The plant-derived glucocorticoid receptor agonist Endiandrin A acts as co-stimulator of colonic epithelial sodium channels (ENaC) via SGK-1 and MAPKs.

In a search for secondary plant compounds that bind to the glucocorticoid receptor (GR), the cyclobutane lignan endiandrin A was discovered from the rainforest tree Endiandra anthropophagorum Domin. Our present study aims to characterize the effect of endiandrin A on GR-dependent induction of colonic sodium transport. The effect of endiandrin A was analyzed in GR-expressing colonic HT-29/B6 cells (HT-29/B6-GR). GR transactivation and subcellular localization were investigated by reporter gene assay and immunofluorescence. Epithelial sodium channel (ENaC) was analyzed by qRT-PCR and by measuring amiloride-sensitive short-circuit current (I(sc)) in Ussing chambers. Endiandrin A (End A) has been identified as GR receptor binder. However, it did not cause significant GR transactivation as pGRE-luciferase activity was only 7% of that of the maximum effect of dexamethasone. Interestingly, endiandrin A had a significant impact on dexamethasone-dependent sodium absorption in cells co-exposed to tumor necrosis factor (TNF)-α. This was in part due to up-regulation of β- and γ-ENaC subunit expression. Endiandrin A potentiated GR-mediated transcription by increasing GR protein expression and phosphorylation. It inhibited c-Jun N-terminal kinase (JNK) activation induced by dexamethasone and/or TNF-α and increased levels of GR localized to the nucleus. Additionally, endiandrin A increased the serum- and glucocorticoid-induced kinase (sgk)-1 via activation of p38. Finally, the regulation of ENaC function by endiandrin A was confirmed in rat native colon. In conclusion, endiandrin A potentiates glucocorticoid-driven activation of colonic epithelial sodium channels via JNK inhibition and p38 activation due to transcriptional up-regulation of β- and γ-ENaC-subunits along with induction of sgk-1

Enhanced nuclear GR translocation through inhibition of TNF-α-induced JNK phosphorylation by endiandrin A in HT-29/B6-GR cells.

<p>(<b>A</b>) HT-29/B6-GR cells were pre-incubated with the JNK inhibitor SP 600125 (10 µM) for 1 hour before addition of dexamethasone (1 µM) and/or TNF-α (500 IU/ml) and/or endiandrin A (20 µM) for 24 hours. Measurement of ENaC-dependent Na⁺ absorption occurred as the drop in I_SC after amiloride (100 µM). Data are given as means ± s.e.m., n = 10, ***<i>P</i><0.001 compared with dexamethasone + TNF-α 500 IU/ml incubation, ^###<i>P</i><0.001 compared to dexamethasone + TNF-α 500 IU/ml + endiandrin A. (<b>B</b>) Western blot analysis of JNK and pJNK protein (∼46/54 kDa) of HT-29/B6-GR cell lysates incubated with dexamethasone (1 µM) and/or TNF-α (500 IU/ml) w/wo endiandrin A (20 µM) for 15 minutes. Human β-actin (∼42 kDa) served as a loading control. (<b>C</b>) Densitometry of endiandrin A-induced effects on phosphorylated JNK levels normalized to total JNK. HT-29/B6-GR cells were incubated with dexamethasone (1 µM) and/or TNF-α (500 IU/ml) w/wo endiandrin A (20 µM) for 15 minutes. Shown are means ± s.e.m., n = 4, **<i>P</i><0.01 compared to TNF-α exposure,^##<i>P</i><0.01 compared to dexamethasone + TNF-α 500 IU/ml. (<b>D</b>) Immunofluorescence staining of GR. HT-29/B6-GR cells were incubated with dexamethasone (1 µM) and TNF-α (500 IU/ml) and/or endiandrin A (20 µM) for 3 hours. Cells were stained with anti-GR antibody (green), nuclei were DAPI stained (blue).</p

Effect of endiandrin A on GR transactivation in HT-29/B6-GR cells.

<p>(<b>A</b>) Structure of endiandrin A.</p

Enhancement of ENaC-dependent Na⁺ absorption and β- and γ-ENaC transcription by endiandrin A in HT-29/B6-GR cells.

<p>HT-29/B6-GR cells were incubated with dexamethasone (1 µM) and/or TNF-α (500 IU/ml) and/or endiandrin A (20 µM) for 24 hours. (<b>A</b>) Measurement of ENaC-dependent Na⁺ absorption was determined as the drop in I_SC after amiloride (100 µM) after 24 hours. Data are means ± s.e.m., n = 6–15, ^*<i>P</i><0.05 compared with control, ^###<i>P</i><0.001 compared with dexamethasone alone, ^°°<i>P</i><0.01 compared with dexamethasone + TNF-α 500 IU/ml. (<b>B</b>)/(<b>C</b>) Measurement of (<b>B</b>) β- or (<b>C</b>) γ-ENaC-mRNA. GAPDH was used for normalization of mRNA expression. (<b>B</b>) Real-time PCR of β-ENaC mRNA after 24 hours. Data are means ± s.e.m., n = 4–13, ^***<i>P</i><0.001 compared with dexamethasone alone, ^##<i>P</i><0.01 compared with dexamethasone + TNF-α 500 IU/ml. (<b>C</b>) Real-time PCR of γ-ENaC mRNA after 24 h. Data are means ± s.e.m., n = 4–12, ^*<i>P</i><0.05 compared with dexamethasone alone, ^#<i>P</i><0.05 compared with dexamethasone + TNF-α 500 IU/ml.</p

Endiandrin A increased activation of a GRE-driven luciferase construct and GR protein levels in HT-29/B6-GR cells.

