1. Volume 136, Issue 3, Pages 933-942.e2 (March 2009)
Glucocorticoids and Tumor Necrosis Factor-α Synergize to Induce Absorption by the Epithelial Sodium Channel in the Colon 
Theresa Bergann, Sebastian Zeissig, Anja Fromm, Jan F. Richter, Michael Fromm, Joerg-Dieter Schulzke 
Gastroenterology 
Volume 136, Issue 3, Pages e2 (March 2009)
DOI: /j.gastro
Copyright © 2009 AGA Institute Terms and Conditions

2. Figure 1
Dexamethasone and TNF-α synergistically induce ENaC-dependent Na+ absorption in HT-29/B6-GR cells and increase β- and γ-ENaC mRNA levels. (A) Filter-grown, confluent HT-29/B6-GR cells were stimulated for 24 hours with dexamethasone (1 μmol/L) and/or TNF-α (10,000 U/mL). In Ussing chambers, ENaC-dependent Na+ absorption was determined as the drop in ISC after amiloride (10−4 mol/L). Results are means ± SEM of n = 6, ***P < .001 compared with dexamethasone. (B and C) Real-time PCR of β-ENaC and γ-ENaC mRNA. HT-29/B6-GR cells were stimulated for 24 hours with dexamethasone (1 μmol/L) and/or TNF-α (10,000 U/mL). GAPDH was used for normalization of mRNA expression. Data are means ± SEM of x-fold induction over controls (n = 9, **P < .01, ***P < .001 compared with dexamethasone). (D) Western Blot analysis of β-ENaC. β-ENaC protein (∼60 kilodaltons) expression was monitored in HT-29/B6-GR lysates after 24 hours of incubation with dexamethasone and/or TNF-α. Human β-actin (∼42 kilodaltons) served as a loading control.
Gastroenterology  , e2DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions

3. Figure 2
Transcriptional activation of a glucocorticoid response element (GRE)-driven luciferase construct by dexamethasone is enhanced by TNF-α because of elevation of GR protein levels. (A) HT-29/B6-GR cells were transfected with pGRE-Luc and incubated for 24 hours with dexamethasone (1 μmol/L) and/or TNF-α (10,000 U/mL). Results are given as normalized relative luciferase activity. Means ± SEM, n = 6, ***P < .001 compared with dexamethasone, ###P < .001 compared with control, NS compared with control. (B) Western blot analysis of GR and p-GR (p-Ser211) in HT-29/B6-GR cell lysates after 3 hours of incubation with dexamethasone and/or TNF-α. One representative blot is shown. (C) Densitometry of total GR chemiluminescence signals normalized to β-actin. Shown are means ± SEM, n = 4, *P < .05 compared with dexamethasone. (D) Confocal laser scanning microscopy of GR. HT-29/B6-GR cells were incubated for 3 hours with dexamethasone and/or TNF-α. Cells were stained with anti-GR antibody (red); nuclei were DAPI stained (blue).
Gastroenterology  , e2DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions

4. Figure 3
TNF-α causes an increase in GR-α mRNA expression and mRNA half-life. (A) GR mRNA expression in HT-29/B6-GR cells. Cells were incubated with dexamethasone (1 μmol/L) and/or TNF-α (10,000 U/mL) for 3 hours. GR-α mRNA level was analyzed by real-time PCR. Data are means ± SEM of x-fold induction over controls (n = 4, *P < .05 compared with dexamethasone). (B) Gene reporter assay with pcDNA3.1(+)-Luc. HT-29/B6-GR cells were transfected with pcDNA3.1(+)-Luc and incubated for 24 hours with dexamethasone and/or TNF-α. Shown is normalized relative luciferase activity. Means ± SEM, n = 4. (C) GR-α mRNA half-life in HT-29/B6-GR cells. mRNA synthesis was blocked by preincubation with actinomycin D (7 μg/mL) for 1 hour. Dexamethasone and/or TNF-α were added, and total RNA was isolated after 0, 2, 4, and 7 hours. Degradation of GR mRNA was monitored by real-time PCR. Shown are means ± SEM, n = 5, *P < .05.
Gastroenterology  , e2DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions

