Pentacyclic triterpenes modulate farnesoid X receptor expression in colonic epithelial cells: Implications for colonic secretory function

Abstract

The nuclear bile acid receptor, farnesoid X receptor (FXR), is an important regulator of intestinal and metabolic function. Previous studies suggest that pentacyclic triterpenes (PCTs), a class of plant-derived bioactive phytochemical, can modulate FXR activity and may therefore offer therapeutic benefits. Here, we investigated the effects of a prototypical PCT, hederagenin (HG), on FXR expression, activity, and antisecretory actions in colonic epithelial cells. T₈₄ cells and murine enteroid-derived monolayers were employed to assess HG effects on FXR expression and activity in colonic epithelia. We measured mRNA levels by qRT-PCR and protein by ELISA and immunoblotting. Transepithelial Cl^- secretion was assessed as changes in short circuit current in Ussing chambers. We determined HG treatment (5-10 μM) alone did not induce FXR activation but significantly increased expression of the receptor, both in T₈₄ cells and murine enteroid-derived monolayers. This effect was accompanied by enhanced FXR activity, as assessed by FGF-15/19 induction in response to the synthetic, GW4064, or natural FXR agonist, chenodeoxycholic acid. Effects of HG on FXR expression and activity were mimicked by another PCT, oleanolic acid. Furthermore, we found FXR-induced downregulation of cystic fibrosis transmembrane conductance regulator Cl^- channels and inhibition of transepithelial Cl^- secretion were enhanced in HG-treated cells. These data demonstrate that dietary PCTs have the capacity to modulate FXR expression, activity, and antisecretory actions in colonic epithelial cells. Based on these data, we propose that plants rich in PCTs, or extracts thereof, have excellent potential for development as a new class of "FXR-targeted nutraceuticals".

Keywords: bile acid; cystic fibrosis transmembrane conductance regulator; farnesoid X receptor; intestinal epithelium; natural product; nuclear receptor.

Figure 1

Chemical structures of the compounds of interest throughout this study. Carbon backbone and side chain structures of the bile acid chenodeoxycholic acid (1), the pentacyclic triterpenes hederagenin (2), oleanolic acid (3), the hederagenin-related glycosides, α-hederin (4), and hederacoside C (5).

Figure 2

Hederagenin is not an agonist of FXR. T₈₄ cells were serum-starved for 24 h and bilaterally treated with DMSO (1%), GW4064 (5 μM), or HG (5–100 μM) for 24 h. A, basolateral supernatants were collected, and FGF-19 protein was analyzed by ELISA (n = 4; ∗p < 0.05). B, FGF-19 mRNA expression was analyzed by qRT-PCR (n = 3). C, TEER was assessed pretreatment and posttreatment (n = 7; ∗∗∗p < 0.001). D, FXR reporter cells were treated with DMSO (0.1%), GW4064 (5 μM), or HG (1–10 μM) for 24 h. FXR activation was assessed by luminescence measurement (n = 3; ∗∗p < 0.01). Data are expressed as mean ± SD. Statistical analysis was performed using either repeated measures one-way ANOVA with Dunnett’s post hoc test or two-way ANOVA with Sidak’s post hoc test, as appropriate. FXR, farnesoid X receptor; HG, hederagenin; qRT-PCR, quantitative real-time PCR.

Figure 3

Hederagenin significantly upregulates FXR expression in colonic epithelial cells. T₈₄ cells were serum-starved for 24 h and bilaterally treated with DMSO (0.05%–0.1%) or HG (5–10 μM) for 24 h. A, FXR mRNA expression was analyzed by qRT-PCR (n = 4; ∗∗p < 0.01, ∗p < 0.05). T₈₄ cells were serum-starved for 24 h and bilaterally treated with DMSO (0.05%) or HG (5 μM) for a range of times. B, FXR mRNA expression was analyzed by qRT-PCR (n = 3–7; ∗p < 0.05). C, representative Western blot of FXR protein expression with β-actin being used as the loading control. FXR protein expression was assessed by densitometry. All values were normalized to β-actin protein expression and expressed as fold change over vehicle-treated controls (n = 7; ∗p < 0.05). D, human colonic biopsies were treated with DMSO (0.05%) or HG (5 μM) for 6 h. Tissues were collected and stored in RNA later. FXR mRNA expression was analyzed by qRT-PCR (n = 6; ∗p < 0.05). E, T₈₄ cells were serum-starved for 24 h prior to 6 h treatment with DMSO (0.05%), HG (5 μM), HC (5 μM), or α-H (5 μM). FXR mRNA expression was analyzed by qRT-PCR (n = 4; ∗p < 0.05). F, T₈₄ cells were serum-starved for 24 h and bilaterally treated with DMSO (0.05%) or HG (5 μM) for the times indicated. VDR protein expression was assessed by Western blotting. Representative Western blot image of VDR protein expression with β-actin being used as a loading control. VDR protein expression was assessed by densitometry. All values were normalized to β-actin protein expression and expressed as fold change over vehicle-treated controls (n = 3). Data are expressed as mean ± SD. Statistical analysis was performed using either the paired Student t test, the repeated measures one-way ANOVA with Dunnett’s post hoc test, or mixed effect analysis with Dunnett’s post hoc test, as appropriate. FXR, farnesoid X receptor; HG, hederagenin; VDR, vitamin D receptor; qRT-PCR, quantitative real-time PCR.

