Epigenetics and MicroRNAs in Pharmacogenetics.

Germline pharmacogenetics has so far mainly studied common variants in "pharmacogenes," i.e., genes encoding drug metabolizing enzymes and transporters (DMET genes), certain auxiliary and regulatory genes, and drug target genes. Despite remarkable progress in understanding genetically determined differences in pharmacokinetics and pharmacodynamics of drugs, currently known common variants even in important pharmacogenes explain genetic variability only partially. This suggests "missing heritability" that may in part be due to rare variants in the classical pharmacogenes, but current evidence suggests that largely unexplored resources with potential for pharmacogenetics exist, both within already known pharmacogenes and in entirely new areas. In particular, recent studies suggest that epigenetic processes and noncoding RNAs, including mostly microRNAs (miRNAs), represent important and largely unexplored layers of DMET gene regulation that may fill some of the gaps in understanding interindividual variability and lead to new biomarkers. In this chapter we summarize recent advances in the understanding of genetic variability in epigenetic and miRNA-mediated processes with focus on their significance for DMET regulation and pharmacokinetic or pharmacological endpoints.

Coordinate regulation of Cyp2e1 by ß-catenin- and hepatocyte nuclear factor 1α-dependent signaling.

Depending on their position within the liver lobule, hepatocytes fulfill different metabolic functions. Cytochrome P450 (CYP) 2E1 is a drug-metabolizing enzyme which is exclusively expressed in hepatocytes surrounding branches of the hepatic central vein. Previous publications have shown that signaling through the Wnt/ß-catenin pathway, a major determinant of liver zonation, and the hepatocyte-enriched transcription factor HNF (hepatocyte nuclear factor) 1α participate in the regulation of the gene. This study was aimed to decipher the molecular mechanisms by which the two transcription factors, ß-catenin and HNF1α, jointly regulate CYP2E1 at the gene promoter level. Chromatin immunoprecipitation identified a conserved Wnt/ß-catenin-responsive site (WRE) in the murine Cyp2e1 promoter adjacent to a known HNF1α response element (HNF1-RE). In vitro analyses demonstrated that both, activated ß-catenin and HNF1α, are needed for the full response of the promoter. The WRE was dispensable for ß-catenin-mediated effects on the Cyp2e1 promoter, while activity of ß-catenin was integrated into the promoter response via the HNF1-RE. Physical interaction of ß-catenin and HNF1α was demonstrated by co-immunoprecipitation. In conclusion, present data the first time identify and characterize the interplay of HNF1α and ß-catenin and elucidate molecular determinants of CYP2E1 expression in the liver.

Inhibition of ß-catenin signaling by phenobarbital in hepatoma cells in vitro.

The antiepileptic drug phenobarbital (PB) exerts hepatic effect based on indirect activation of the constitutive androstane receptor (CAR) via inhibition of the epidermal growth factor receptor (EGFR) and the kinase Src. It has furthermore been observed that in mice PB suppresses the growth of hepatocellular carcinoma with overactive signaling through the oncogenic Wnt/ß-catenin pathway, thus suggesting an interference of PB with ß-catenin signaling. The present work was aimed to characterize effects of PB on ß-catenin signaling at different cellular levels and to elucidate molecular details of the interaction of PB and ß-catenin in an in vitro system of mouse hepatoma cells. PB efficiently inhibited signaling through ß-catenin. This phenomenon was in-depth characterized at the levels of ß-catenin protein accumulation and transcriptional activity. Mechanistic analyses revealed that the effect of PB on ß-catenin signaling was independent of the activation of CAR and also independent of the cytosolic multi-protein complex responsible for physiological post-translation control of the ß-catenin pathway via initiation of ß-catenin degradation. Instead, evidence is provided that PB diminishes ß-catenin protein production by inhibition of protein synthesis via signal transduction through EGFR and Src. The proposed mechanism is well in agreement with previously published activities of PB at the EGFR and Src-mediated regulation of ß-catenin mRNA translation. Inhibition of ß-catenin signaling by PB through the proposed mechanism might explain the inhibitory effect of PB on the growth of specific sub-populations of mouse liver tumors. In conclusion, the present data comprehensively characterize the effect of PB on ß-catenin signaling in mouse hepatoma cells in vitro and provides mechanistic insight into the molecular processes underlying the observed effect.