1. 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 (January 2007)
DOI: /j.gastro
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2. 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  , DOI: ( /j.gastro )
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3. 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  , DOI: ( /j.gastro )
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4. 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  , DOI: ( /j.gastro )
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5. 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  , DOI: ( /j.gastro )
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6. 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  , DOI: ( /j.gastro )
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7. 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  , DOI: ( /j.gastro )
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8. 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  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions