title: U0126, a mitogen-activated protein kinase kinase 1 and 2 (MEK1 and 2) inhibitor, selectively up-regulates main isoforms of CYP3A subfamily via a pregnane X receptor (PXR) in HepG2 cells.
abstract: Hepatocyte tumor cell lines lack the expression or induction properties of major cytochrome P450 (CYP) enzymes compared to primary human hepatocytes. The Ras/Raf/MEK/ERK signaling cascade contributes to hepatocarcinogenesis, dedifferentiation and loss of hepatocyte drug metabolism in hepatocyte tumors. In the present study, we examined whether MEK1/2 inhibitors can restore the expression of CYP genes in hepatocarcinoma HepG2 cells. We found that U0126, a prototype dual MEK1/2 inhibitor, is a potent inducer of CYP3A4, CYP3A5 and CYP3A7 mRNA expression (>100-fold) in HepG2 cells and CYP3A4 mRNA expression in primary human hepatocytes. This U0126-mediated induction is sensitive to the transcriptional inhibitor actinomycin D and was not detected for CYP2B6 or MDR1 mRNA expression. In gene reporter assays, U0126 activates a CYP3A4 promoter luciferase reporter construct containing PXR response elements (PXREs), but not a construct containing mutated PXREs. Based on a ligand binding assay and the examination of a PXR mutant expressing an obstructed ligand binding pocket, we found that U0126 is a ligand of PXR. We also found that U0126 up-regulates the mRNA expression of the nuclear receptors HNF4α, CAR, VDR and PXR but abolishes small heterodimer partner (SHP) corepressor expression in HepG2 cells. The MEK1/2 inhibitors PD0325901 and PD184352, as well as dominant-negative MEK1 expression, also down-regulate SHP mRNA expression. In contrast, dominant-negative MEK1 expression does not significantly induce CYP3A4 gene in HepG2 cells. In conclusion, we found that U0126 is an atypical PXR ligand that via direct (binding and activation of PXR) and indirect (SHP dowregulation) mechanisms selectively restores CYP3A genes in HepG2 cells.
authors.affiliation: Department of Pharmacology and Toxicology, Faculty of Pharmacy in Hradec Kralove, Charles University in Prague, Heyrovskeho 1203, 500 05, Hradec Kralove, Czech Republic.
authors.forename: Tomas
authors.initials: T
authors.lastname: Smutny
authors.forename: Michal
authors.initials: M
authors.lastname: Bitman
authors.forename: Michal
authors.initials: M
authors.lastname: Urban
authors.forename: Michaela
authors.initials: M
authors.lastname: Dubecka
authors.forename: Radim
authors.initials: R
authors.lastname: Vrzal
authors.forename: Zdeněk
authors.initials: Z
authors.lastname: Dvorak
authors.forename: Petr
authors.initials: P
authors.lastname: Pavek
mesh_terms: D002070:Butadienes / Q000494:pharmacology*; D051544:Cytochrome P-450 CYP3A / Q000235:genetics*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D056945:Hep G2 Cells; D022781:Hepatocytes / Q000187:drug effects / Q000201:enzymology; D006801:Humans; D007527:Isoenzymes; D008024:Ligands; D049410:Luciferases, Renilla / Q000235:genetics; D048369:MAP Kinase Kinase 1 / Q000037:antagonists & inhibitors*; D048370:MAP Kinase Kinase 2 / Q000037:antagonists & inhibitors*; D020935:MAP Kinase Signaling System / Q000187:drug effects; D009570:Nitriles / Q000494:pharmacology*; D010957:Plasmids; D000077297:Pregnane X Receptor; D061251:Primary Cell Culture; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011987:Receptors, Steroid / Q000378:metabolism*; D014162:Transfection; D015854:Up-Regulation
chemical_list: D002070:Butadienes; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D008024:Ligands; D009570:Nitriles; D000077297:Pregnane X Receptor; D011987:Receptors, Steroid; C113580:U 0126; D049410:Luciferases, Renilla; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; C482128:MAP2K2 protein, human; D048369:MAP Kinase Kinase 1; D048370:MAP Kinase Kinase 2; C482125:MAP2K1 protein, human