Figure 4

Pentacyclic triterpenes potentiate agonist-induced FXR signaling in T₈₄colonic epithelial cells. T₈₄ cells were serum-starved for 24 h and bilaterally pretreated with HG (5 μM) for 1 h prior to 24 h treatment with varying concentrations of GW4064 (0.5–5 μM). A, FGF-19 mRNA expression was analyzed by qRT-PCR (n = 8; ∗∗p < 0.01, ∗p < 0.05, ^#p < 0.05). B, basolateral supernatants were collected and FGF-19 protein expression was assessed by ELISA (n = 9, ∗p < 0.05). C, T₈₄ cells were bilaterally pretreated with HG (5 μM) for 1 h prior to 24 h treatment with CDCA (50 μM). Basolateral supernatants were collected, and FGF-19 protein expression was assessed by ELISA (n = 8, ∗p < 0.05, ∗∗p < 0.01, ^#p < 0.05). D, T₈₄ cells were bilaterally pretreated with OA (5 μM) for 1 h prior to 24 h treatment with GW4064 (5 μM). Basolateral supernatants were collected, and FGF-19 protein expression was assessed by ELISA (n = 4, ∗p < 0.05, ^#p < 0.05). Data are expressed as mean ± SD. Statistical analysis was performed using repeated measures one-way ANOVA with Tukey’s post hoc test. ∗denotes significant differences compared to untreated controls, and ^#denotes significant differences compared to GW4064-treated cells. FXR, farnesoid X receptor; HG, hederagenin; OA, oleanolic acid; qRT-PCR, quantitative real-time PCR.

Figure 5

Hederagenin enhances FXR expression and signaling in murine colonic enteroids. Mouse colonic enteroids, grown as monolayers on permeable supports, were treated with HG (5 μM) for 1 h, prior to treatment with GW4064 (5 μM) for 24 h (A) FXR and (B) FGF-15 mRNA expression were analyzed by qRT-PCR (n = 4; ∗p < 0.05, ∗∗p < 0.01, ^#p < 0.05). Data are expressed as mean ± SD. Statistical analysis was performed using paired Student t test or repeated measures one-way ANOVA with Tukey’s post hoc test. ∗denotes significant differences compared to untreated controls, and ^#denotes significant differences compared to GW4064-treated cells. FXR, farnesoid X receptor; HG, hederagenin; qRT-PCR, quantitative real-time PCR.

Figure 6

Hederagenin significantly enhances FXR-inhibition of CFTR expression and chloride secretion. T₈₄ cells were serum-starved for 24 h and bilaterally pretreated with HG (5 μM) for 1 h prior to 24 h treatment with varying concentrations of GW4064 (0.5–5 μM). A, CFTR mRNA expression was analyzed by qRT-PCR (n = 8; ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05, ^##p < 0.01, ^#p < 0.05). B, representative Western blot of CFTR protein expression with β-actin being used as the loading control. CFTR protein expression was assessed by densitometry. All values were normalized to β-actin protein expression and expressed as fold change over vehicle-treated controls (n = 9; ∗∗∗p < 0.001, ^##p < 0.01). C, murine colonic enteroids were pretreated with either HG (5 μM) or DMSO (0.05%) for 1 h, prior to 24 h treatment with GW4064 (5 μM). CFTR mRNA expression was analyzed by qRT-PCR (n = 4; ∗∗∗p < 0.001, ∗∗p < 0.01, ^#p < 0.05). D, T₈₄ cells monolayers were bilaterally pretreated with HG (5 μM) for 1 h prior to 24 h treatment with GW4064 (5 μM). Cells were then mounted in Ussing chambers and voltage-clamped to zero potential difference for measurements of short circuit current (Ӏ_sc). Cells were apically treated with FSK (10 μM) and changes in Ӏ_sc were monitored using Acquire and Analyse software (n = 7; ∗∗∗p < 0.001, ∗∗p < 0.01, ^##p < 0.01). E, representative I_sc traces of the data shown in panel (D). Data are expressed as mean ± SD. Statistical analysis was performed using repeated measures one-way ANOVA with Tukey’s post hoc test. ∗denotes significant differences compared to untreated controls, and ^#denotes significant differences compared to GW4064-treated cells. CFTR, cystic fibrosis transmembrane conductance regulator; FSK, forskolin; FXR, farnesoid X receptor; HG, hederagenin; qRT-PCR, quantitative real-time PCR.