Role of Human Hepatocyte Nuclear Factor 4α in the Expression of Drug-Metabolizing Enzymes and Transporters in Human Hepatocytes Assessed by Use of Small Interfering RNA

Abstract

Hepatocyte nuclear factor 4alpha (HNF4alpha) is an important transcription factor in hepatic gene expression. Here, we have investigated the role of HNF4alpha in the expression of drug-metabolizing enzymes and transporters in human hepatocytes using an adenovirus expressing human HNF4alpha-small interfering RNA (hHNF4alpha-siRNA). The hHNF4alpha-siRNA effectively reduced the mRNA and nuclear protein levels of hHNF4alpha in a concentration-dependent manner. The hHNF4alpha-siRNA also decreased the mRNA levels of CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, UGT1A1, UGT1A9, SULT2A1, ABCB1, ABCB11, ABCC2, OATP1B1 and OCT1, as well as those of PXR and CAR. To discern the role of these nuclear receptors, we co-infected hepatocytes with hHNF4alpha-siRNA and PXR- or CAR-expressing adenovirus. The hHNF4alpha-siRNA-induced reductions of the enzyme and transporter mRNA levels were not restored except CYP2B6 mRNA levels, which were returned to the control level by overexpressing CAR. Furthermore, although hHNF4alpha-siRNA did not significantly affect the fold-induction of CYP2B6, CYP2C8, CYP2C9, or CYP3A4 mRNA levels following treatment with CYP inducers, the levels in hHNF4alpha-suppressed cells fell significantly compared to the control. These results suggest that HNF4alpha plays a dominant role in the expression of drug-metabolizing enzymes and transporters in human hepatocytes, and that HNF4alpha expression levels is a possible determinant for inter-individual variations in the expression of these enzymes and transporters.

Hepatocyte Nuclear Factor 4α Regulates Rifampicin-Mediated Induction ofCYP2CGenes in Primary Cultures of Human Hepatocytes

“…Our conclusions regarding the involvement of HNF4␣ in the PXRmediated induction of CYP2C8 and CYP2C9 differ somewhat from those of another laboratory (Kamiyama et al, 2007), which reported that adenoviral constructs expressing HNF4␣-siRNA had no effect on xenobiotic-mediated induction of CYP2C8 and CYP2C9 mRNA in cultures of primary hepatocytes. However, their interpretations were based on the observations that constitutive as well as xenobioticinduced levels of CYP2C8 and CYP2C9 mRNA were decreased by HNF4␣-siRNA; subsequently, the magnitude of the induction by CAR/PXR ligands remained unchanged in their hands.…”

“…PXR and CAR mRNA levels were moderately decreased (40 and 60%, respectively) by silencing HNF4␣, as also noted by Yamazoe and coworkers (Kamiyama et al, 2007). Although this decrease in PXR could conceivably contribute to the decreased response of CYP2C genes to the PXR ligand rifampicin, their study showed that infection of primary human hepatocytes with adenoviral constructs containing PXR or CAR simultaneously with siHNF4␣ restored CAR/ PXR levels but did not restore induction by xenobiotics (Kamiyama et al, 2007). Although mRNA levels of the coactivator PGC-1 were also decreased by 50% in our studies, those of SRC-1 and peroxisome proliferator-activated receptor-binding protein were not affected.…”

“…However, the newly identified HNF4␣-binding site at Ϫ211 bp in the CYP2C9 promoter is not present in the CYP2C19 promoter, and the absence of this site could possibly contribute to reports of a lack of HNF4␣ transactivation of the CYP2C19 promoter in cell lines (Kawashima et al, 2006). PXR and CAR mRNA levels were moderately decreased (40 and 60%, respectively) by silencing HNF4␣, as also noted by Yamazoe and coworkers (Kamiyama et al, 2007). Although this decrease in PXR could conceivably contribute to the decreased response of CYP2C genes to the PXR ligand rifampicin, their study showed that infection of primary human hepatocytes with adenoviral constructs containing PXR or CAR simultaneously with siHNF4␣ restored CAR/ PXR levels but did not restore induction by xenobiotics (Kamiyama et al, 2007).…”

“…Low levels of CYP2Cs are also found in extrahepatic tissues such as intestine, lung, kidney, and brain (Krishna and Klotz, 1994). Constitutive expression of CYP2C genes in the liver is believed to be under the control of endogenous regulatory signals such as HNF4␣ (Jover et al, 2001;Kamiyama et al, 2007), HNF3␥ (Bort et al, 2004), and the CCAAT/enhancer-binding protein ␣ (Jover et al, 1998). However, exposure to numerous structurally unrelated xenobiotics, including rifampicin, hyperforin, phenobarbital, and dexamethasone (Raucy et al, 2002;Madan et al, 2003;Komoroski et al, 2004), up-regulates CYP2C enzyme expression.…”

Transcriptional regulation of urate transportosome member SLC2A9 by nuclear receptor HNF4α

“…Interestingly, it has been previously reported that HNF4␣ is important for the above-mentioned PXR-mediated transactivation of CYP3A4 (45). In addition, HNF4␣ has been shown to be a major modulator of hepatic gene expression and to directly regulate several drug transporters and metabolizing enzymes, indicating its regulatory role in drug disposition gene expression (20).…”