Figure 6.

Farnesoid X receptor (FXR) and constitutive androstane receptor (CAR) modulate bilirubin-mediated hBD-1 induction. (A) Quantitative luciferase reporter gene assays with a construct containing 1,100 bp upstream region of hBD-1 promoter were performed in human HuH7 cells 48 h after indicated treatments with known agonists of nuclear receptors (ordered alphabetically) 5 µM CITCO (CAR), 10 µM GW4064 (FXR), 10 µM TO-90 (LXRα), 100 µM WY14,643 (PPARα), 10 µM Rosiglitazone (PPARγ, Rosi), and 10 µM of Rifampicin (PXR) in total 72 h after the transfection with the luciferase-containing constructs. The cells were co-transfected with the vector containing Renilla luciferase for normalization of transfection efficiency. The bars represent average fold change lucifearse induction normalized to solvent, DMSO, and control. Error bars indicate SD between three independent experiments. ****p < 0.001. (B) qRT-PCR assessment of hBD-1 mRNA expression in HepaRG lysates following transfections with the indicated small interfering RNAs (siRNAs), *p < 0.05. (C) qRT-PCR analysis of hBD-1 expression in HepaRG cells upon treatment with siRNAs targeting PPARα (siPPARα), FXR (siFXR), CAR (siCAR), and non-targeting scramble siRNA (siCTR) (black bars) and in combination with bilirubin treatment for 48 h (grey bars). The bars represent average fold change expression normalized to hBD-1 expression in the cells treated with the solvent, DMSO, control, and siCTR. Error bars indicate SD between three independent experiments. *p < 0.05 compared to DMSO/siCTR control condition.