title: Up-regulation of CYP expression in hepatoma cells stably transfected by chimeric nuclear receptors.
abstract: Most hepatoma cell lines lack proper expression and induction of cytochrome P450 (CYP) enzymes and this deficiency hampers their use as in vitro models for drug and xenobiotic metabolism. According to previous studies, the poor expression of CYP enzymes may be due to decreases in CYP gene transcription. Two nuclear receptors (NRs), the pregnane X receptor (PXR) and the constitutive androstane receptor (CAR), are known to regulate many genes involved in xenobiotic metabolism and disposition. Here, we studied the expression of different CYP, NR and NR co-regulator genes in hepatoma cell lines. Next, we created "chimeric NR" constructs by cloning the strong activation domain from the p65 subunit of transcription factor NF-kappaB and appending it to either N- or C-termini of the human CAR or PXR. We established that these chimeric NRs displayed enhanced trans-activation potential as compared to the unmodified NRs, and showed that transient transfection of a single chimeric NR increased the expression of several CYPs simultaneously. Finally, stable cell lines expressing a chimeric NR had elevated levels of CYP3A4, CYP2B6 and CYP2C9 mRNAs and CYP3A4-mediated metabolism when compared to the wild-type hepatoma cells. These findings establish a proof of principle how improved metabolic cell models could be designed.
authors.affiliation: School of Pharmacy, Faculty of Health Sciences and Biocenter Kupio, University of Eastern Finland, Yliopistonranta 1C, P.O. Box 1627, FI-70211 Kuopio, Finland. Jenni.Kublbeck@uef.fi
authors.forename: Jenni
authors.initials: J
authors.lastname: Küblbeck
authors.forename: Mika
authors.initials: M
authors.lastname: Reinisalo
authors.forename: Riitta
authors.initials: R
authors.lastname: Mustonen
authors.forename: Paavo
authors.initials: P
authors.lastname: Honkakoski
mesh_terms: D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics / Q000378:metabolism; D006528:Carcinoma, Hepatocellular / Q000378:metabolism; D045744:Cell Line, Tumor; D056970:Co-Repressor Proteins / Q000235:genetics / Q000378:metabolism; D000090702:Constitutive Androstane Receptor; D065702:Cytochrome P-450 CYP2B6; D065729:Cytochrome P-450 CYP2C9; D051544:Cytochrome P-450 CYP3A / Q000235:genetics / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D017930:Genes, Reporter; D056945:Hep G2 Cells; D006801:Humans; D008099:Liver / Q000378:metabolism*; D056918:Nuclear Receptor Coactivators / Q000235:genetics / Q000378:metabolism; D010089:Oxidoreductases, N-Demethylating / Q000235:genetics / Q000378:metabolism; D000077297:Pregnane X Receptor; D054730:Protein Interaction Domains and Motifs / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism*; D011987:Receptors, Steroid / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D051996:Transcription Factor RelA / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation; D015854:Up-Regulation; D015262:Xenobiotics / Q000378:metabolism*
chemical_list: D056970:Co-Repressor Proteins; D000090702:Constitutive Androstane Receptor; D056918:Nuclear Receptor Coactivators; D000077297:Pregnane X Receptor; C498143:RELA protein, human; D012333:RNA, Messenger; D018160:Receptors, Cytoplasmic and Nuclear; D011987:Receptors, Steroid; D011993:Recombinant Fusion Proteins; D051996:Transcription Factor RelA; D015262:Xenobiotics; D003577:Cytochrome P-450 Enzyme System; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; C585599:CYP2B6 protein, human; D065702:Cytochrome P-450 CYP2B6; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D010089:Oxidoreductases, N-Demethylating