Introduction: The nuclear bile acid receptor, farnesoid X receptor (FXR), is an important regulator of colonic epithelial function and the therapeutic potential for targeting FXR in the treatment of chronic intestinal disease is well documented. We have previously shown that bile acids, acting via FXR, inhibit colonic epithelial Cl– secretion by downregulating expression of the CFTR Cl– channel. We have also shown that the pentacyclic triterpene (PCT), hederagenin (HG), a plant-derived bioactive phytochemical, enhances the expression and activity of FXR in colonic epithelial cells and potentiates the effects of FXR on CFTR. Peroxisome proliferator-activated receptors (PPARs), described as nutrient sensors, have recently been shown to modulate FXR expression. Here, we investigated molecular mechanisms underlying FXR regulation of CFTR expression in colonic epithelial cells, and a potential role for PPARs in mediating HG actions on FXR expression and activity.
Methods: Monolayers of T84 colonic epithelial cells were treated with HG, PPARγ, PPARα, or PPARδ agonists, rosiglitazone, WY14643, and GW501516, and antagonists, GW9662, GW6471, and GSK3787, respectively. GW4064 was employed as an FXR agonist and FGF19 used as an index of FXR activation. Levels of FGF19, NF-κB p65, FXR, and ANGPTL4, a PPAR target gene, were measured by qRT-PCR, western blotting, or ELISA. FXR binding to the CFTR promoter was investigated by chromatin immunoprecipitation (ChIP).
Results: FXR activation did not alter expression, phosphorylation, or nuclear translocation of the NF-κB p65 subunit, while preliminary ChIP-qPCR analysis demonstrated enhanced binding of FXR to the CFTR promoter upon GW4064 treatment. Treatment with HG significantly increased mRNA expression of the PPAR target gene, ANGPTL4, to 5.7 ± 1 fold (n = 5; p < 0.05) of untreated cells after 3 hours. The effects of HG on FXR expression were mimicked by rosiglitazone (1 µM), WY14643 (10 µM), and GW501516 (0.5 µM), respectively. The PPARα antagonist, GW6471 (1 µM), reduced HG-induced FXR mRNA and protein expression from 2.8 ± 0.4 to 0.9 ± 0.1 (n = 5; p < 0.05) and 1.8 ± 0.3 to 1 ± 0.1 (n = 3) fold of untreated controls, respectively, whereas the PPARγ and PPARδ antagonists, GW9662 (5 µM) and GSK3787 (1 µM), did not prevent upregulation of FXR by HG.
Conclusion: FXR regulates colonic epithelial CFTR expression and function by a mechanism which appears to involve direct binding of FXR to the CFTR promoter. The bioactive phytochemical, HG, regulates colonic epithelial FXR expression through mechanisms that involve activation of PPARα. By virtue of their ability to upregulate FXR expression, and thereby enhance its antisecretory actions, plant extracts containing HG have excellent potential to be developed as FXR-targeted nutraceuticals for the treatment and prevention of intestinal disease.