Up-regulation of CYP expression in hepatoma cells stably transfected by chimeric nuclear receptors

Jenni Küblbeck; Mika Reinisalo; Riitta Mustonen; Paavo Honkakoski

doi:10.1016/j.ejps.2010.03.022

Up-regulation of CYP expression in hepatoma cells stably transfected by chimeric nuclear receptors

Eur J Pharm Sci. 2010 Jul 11;40(4):263-72. doi: 10.1016/j.ejps.2010.03.022. Epub 2010 Apr 8.

Authors

Jenni Küblbeck¹, Mika Reinisalo, Riitta Mustonen, Paavo Honkakoski

Affiliation

¹ 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

PMID: 20381613
DOI: 10.1016/j.ejps.2010.03.022

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.

Publication types

Comparative Study
Research Support, Non-U.S. Gov't

MeSH terms

Aryl Hydrocarbon Hydroxylases / genetics
Aryl Hydrocarbon Hydroxylases / metabolism
Carcinoma, Hepatocellular / metabolism
Cell Line, Tumor
Co-Repressor Proteins / genetics
Co-Repressor Proteins / metabolism
Constitutive Androstane Receptor
Cytochrome P-450 CYP2B6
Cytochrome P-450 CYP2C9
Cytochrome P-450 CYP3A / genetics
Cytochrome P-450 CYP3A / metabolism
Cytochrome P-450 Enzyme System / genetics
Cytochrome P-450 Enzyme System / metabolism*
Genes, Reporter
Hep G2 Cells
Humans
Liver / metabolism*
Nuclear Receptor Coactivators / genetics
Nuclear Receptor Coactivators / metabolism
Oxidoreductases, N-Demethylating / genetics
Oxidoreductases, N-Demethylating / metabolism
Pregnane X Receptor
Protein Interaction Domains and Motifs / genetics
RNA, Messenger / metabolism
Receptors, Cytoplasmic and Nuclear / genetics
Receptors, Cytoplasmic and Nuclear / metabolism*
Receptors, Steroid / genetics
Receptors, Steroid / metabolism*
Recombinant Fusion Proteins / metabolism
Transcription Factor RelA / genetics
Transcription Factor RelA / metabolism
Transcriptional Activation
Up-Regulation*
Xenobiotics / metabolism*

Substances

Co-Repressor Proteins
Constitutive Androstane Receptor
Nuclear Receptor Coactivators
Pregnane X Receptor
RELA protein, human
RNA, Messenger
Receptors, Cytoplasmic and Nuclear
Receptors, Steroid
Recombinant Fusion Proteins
Transcription Factor RelA
Xenobiotics
Cytochrome P-450 Enzyme System
CYP2C9 protein, human
Cytochrome P-450 CYP2C9
Aryl Hydrocarbon Hydroxylases
CYP2B6 protein, human
Cytochrome P-450 CYP2B6
Cytochrome P-450 CYP3A
CYP3A4 protein, human
Oxidoreductases, N-Demethylating