Regulation of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by the Nuclear Bile Acid Receptor, Farnesoid X Receptor.

R6019 --> 731.7 --> Introduction and Aims: CFTR, a Cl‐ channel important in regulating intestinal fluid and electrolyte secretion, is implicated in the pathogenesis of a number of intestinal disorders. We have previously shown that bile acids, acting via the nuclear receptor, farnesoid X receptor (FXR), inhibit colonic epithelial CFTR expression. Dietary phytochemicals have been reported to have the capacity to modulate FXR signalling. Here, we set out to investigate mechanisms underlying FXR regulation of CFTR expression, and the potential for therapeutically targeting the receptor with dietary phytochemicals. Methods: Monolayers of T84 colonic epithelial cells were treated with the FXR agonist, GW4064 (5 µM) in the absence or presence of a plant phytochemical, designated here as KFS1 (5 µM). mRNA and protein expression were measured by qPCR and Western blotting. FXR binding to the CFTR promoter was investigated by chromatin immunoprecipitation (ChIP). Electrophysiological studies of T84cells were conducted in Ussing chambers. Studies on human and murine enteroids were carried out with ethical approval from Johns Hopkins University School of Medicine Institutional Review Board and the Institutional Review Board of the University of California San Diego, respectively. Results: Treatment of T84 monolayers with GW4064 downregulated CFTR mRNA to 0.51 ± 0.06 fold after 12 hrs (n = 12; p < 0.001) and protein levels to 0.28 ± 0.06 fold after 48 hrs, compared to controls (n = 8; p < 0.001). Studies in Ussing chambers showed that GW4064 treatment for 48 hrs inhibited Cl‐ secretory responses to the Ca2+‐ and cAMP‐dependent agonists, carbachol (CCh; 100mM) and forskolin (FSK; 10mM), by 79.9 ± 7.5 % and 74.2 ± 8.9 %, respectively. Transcriptomic analysis of human colonic enteroids revealed FXR to be robustly expressed in secretory (crypt‐like) cells and that its activation also induced CFTR downregulation. FXR activation did not alter expression or phosphorylation of the p65 subunit of NF‐κB, or inhibit its translocation to the nucleus. ChIP‐qPCR analysis appears to show enhanced binding of FXR to the CFTR promoter upon GW4064 treatment. The phytochemical, KFS1 (5 mM; 24hrs), upregulated FXR mRNA and protein expression in T84 cells and enhanced GW4064‐induced downregulation of CFTR mRNA by 0.28 ± 0.05 fold (n = 8; p <0.01) and protein by 0.25 ± 0.11 fold (n = 4) after 24 hours. Similarly, KFS1 significantly upregulated FXR mRNA expression in murine colonic epithelial enteroids (2.3 ± 0.2; n = 4; p < 0.01) and enhanced GW4064‐induced downregulation of CFTR mRNA by 0.5 ± 0.1 fold (n = 4; p < 0.05) compared to GW4064 alone. Finally, KFS1 enhanced FXR inhibition of agonist‐induced Cl‐ secretory responses across T84 cells mounted in Ussing chambers. Conclusion: FXR regulates colonic epithelial CFTR expression and function by a mechanism which appears to involve direct binding of FXR to the CFTR promoter. By virtue of their ability to upregulate FXR expression, and thereby enhance its antisecretory actions, plant extracts containing KFS1 have excellent potential to be developed as FXR‐targeted nutraceuticals for the treatment and prevention of intestinal disease. [ABSTRACT FROM AUTHOR]