Vitamin D Receptor Activation Exerts Anti‐Secretory Actions In Colonic Epithelial Cells

The vitamin D receptor (VDR) is a nuclear receptor that is expressed in many tissues throughout the body, but is particularly abundant within the colon. VDR is classically known to be activated by the active form of vitamin D, calcitriol, but is appreciated to also serve as a receptor for luminal bile acids. VDR has previously been shown to have anti-inflammatory and barrier promoting actions in the large intestine. However, there is little known of the role VDR plays in regulating colonic epithelial transport function. Interestingly, previous studies from our laboratory have shown the nuclear bile acid receptor, farnesoid X receptor (FXR), to exert anti-secretory actions in colonic epithelial cells. The current study aims to investigate a potential role for VDR in regulating colonic Cl- secretion and whether it may be a target for the treatment of diarrhoeal diseases.To investigate the effects of VDR activation by calcitriol on colonic epithelial Cl- secretion.Polarised monolayers of T84 cells, grown on permeable supports, were used as a model of the colonic epithelium. Protein expression of VDR and CFTR was measured by western blotting. Calcitriol (1 - 20 nM) was used to activate VDR and the VDR target protein, CYP24A1, was used to verify activation of the receptor by qRT-PCR. The specificity of calcitriol as a VDR agonist was determined by investigating FXR activation using the FXR target protein, FGF19, and a FXR-luciferase reporter cell line. The effects of VDR activation by calcitriol (10 nM) on chloride secretion over a timecourse (6 - 48 hr) was determined using the Ussing chamber technique. Changes in short-circuit current (Isc) induced by the Ca2+-dependent agonist, carbachol (CCh; 100 µM) were assessed.VDR was found to be expressed in T84 cell monolayers as determined by western blotting. Treatment of T84 cells with calcitriol (5 - 20 nM) significantly (n = 3, *p ≤ 0.05) increased expression of CYP24A1, indicating activation of VDR by the agonist. Calcitriol did not alter the activity of FXR, either in an FXR-luciferase reporter cell line or in T84 cells, indicating specificity of the agonist for VDR. In Ussing chambers, the FXR agonist, GW4064 (5 µM; 24 hrs), inhibited Cl- secretory responses to CCh, as previously reported (n = 5, **p ≤ 0.01). Calcitriol (10 nM) was also found to significantly inhibit CCh-stimulated Cl- secretory responses by 39.9 ± 5.1% (6 hr), 40.4 ± 4.3% (18 hr) and 62.0 ± 6.1% (24 hr) (n = 5, **p ≤ 0.01). In addition to their effects on CCh-induced chloride secretion, GW4064 (5 µM; 24 hrs) downregulated the protein expression of CFTR by 0.5 ± 0.1 (n = 8, ***p ≤ 0.001) (as previously reported) and calcitriol (10 nM) downregulated CFTR by 0.35 ± 0.1 (n=8, *p≤ 0.05).Similar to the bile acid receptor, FXR, VDR is expressed in colonic epithelial cells where activation by its natural ligand, calcitriol, downregulates CFTR expression and inhibits agonist-induced Cl- secretory responses. Since Cl- secretion is the primary driving force for intestinal fluid secretion, our data suggest that VDR may serve as a target for the development of new anti-diarrhoeal agents. Future studies will aim to determine the molecular mechanisms involved and investigate a possible role for VDR in mediating anti-secretory actions of luminal bile acids.