Related Experiment Videos
Cytochrome P450 regulation by hepatocyte nuclear factor 4 in human hepatocytes: a study using adenovirus-mediated
R Jover1, R Bort, M J Gómez-Lechón
1Unidad de Hepatología Experimental, Centro de Investigación, Hospital Universitario La Fe, SVS, Valencia, Spain.
Abstract:
Hepatocyte nuclear factor 4 (HNF4) is a member of the nuclear receptor super-family that has shown activating effects on particular cytochrome P450 (CYP) promoters from several species. However, its role in the regulation of human CYPs in the liver is still poorly understood, as no comprehensive studies in human-relevant models have been performed. In the present study, we have investigated whether HNF4 plays a general role in the expression of 7 major CYP genes in primary cultured human hepatocytes. To this end, we developed an adenoviral vector for efficient expression of HNF4 antisense RNA. Transduction of human hepatocytes with the recombinant adenovirus resulted in a time-dependent increase in the antisense transcript, followed by a concomitant decrease in apolipoprotein C III mRNA (a target gene of HNF4). Specificity was confirmed by showing that increasing levels of HNF4 antisense RNA resulted in the reduction of HNF4 protein, whereas retinoic X receptoralpha-(RXRalpha), the closest homologous member of the nuclear receptor super-family, was unaffected. Analysis of CYP gene expression in human hepatocytes transfected with HNF4 antisense RNA revealed singular behaviors: (1) CYP3A4, CYP3A5, and CYP2A6 showed an important, dose-dependent down-regulation on blockage of HNF4 translation; (2) a moderate inhibition of CYP2B6, CYP2C9, and CYP2D6 expression was observed (40%-45% reduction); (3) the levels of CYP2E1 were not affected even in the absence of this transcription factor. In conclusion, using an original strategy (efficient antisense RNA expression vector), our study shows that HNF4 is a general regulator supporting the expression of major drug-metabolizing CYPs in human hepatocytes.
Insights
Hepatocyte nuclear factor 4 (HNF4) regulates major drug-metabolizing cytochrome P450 (CYP) genes in human liver cells. This study demonstrates HNF4
Area of Science:
- Hepatology
- Molecular Biology
- Pharmacology
Background:
- Hepatocyte nuclear factor 4 (HNF4) is a nuclear receptor with known effects on cytochrome P450 (CYP) gene promoters in various species.
- The precise role of HNF4 in regulating human liver CYP genes remains incompletely understood, lacking comprehensive studies in human-relevant models.
Purpose of the Study:
- To investigate the general role of HNF4 in the expression of seven major CYP genes in primary cultured human hepatocytes.
- To elucidate the regulatory function of HNF4 on human drug-metabolizing enzymes.
Main Methods:
- Development of an adenoviral vector for efficient expression of HNF4 antisense RNA.
- Transduction of primary human hepatocytes with the HNF4 antisense RNA vector.
- Analysis of CYP gene expression and HNF4 protein levels following antisense RNA expression.
Main Results:
- HNF4 antisense RNA expression led to a dose-dependent decrease in CYP3A4, CYP3A5, and CYP2A6 mRNA levels.
- Moderate inhibition (40%-45%) of CYP2B6, CYP2C9, and CYP2D6 expression was observed.
- CYP2E1 expression levels remained unaffected by HNF4 knockdown.
Conclusions:
- HNF4 acts as a general regulator supporting the expression of several major drug-metabolizing CYP genes in human hepatocytes.
- The study highlights differential regulation of CYP genes by HNF4, with CYP2E1 being an exception.
- An efficient antisense RNA expression vector strategy was successfully employed to study HNF4 function.