<p>(<b>A</b>) HT-29/B6-GR cells were transfected with pGRE-Luc and incubated with dexamethasone (1 µM) and/or TNF-α (500 IU/ml) and/or endiandrin A (20 µM) for 24 hours. Data are given as normalized relative luciferase activity and as means ± s.e.m., n = 6–14 ^*<i>P</i><0.05, ^**<i>P</i><0.01 compared with dexamethasone alone, ^##<i>P</i><0.01 compared with dexamethasone + TNF-α 500 IU/ml. (<b>B</b>) Western blot analysis of total GR and phospho-GR protein (∼95 kDa) of HT-29/B6-GR cell lysates incubated with dexamethasone (1 µM) and/or TNF-α (500 IU/ml) and/or endiandrin A (20 µM) for 3 hours. Human β-actin (∼42 kDa) served as a loading control. (<b>C</b>) Densitometry of total GR chemiluminescence signals. Shown are means ± s.e.m., n = 5, *<i>P</i><0.05 compared to TNF-α alone, ^#<i>P</i><0.05 compared with dexamethasone + TNF-α 500 IU/ml. (<b>D</b>) Densitometry of phospho-GR chemiluminescence signals. Shown are means ± s.e.m., n = 5, *<i>P</i><0.05 compared with dexamethasone + TNF-α 500 IU/ml. (<b>E</b>) Densitometry of phospho-glucocorticoid receptor chemiluminescence signals normalized to total glucocorticoid receptor. Shown are means ± s.e.m., n = 5, *P<0.05 compared with dexamethasone. (<b>F</b>) GR mRNA expression of HT-29/B6-GR cells incubated with dexamethasone (1 µM) and/or TNF-α (500 U/ml) and/or endiandrin A (20 µM) for 3 hours. GR mRNA level was analyzed by real-time PCR. Data are means ± s.e.m. of x-fold induction of GR expression over that of untreated controls, n = 7–8, *<i>P</i><0.05 compared to TNF-α alone, ^#<i>P</i><0.05 compared with dexamethasone + TNF-α 500 IU/ml.</p

Synergistic ENaC induction by endiandrin A is linked to an activation of p38 and ERK in HT-29/B6-GR cells.

<p>HT-29/B6-GR cells were pre-incubated with the indicated MAPK inhibitors (10 µmol/L) for 1 hour before addition of dexamethasone (1 µM) and/or TNF-α (500 U/ml) and/or endiandrin A (20 µM) for 24 hours. (<b>A</b>) Measurement of ENaC-dependent Na⁺ absorption as the drop in I_SC after amiloride (100 µM) and measurement of γ-ENaC-mRNA after 24 hours. GAPDH was used for normalization of mRNA expression. Data are given as means ± s.e.m., n = 8–11 and n = 5–8, ***<i>P</i><0.001 compared with dexamethasone incubation,^#<i>P</i><0.05, ^##<i>P</i><0.01 and ^###<i>P</i><0.001 compared to dexamethasone + TNF-α 500 IU/ml, ^°°<i>P</i><0.01 and ^°°°<i>P</i><0.001 compared with dexamethasone + TNF-α 500 IU/ml + endiandrin A. (<b>B</b>) Western blot analysis of total GR protein (∼95 kDa) of HT-29/B6-GR cell lysates. Cells were pre-incubated for 1 hour with SB202190 (10 µM) before incubation with dexamethasone (1 µM) and/or TNF-α (500 IU/ml) and/or endiandrin A (20 µM) for 3 hours. Human β-actin (∼42 kDa) served as a loading control. (<b>C</b>) Densitometry of SB202190 induced effects on total GR levels. Shown are means ± s.e.m., n = 7, *<i>P</i><0.05 compared to dexamethasone, TNF-α 500 IU/ml and endiandrin A, ^#<i>P</i><0.05 compared to TNF-α 500 IU/ml and endiandrin A. (<b>D</b>) Western blot analysis of HT-29/B6-GR cell lysates incubated with dexamethasone (1 µM) and/or TNF-α (500 IU/ml) w/wo endiandrin A (20 µM). Shown are p38 and pp38 (∼38 kDa) after 120 minutes stimulation as well as ERK and p-ERK MAPK protein (∼42/44 kDa) after 5 minutes stimulation. Human β-actin (∼42 kDa) served as a loading control.</p

Influence of Sgk-1 on the synergistic ENaC induction in HT-29/B6-GR cells.

<p>(<b>A</b>) HT-29/B6-GR cells were transfected with psgk-1-short-Luc and incubated with dexamethasone (1 µM) and/or TNF-α (500 IU/ml) and/or endiandrin A (20 µM) for 24 hours. Data are given as normalized relative luciferase activity and as means ± s.e.m., n = 6–14 ^*<i>P</i><0.05 compared to dexamethasone alone,^#<i>P</i><0.05 compared with dexamethasone + TNF-α 500 IU/ml. (<b>B</b>) Measurement of sgk-1 mRNA after 24 h. GAPDH was used for normalization of mRNA expression. Data are means ± s.e.m., n = 5–8, ^**<i>P</i><0.01 compared with dexamethasone treatment, ^##<i>P</i><0.01 compared with dexamethasone + TNF-α 500 IU/ml, ^°°°<i>P</i><0.001 compared with TNF-α 500 IU/ml alone. (<b>C</b>) Measurement of sgk-1 mRNA pre-incubation with the indicated inhibitors (10 µM) for 1 hour before addition of dexamethasone (1 µM) and/or TNF-α (500 IU/ml) and/or endiandrin A (20 µM) for 24 hours. Data are means ± s.e.m., n = 5–7, ^***<i>P</i><0.001 compared with dexamethasone + TNF-α 500 IU/ml, ^#<i>P</i><0.05 compared to dexamethasone + TNF-α 500 IU/ml + U0126 inhibitor.</p