The WNT/b-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/b-catenin pathway on the regulation and inducibility of major human P450 enzymes, HepaRG cells were treated with either the WNT/b-catenin signaling pathway agonist, WNT3a, or with small interfering RNA directed against b-catenin, alone or in combination with a panel of activating ligands for AhR [2,3,7,8-tetrachlorodibenzo-p-

BackgroundThe 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.MethodsThe 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.ResultsBioinformatic 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-1β, 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/β-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.ConclusionsOur 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1500-x) contains supplementary material, which is available to authorized users.

Mantle cell lymphoma (MCL) cells are characterized by a discrepancy between high mRNA and low protein levels of the pro-apoptotic BH3-only protein NOXA. Modulation of NOXA protein expression has been described as a major mechanism of cell death induction in MCL cells. However, the efficiency of induction of apoptosis at lower concentrations is limited despite effective stabilization of NOXA. We therefore investigated whether the main binding partner of NOXA, the anti-apoptotic protein MCL-1 is co-regulated by these agents and whether dual targeting of NOXA and MCL-1 could be a promising strategy to enhance effectiveness of cell death induction in MCL cell lines. Screening a panel of compounds supposed to lead to NOXA stabilization in MCL, we identified the proteasome inhibitor Bortezomib, the fatty acid synthase inhibitor Orlistat and the ROS inducing agents Helenalin, a sesquesterpenone lactone from Arnica and the naphtoquinone derivative Menadione to kill MCL cells very effectively in a NOXA -dependent manner. Investigating the NOXA-/MCL-1-protein expression upon treatment with these agents, we could observe that at lower, sublethal concentrations of Bortezomib and Orlistat, NOXA protein was increased to a certain extent but also the anti-apoptotic MCL-1 was highly induced, counteracting induction of apoptosis. From this observation it has to be concluded that MCL-1 limits the apoptotic activity of NOXA stabilizing agents and dual targeting of MCL-1 and NOXA is required for optimal killing of MCL cells. In search for MCL-1 regulating agents we could identify the cdk-inhibitor Dinaciclib to be the most effective one. This compound rapidly downregulates Mcl-1 protein in a dose- and time-dependent manner presumably due to cdk 9-mediated inhibition of phosphorylation of the RNA-Polymerase II-subunit RPB1. To study the efficiency of dual targeting of NOXA/MCL-1 in killing of MCL cells we combined sublethal doses of Dinaciclib with NOXA stabilizing agents and observed a synergistic induction of apoptosis in MCL cells. Western Blot analysis showed that combination treatment decreased Mcl-1 and increased NOXA protein expression compared to single-agents. It could be shown that induction of cell death by treatment with the combined agents depends on NOXA as transfection of cells with NOXA-siRNA rescued cells from induction of apoptosis. Cell death upon treatment with Dinaciclib and ROS-inducing agents Helenalin and Menadione could furthermore be rescued by preincubation with the antioxidant GSH. Interestingly, combined treatment of cells with Orlistat and Dinaciclib killed most effectively when Dinaciclib was added for 8 hours after 16 hours of preincubation with the NOXA stabilizing agent. This observation contributes to the hypothesis that stabilization of NOXA protein leads to priming of MCL cells to induction of apoptosis by pharmacological downregulation of Mcl-1 with Dinaciclib. In summary, the NOXA-MCL-1 balance is critical for survival of MCL cells. Dual targeting of MCL-1 and NOXA efficiently kills MCL cells and appears to be of particular importance especially at lower concentrations of these compounds. These culture conditions most likely resemble the limited exposure after in vivo treatment. Therefore the combination of NOXA stabilizing agents with Dinaciclib appears to be a promising strategy to be tested in clinical trials. Disclosures No relevant conflicts of interest to declare.