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Up-regulating CYP3A4 expression in C3A cells by transfection with a novel chimeric regulator of hPXR-p53-AD
Feng Chen1, Xiao-Hui Rao2, Jin-Lian Yang1
1Institute of Regenerative Medicine, Southern Medical University Zhujiang Hospital, Guangzhou, China.
Abstract:
Most hepatoma cell lines lack proper expression and induction of CYP3A4 enzyme, which limits their use for predicting drug metabolism and toxicity. Nuclear receptor pregnane X receptor (PXR) has been well recognized for its critical role in regulating expression of CYP3A4 gene. However, its physiological activity of binding to the particular site of promoter is significantly weakened in hepatic cell lines. To address this problem, we created "chimeric PXR" constructs by appending a strong activation domain (AD) from p53 subunit to either N- or C- termini of the human PXR (hPXR), that is, hPXR-p53 and p53-hPXR. C3A, a hepatoma cell line, was used as the cell model to test the regulation effect of chimeric hPXR over wild type (WT) hPXR on CYP3A4 expression at gene, protein, and metabolism levels, respectively. Compared with C3A cells transiently transfected with WT hPXR, the activity of CYP3A4.XREM.luc reporter gene in C3A cells transfected with hPXR-p53 or p53-hPXR increased 5- and 9-fold respectively, and the levels of CYP3A4 mRNA expression increased 3.5- and 2.6-fold, respectively. C3A cells stably transfected with hPXR-p53-AD exhibited an improved expression of CYP3A4 at both gene (2-fold) and protein (1.5-fold) levels compared to WT C3A cells. Testosterone, a CYP3A4-specific substrate, was used for detecting the metabolism activity of CYP3A4. No testosterone metabolite could be detected in microsomes from WT C3A cells and WT C3A cells-based array, while the formation of 6β-hydroxytestosterone metabolite in the transfected cells was 714 and 55 pmol/mg protein/min, respectively. In addition, all the above expression levels in the transfected cell models could be further induced with additional treatment of Rifampicin, a specific inducer for CYP3A4. In conclusion, our study established a proof-of-principle example that genetic modification with chimeric hPXR-p53-AD could improve CYP3A4 metabolism ability in hepatic cell line.
Insights
Hepatoma cell lines struggle with drug metabolism prediction due to low CYP3A4 enzyme activity. Genetic modification using chimeric pregnane X receptor (PXR) constructs significantly enhanced CYP3A4 expression and drug metabolism in these cells.
Area of Science:
- Pharmacology
- Molecular Biology
- Hepatology
Background:
- Hepatoma cell lines often exhibit insufficient CYP3A4 enzyme expression and induction, limiting their utility in drug metabolism and toxicity studies.
- The pregnane X receptor (PXR) is crucial for regulating CYP3A4, but its promoter binding activity is diminished in hepatic cell lines.
Purpose of the Study:
- To engineer chimeric human PXR (hPXR) constructs by fusing the p53 activation domain (AD) to hPXR.
- To evaluate the efficacy of these chimeric hPXR constructs (hPXR-p53 and p53-hPXR) in enhancing CYP3A4 expression and function in the C3A hepatoma cell line.
Main Methods:
- Creation of chimeric hPXR constructs (hPXR-p53 and p53-hPXR) by appending the p53 AD.
- Transfection of C3A hepatoma cells with wild-type (WT) hPXR and chimeric hPXR constructs.
- Assessment of CYP3A4 reporter gene activity, mRNA and protein levels, and metabolic activity using testosterone as a substrate.
Main Results:
- Chimeric hPXR constructs (hPXR-p53 and p53-hPXR) significantly increased CYP3A4 reporter gene activity (5- and 9-fold) and mRNA levels (3.5- and 2.6-fold) compared to WT hPXR.
- Stable transfection with hPXR-p53-AD improved CYP3A4 gene (2-fold) and protein (1.5-fold) expression.
- Significant testosterone metabolism (formation of 6β-hydroxytestosterone) was detected in cells expressing chimeric hPXR, unlike WT hPXR cells.
- Rifampicin treatment further induced CYP3A4 expression in transfected cells.
Conclusions:
- Genetic modification using chimeric hPXR-p53-AD is a viable strategy to enhance CYP3A4 expression and metabolic capacity in hepatoma cell lines.
- This approach offers a promising tool for improving in vitro models for drug metabolism and toxicity prediction.
- The developed chimeric PXR system demonstrates improved drug-metabolizing enzyme activity, addressing limitations of current hepatoma cell models.
