electrogenic sodium transport in rat distal colon induced by dexamethasone and/or butyrate. Rat distal colon was mounted in Ussing chambers, and dexamethasone (5 × 10−9 mol/L) and/or sodium butyrate (1.25 mmol/L) were added after 1 hour and were incubated for 17 hours. Transepithelial resistance (Rt) was constantly registered to confirm viability of specimens. Electrogenic sodium absorption via ENaC was determined by addition of 10−4 mol/L amiloride at the end of the experiment. (B) Electrogenic sodium absorption in rat distal colon induced by dexamethasone and/or butyrate. Experiments were performed as described above and 10−4 mol/L amiloride was added at the end of the experiment to determine the electrogenic sodium absorption via ENaC. The results are the mean ± SEM of n = 7 tissues of independent animals. ***P < .001 compared with dexamethasone treatment. Gastroenterology 2007 132, 236-248DOI: (10.1053/j.gastro.2006.10.033) Copyright © 2007 AGA Institute Terms and Conditions