Pregnane X receptor is required for interleukin-6-mediated down-regulation of cytochrome P450 3A4 in human hepatocytes

Abstract

Cytochrome P450 3A4 (CYP3A4) is the most abundant cytochrome P450 enzyme in human liver and metabolizes more than 60% of prescribed drugs in human body. Patients with liver conditions such as cirrhosis show increased secretion of cytokines (e.g., interleukin-6) and decreased capacity of oxidation of many drugs. In this study, we provided molecular evidence that cytokine secretion directly contributed to the decreased capacity of oxidative biotransformation in human liver. After human hepatocytes were treated with IL-6, the expression of CYP3A4 decreased at both mRNA and protein levels, so did the CYP3A4 enzymatic activity. Meanwhile, the repression of CYP3A4 by IL-6 occurred after the decrease of pregnane X receptor (PXR) in human hepatocytes. The PXR-overexpressed cells (transfected with human PXR) increased the CYP3A4 mRNA level, and the repression of CYP3A4 by IL-6 was greater in the PXR-overexpressed cells than in the control cells. Further, PXR knockdown (transfected with siPXR construct) decreased the CYP3A4 mRNA level with less repression by IL-6 than in the control cells transfected with corresponding vector. Collectively, our study suggests that PXR is necessary for IL-6-mediated repression of the CYP3A4 expression in human hepatocytes.

Fig. 1. Repression effects of IL-6 on CYP3A4 expression and enzymatic activity in primary human hepatocytes and HepG2 cells

A, effect of IL-6 treatment on the level of CYP3A4 mRNA in five donors’s primary hepatocytes. Human primary hepatocytes were treated with IL-6(10ng/ml) or the same volume of PBS for 24 h. Total RNA was isolated and subjected to the qRT-PCR analysis for the level of CYP3A4 mRNA probe as described under Materials and Methods. The qPCR Cts were 24 for CYP3A4 and 20 for GAPDH. B, effects of IL-6 treatment on the CYP3A4 enzymatic activity (top) and the CYP3A4 protein expression (bottom) in human primary hepatocytes. C, effects of IL-6 treatment on the CYP3A4 enzymatic activity (top) and the CYP3A4 protein expression (bottom) in HepG2 cell. Human hepatocytes or HepG2 cells were treated with IL-6(10ng/ml) or the same volume of PBS for 48 h, and cell lysates or microsomes were prepared and assayed for the activity of CYP3A4 as described in Materials and Methods. To determined the content of CYP3A4 protein, human hepatocytes lysates(8µg) or HepG2 cell microsomes(20µg) was subjected to Western analyses with an antibody against CYP3A4 or GAPDH. All the experiments were repeated at least three times, and the data were expressed as mean ± S.D. * p<0.05, a statistically significant decrease by IL-6 treatment.

Fig. 2. Transcriptional involvement in suppression of CYP3A4 by IL-6

A, effect of DRB on the suppression of CYP3A4 mRNA in primary human hepatocytes. Human hepatocytes were treated with 10ng/ml IL-6 for 9 h in the absence or presence of 5µM DRB. Total RNA was prepared and analyzed for the levels of CYP3A4 and GAPDH by qRT-PCR. The qPCR Cts were 24 for CYP3A4 and 20 for GAPDH. B, repression of CYP3A4-DP-Luc promoter reporter. HepG2 cells were transiently transfected by FuGENE HD with a mixture containing 50 ng of CYP3A4-DP-Luc, along with 5 ng of the Null-Renilla reniformis luciferase plasmid in the presence and absence of PXR. The transfected cells were treated with 10 ng/ml IL-6 or the same volume of PBS for 24 h. Luciferase activities were determined with a Dual-Luciferase reporter assay system, and the reporter activity was normalized based on the Null-Renilla reniformis luminescence signal. C, differential repression of CYP3A4-DP-Luc and CYP3A4-P-Luc reporters mediated by IL-6 in the presence of PXR. HepG2 cells were transfected and treated with various concentrations of IL-6(0–10ng/ml) for 24 h. The reporter activities were determined as described above. All the experiments were repeated at least three times, and the data were expressed as mean ± S.D. p<0.05, a statistically significant decrease by IL-6 treatment.

Fig. 3. Repression effect of IL-6 on PXR expression in primary human hepatocytes

A, a time-course effect of IL-6 on the levels of PXR and CYP3A4 in primary human hepatocytes. Human hepatocytes were treated with IL-6(10ng/ml) or the same volume of PBS for 24 h. Total RNA was isolated and subjected to the qRT-PCR analysis for the levels of PXR and CYP3A4. The qPCR Cts were 25 for PXR, 24 for CYP3A4 and 20 for GAPDH. B, repression effect of IL-6 on the PXR protein level in primary hepatocytes. Human hepatocytes were treated with IL-6(10ng/ml) or the same volume of PBS for 48 h, and cell lysates(10µg) were prepared and analyzed by Western blotting. All the experiments were repeated at least three times, and the data were expressed as mean ± S.D. p<0.05, a statistically significant decrease by IL-6 treatment.

Fig. 4. Suppression of CYP3A4 as a function of PXR

A, effect of SiPXR on the suppression of CYP3A4. HepG2 cells were transiently transfected with the siPXR construct or the corresponding vector for 72 h, and the transfected cells were treated with IL-6(10ng/ml) or the same volume of PBS for 24 h. Total RNA was prepared and analyzed for levels of CYP3A4, PXR and GAPDH by qRT-PCR and RT-PCR. B, effect of PXR overexpression on the repression of CYP3A4 mediated by IL-6. The same procedure as the above was used for overexpression experiment except for the replacement siPXR construct with the human PXR construct or the corresponding vector for 48h. The qPCR Cts were 27 for PXR, 32 for CYP3A4 and 16 for GAPDH. All the experiments were repeated at least three times, and the data were expressed as mean ± S.D. p<0.05, a statistically significant decrease by IL-6 treatment.

Fig. 5. The repression of wild type and 15 natural human PXR variants mediated by IL-6

HepG2 cells were transiently transfected with a mixture containing 50 ng of CYP3A4-DP-Luc, 50 ng wide type or nature human PXR variant, along with 5ng of the Null-Renilla reniformis luciferase plasmid. The transfected cells were treated with 10ng/ml IL-6 or the same volume of PBS for 24 h. Luciferase activities were determined as described in the above. To determine whether the expression is altered with certain variants, cell lysates(8µg) were analyzed by Western blotting(Anti-hPXR) for the expression level(bottom). Three independent experiments were performed, and the data were expressed as mean ± S.D.