Activating and Inhibitory Functions of WNT/ß-Catenin in the Induction of Cytochromes P450 by Nuclear Receptors in HepaRG Cells.

The WNT/ß-catenin signaling pathway has been identified as an important endogenous regulator of hepatic cytochrome P450 (P450) expression in mouse liver. In particular, it is involved in the regulation of P450 expression in response to exposure to xenobiotic agonists of the nuclear receptors constitutive androstane receptor (CAR), aryl hydrocarbon receptor (AhR), and Nrf2. To systematically elucidate the effect of the WNT/ß-catenin pathway on the regulation and inducibility of major human P450 enzymes, HepaRG cells were treated with either the WNT/ß-catenin signaling pathway agonist, WNT3a, or with small interfering RNA directed against ß-catenin, alone or in combination with a panel of activating ligands for AhR [2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)], CAR [6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime (CITCO)], pregnane X receptor (PXR) [rifampicin], and peroxisome proliferator-activated receptor (PPAR) α [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthioacetic acid (WY14,643)]. Assessment of P450 gene expression and enzymatic activity after downregulation or activation of the WNT/ß-catenin pathway revealed a requirement of ß-catenin in the AhR-, CAR-, and PXR-mediated induction of CYP1A, CYP2B6 and CYP3A4 (for CAR and PXR), and CYP2C8 (for PXR) gene expression. By contrast, activation of the WNT/ß-catenin pathway prevented PPARα-mediated induction of CYP1A, CYP2C8, CYP3A4, and CYP4A11 genes, suggesting a dominant-negative role of ß-catenin in PPARα-mediated regulation of these genes. Our data indicate a significant effect of the WNT/ß-catenin pathway on the regulation of P450 enzymes in human hepatocytes and reveal a novel crosstalk between ß-catenin and PPARα signaling pathways in the regulation of P450 expression.

The truncated splice variant of peroxisome proliferator-activated receptor alpha, PPARα-tr, autonomously regulates proliferative and pro-inflammatory genes.

BACKGROUND: The peroxisome proliferator-activated receptor alpha (PPARα) controls lipid/energy homeostasis and inflammatory responses. The truncated splice variant PPARα-tr was suggested to exert a dominant negative function despite being unable to bind consensus PPARα DNA response elements. METHODS: The distribution and variability factor of each PPARα variant were assessed in the well-characterized cohort of human liver samples (N = 150) on the mRNA and protein levels. Specific siRNA-mediated downregulation of each transcript as well as specific overexpression with subsequent qRT-PCR analysis of downstream genes was used for investigation of specific functional roles of PPARα-wt and PPARα-tr forms in primary human hepatocytes. RESULTS: Bioinformatic analyses of genome-wide liver expression profiling data suggested a possible role of PPARα-tr in downregulating proliferative and pro-inflammatory genes. Specific gene silencing of both forms in primary human hepatocytes showed that induction of metabolic PPARα-target genes by agonist WY14,643 was prevented by PPARα-wt knock-down but neither prevented nor augmented by PPARα-tr knock-down. WY14,643 treatment did not induce proliferative genes including MYC, CDK1, and PCNA, and knock-down of PPARα-wt had no effect, while PPARα-tr knock-down caused up to 3-fold induction of these genes. Similarly, induction of pro-inflammatory genes IL1B, PTGS2, and CCL2 by IL-6 was augmented by knock-down of PPARα-tr but not of PPARα-wt. In contrast to human proliferative genes, orthologous mouse genes were readily inducible by WY14,643 in PPARα-tr non-expressing AML12 mouse hepatocytes. Induction was augmented by overexpression of PPARα-wt and attenuated by overexpression of PPARα-tr. Pro-inflammatory genes including IL-1ß, CCL2 and TNFα were induced by WY14,643 in mouse and human cells and both PPARα forms attenuated induction. As potential mechanism of PPARα-tr inhibitory action we suggest crosstalk with WNT/ß-catenin pathway. Finally, treatment with WY14,643 in the presence of PPARα-tr resulted in the significant reduction of cell viability of AML12 and human ovarian cancer cell line, SKOV3. CONCLUSIONS: Our data suggest that the truncated PPARα splice variant functions as an endogenous inhibitor of proliferative and pro-inflammatory genes in human cells and that its absence in mouse may explain species-specific differences in fibrate-induced hepatocarcinogenesis.