Impact of Transcription Factor Profile and Chromatin Conformation on Human Hepatocyte Cyp3a Gene Expression

Abstract

ABSTRACT:Recent data have made it increasingly clear that the gene expression profile of a cell system, and its alteration in response to external stimuli, is highly dependent on both the higher order chromatin structure of the genome and the interaction of gene products in interpreting stimuli. To further explore this phenomenon, we have examined the role of both of these factors in controlling xenobiotic-mediated gene expression changes in primary and transformed human hepatocytes (HuH7). Using quantitative polymerase chain reaction, expression levels of several transcription factors implicated in the liver-specific regulation of the CYP3A gene family were examined in human adult and fetal liver RNA samples. These expression profiles were then compared with those obtained from both primary and transformed human hepatocytes, showing that, in general, cultured cells exhibit a distinct profile compared with either the fetal or adult samples. Transcriptome profiles before and after exposure to the CYP3A transcriptional activators rifampicin, dexamethasone, pregnane-16␣-carbonitrile, and phenobarbital were subsequently examined. Whereas exposure to these compounds elicited a dose-dependent increase in CYP3A transcription in primary hepatocytes, no alteration in expression levels was observed for the hepatoma cell line HuH7. Alteration in the expression levels of pregnane X receptor and chicken ovalbumin upstream promoter transcription factor I, and the disruption of higher order chromatin within HuH7 cells altered CYP3A expression and/or activation by xenobiotics toward that observed in primary hepatocytes. These data provide potential roles for these two processes in regulating CYP3A expression in vivo.

Response: Comments on “Glycogen Synthase Kinase-3 Mediates Acetaminophen-Induced Apoptosis in Human Hepatoma Cells”

“…To circumvent this problem, we have used a human hepatoma cell line, and this model shares many of the events seen under our in vivo conditions, including the loss of mitochondrial proapoptotic proteins and the sensitivity to caspase inhibitors. HuH7 cells also express significant levels of CYP3A (Phillips et al, 2005), which can efficiently metabolize AAP to N-acetyl-pbenzoquinone imine (Sinclair et al, 1998;Guo et al, 2004), and this is in line with the observed depletion of glutathione in our study (Macanas-Pirard et al, 2005).…”

TRANSCRIPTIONAL REGULATION OF THEPXRGENE: IDENTIFICATION AND CHARACTERIZATION OF A FUNCTIONAL PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR α BINDING SITE WITHIN THE PROXIMAL PROMOTER OFPXR

“…Whereas this may, to some extent, limit the extent of the responses observed, it should not alter their effect/presence. Indeed, previous evidence has shown that many of the ligand-activated and liver-enriched transcription factors are expressed in Huh7, albeit at reduced levels compared with in vivo, supportive of the presence of all the necessary factors within these cells for functioning of these transcription factors (Phillips et al, 2005).…”

HepaRG Cells as Human-Relevant In Vitro Model to Study the Effects of Inflammatory Stimuli on Cytochrome P450 Isoenzymes

“…A separate level of control lies in chromatin conformation, which dictates transcription factor binding (Phillips et al, 2005). Both processes play key roles in regulating P450 expression and alterations in both are important in understanding not only the decline in P450 expression after isolation of hepatocytes from the intact liver but also the disparity in P450 expression between isolated primary hepatocytes and some hepatic cell lines (Phillips et al, 2005;Castell et al, 2006). An important factor in the downregulation of CYP3A4 by IL-6 is the translational induction of a truncated form of CCAATenhancer binding protein b (C/EBPb), the 20-kDa liver-enriched transcriptional inhibitory protein (C/EBPb-LIP).…”

“…The utility of some hepatic cell lines in the study of drug-metabolizing enzymes and hepatic function has been shown to be limited by the deficit in one or more of the extremely complex pathways that make up the fully functioning primary hepatic parenchymal cells (Phillips et al, 2005;Castell et al, 2006;Hariparsad et al, 2008;Martin et al, 2008). On the other hand, HepaRG cells appear to have wide-ranging utility and can express some, but not all, drug-metabolizing enzymes and transporters at levels comparable to primary cells (Kanebratt and Andersson 2008) and have P450-inducer response closely mimicking primary cells (McGinnity et al, 2009).…”