5. Figure 4
MAP kinase p38 is involved in the synergistic ENaC induction by dexamethasone and TNF-α. HT-29/B6-GR cells were preincubated with the indicated MAPK inhibitors (10 μmol/L) for 1 hour before addition of dexamethasone and/or TNF-α for 24 hours. (A) Measurement of ENaC-dependent Na+ absorption. Data are means ± SEM, n = 6, ***P < .001 compared with dexamethasone plus TNF-α, #P < .05 and ###P < .001 compared with dexamethasone, NS compared with dexamethasone + TNF-α. (B) Real-time PCR for γ-ENaC. Data are means ± SEM of x-fold induction over controls (n = 6, **P < .01 compared with dexamethasone + TNF-α, ##P < .01 compared with dexamethasone). (C) Western blot analysis of GR and p-GR (p-Ser211). HT-29/B6-GR cells were preincubated for 1 hour with SB (10 μmol/L) before addition of dexamethasone and/or TNF-α for 3 hours. One representative out of 3 independent experiments is shown.
Gastroenterology  , e2DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions

6. Figure 5
Dexamethasone and TNF-α synergistically induce ENaC-dependent Na+ absorption in rat distal colon. (A) Time course of electrogenic Na+ transport in rat distal colon. Tissue was stimulated with dexamethasone (1 μmol/L) and/or TNF-α (10,000 U/mL) for 14 hours after preincubation with IFN-γ (1,000 U/mL) for 1 hour. ENaC-dependent Na+ absorption was determined as the drop in ISC within 10 minutes after amiloride (10−4 mol/L). (B) Electrogenic Na+ transport in rat distal colon. Experiments were performed as described above. Given are means ± SEM, n = 11, **P < .01 compared with dexamethasone. (C) Influence of TNF-α on aldosterone-induced electrogenic Na+ transport. Rat distal colon was stimulated with TNF-α (10,000 U/mL) for 6 hours (after a preincubation with IFN-γ [1000 U/mL, 1 hour]). Next, aldosterone (3 nmol/L) was added for 8 hours. Results are means ± SEM, n = 5, ***P < .001 compared with control.
Gastroenterology  , e2DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions

7. Figure 6
ENaC-dependent Na+ absorption in rat distal colon is due to an up-regulation of β- and γ-ENaC expression. Real-time PCR of (A) α-ENaC, (B) β-ENaC, and (C) γ-ENaC mRNA. Rat distal colon was incubated as described in Figure 5A. After determination of amiloride-sensitive ISC, total RNA was isolated. Data are means ± SEM of x-fold induction over controls (n = 7, *P < .05 compared with dexamethasone). (D–F) Correlation between the amiloride-sensitive ISC as shown in Figure 5B and the relative mRNA induction of α-ENaC (D), β-ENaC (E), and γ-ENaC (F). R2 indicates the correlation coefficient.
Gastroenterology  , e2DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions

8. Figure 7
Mechanistic model of synergistic action of dexamethasone and TNF-α on γ-ENaC expression. In the proposed model, TNF-α signaling via TNF-R1 (as described earlier36), p38 activator kinase MEK3/6, and p38 MAPK results in a prolongation of GR mRNA lifetime, which in turn leads to elevated levels of GR protein. Dexamethasone also present in the cell induces GR activation. After nuclear translocation, GR binds to GREs located either up- or downstream from the promoter region of the γ-ENaC gene. As a result, γ-ENaC gene transcription is enhanced, and ENaC γ-subunits become synthesized. GR, glucocorticoid receptor; GRE, glucocorticoid response element; MEK3/6, MAP/extracellular signal-regulated kinase 3/6; p38 MAPK, p38 mitogen-activated protein kinase; TNF-α, tumor necrosis factor α; TNF-R1, TNF-receptor 1.
Gastroenterology  , e2DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions

9. Supplementary Figure 1
Determination of the inhibitory specificity of SB Western blot analysis of p-JNK, p-p42/44 and p-MAPKAPK2. HT-29/B6-GR cells were incubated with the p38 MAPK inhibitor SB (10 μmol/L) for 1 hour. Next, dexamethasone (1 μmol/L) and (TNF-α 10,000 U/mL) were added for 20 minutes, and whole cell extracts were prepared. β-actin was used as loading control. Shown is 1 out of 3 independent experiments giving comparable results.
Gastroenterology  , e2DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions