The Effect Of DNA Methylation On The PXR-mediated CYP3A4 Transcriptional Activity

Research backgroundCYP3A4, which is abundant in human liver and intestine, is the most important phase-I enzymes contributing to catalyzes the metabolism of more than half of marketed drugs. It has been well-known for the variability in CYP3A4 expression and activity among individuals. Thus, to understand the function basis of CYP3A4 is helpful to optimize suitable dosages, improve therapeutic efficacy and reduce adverse response. Indeed, to clarify the molecular mechanism underlying the variability in CYP3A4 expression has great significance for clinical individualized medicine.Numbers of reports have demonstrated that genetic polymorphisms are associated with the individual differences of CYP3A4 expression, and more than50 allelic variants have been described for CYP3A4(http://www.cypalleles.ki.se/cyp3a4.htm, date accessed: 4-Jul-2013). However,racial differences in allele frequency of CYP3A4 gene have been reported, with frequencies of less than 1% in the populations studied, genetic factors can only summon a very small proportion of CYP3A4 variation. The expression of CYP3A4 can also be affected by non-genetic factors such as diet, age and sex. In addition, the pregnane X receptor plays a pivotal role in regulating CYP3A4 induction by xenobiotics and hormones. However, a large degree of CYP3A4 variation remains unexplained.In recent years, epigenetics, a cutting-edge field of genetics, is expected to answer a series of questions beyond genetics. Epigenetics investigates heritable changes in gene expression or cellular phenotype that are caused by mechanisms other than changes in the DNA sequence, potentially affected by many other factors, such as age, diet, life style, disease and environment. Several mechanisms of epigenetic regulation are the most studied epigenetic mechanisms in mammals,including DNA methylation, histone modifications, and non-coding RNAs.Although, there has been reported that DNA methylation plays a significant role in influencing CYP3A4, It still remain to be clarified the underlying molecular mechanism.To investigate the effect of DNA methylation on the expression of CYP3A4 and uncover the underlying molecular mechanism, here, we examined the expression of CYP3A4 and PXR in the Hep G2 cells after exposure to 5-Aza-2’-d C and transfection of PSG5-PXR expression plasmid. We also constructed the p Cp GL-CYP3A4-promoter and the p Cp GL-CYP3A4-EP plasmid based on p Cp GL luciferase reporter plasmid. Moreover, We examined the effect of Cp G methylation of the CYP3A4 5?UTR on the CYP3A4 promoter activity using the Dual luciferase assay system Thus, it may provide new insights into the variability in CYP3A4 expression and new strategy for the individual administration.MethodsMethods�'� The Hep G2 cell line was exposed by 5-Aza-2d C in terms of different concentration and active time. Then the CYP3A4 m RNA and PXR m RNA levels were determined by real-time PCR.�'� The cells were transiently transfected with PSG5-PXR expression plasmid or an empty PSG5 plasmid. After 6 hours, the cells were treated with 10μM 5-aza-d C for 96 hours, the CYP3A4 m RNA and PXR m RNA levels were determined by real-time PCR.�'� The pCpGL-CYP3A4-promoter plasmid generated contains the CYP3A45?UTR encompassing constructs spanning(–1675 to +60) ligated into the Cp G dinucleotide-free p Cp GL luciferase reporter plasmid. Plasmids were methylated using Sss I methylases cording to the manufacturer’s recommendation. The completeness of methylation was controlled by digesting both methylated and unmethylated DNA using methylation sensitive restriction enzymes Hpa II. According to the Lipofectamine TM reagents, the Hep G2 cells were transfected with the recombinant plasmid with/without PSG5-PXR, and internal control plasmids.The transfected plasmid were: p Cp GL-CYP3A4-promoter unmethylation, p Cp GL-CYP3A4-promoter methylation, PSG5-PXR,and the control p RL-TK Renilla vector. Luciferase and Renilla activity was assayed using the Dual luciferase assay system(Promega) three independent experiments were performed, located 3 wells.�'� The pCpGL-CYP3A4-EP plasmid generated contains the CYP3A4 5?UTR encompassing constructs spanning(–1675 to +60) and(-11185 to-7644)ligated into the Cp G dinucleotide-free p Cp GL luciferase reporter plasmid.Plasmids were methylated using Sss I methylases cording to the manufacturer’s recommendation. The completeness of methylation was controlled by digesting both methylated and unmethylated DNA using methylation sensitive restriction enzymes Hpa II. According to the Lipofectamine TM reagents, the Hep G2 cells were transfected with the recombinant plasmid with/without PSG5-PXR,and internal control plasmids.The transfected plasmid were:p Cp GL-CYP3A4-EP unmethylation, p Cp GL-CYP3A4-EP methylation, PSG5-PXR, and the control p RL-TK Renilla vector. Luciferase and Renilla activity was assayed using the Dual luciferase assay system(Promega) three independent experiments were performed, located 3 wells.Results1. The effect of 5-Aza-2-d C on the basal expression of PXR and CYP3A4 in the Hep G2 cells The low expression of PXR and CYP3A4 was significantly increased following treatment with 5-Aza-2-d C which showed concentration and time dependence in Hep G2 cells2. The effect of 5-Aza-2-d C treatment and transfection of PSG5-PXR expression plasmid on the expression of CYP3A4 in the Hep G2 cells.To investigate the effect of the DNA methylation status on the CYP3A4 expression, we exogenously expressed PXR in the Hep G2 cells. The PXR m RNA level was dramatically increased by the transfection of the PXR expression plasmid into the Hep G2 cells.In the 5-Aza-2-d C treatment and overexpression of PXR groups, the expression of CYP3A4 was 1.77 folds and 1.85 folds compared with PXR group and 5-Aza-2-d C group in the Hep G2 cells(P<0.05), respectively.3. Construction of p Cp GL-CYP3A4-promoter and p Cp GL-CYP3A4-EP plasmid and in vitro methylation of plasmid DNA.The correct insertion of all constructs was controlled by Agarose gel electrophoresis and sequencing. The completeness of methylation was controlled by digesting both methylated and unmethylated DNA using methylation sensitive restriction enzymes HpaII.4. The effect of methylation in the core-promoter region of the CYP3A4 on the PXR-mediated CYP3A4 transcriptional activity In the Hep G2 cells, the relative luciferase activities of p Cp GL-CYP3A4-promoter unmethylation, p Cp GL-CYP3A4-promoter unmethylation+PSG5-PXR,p Cp GL-CYP3A4-promoter methylation, p Cp GL-CYP3A4-promoter methylation+PSG5-PXR, p Cp GL-CYP3A4-promoter methylation+PSG5-PXR,were 5.01±1.10, 9.82±0.89,4.31±1.06 and 10.19±1.69, respectively. In the p Cp GLCYP3A4-promoter unmethylation groups, the PSG5-PXR group was 1.96 folds than the control group; In the p Cp GL-CYP3A4-promoter groups, the PSG5-PXR group was 2.36 folds than the control group. So, there was no significant difference between plasmid methylation groups and unmethylation groups. The results showed that, the methylation of CYP3A4 promoter had no significantly effect on the PXR-mediated CYP3A4 transcriptional activity.In the Hep G2 cells, the relative luciferase activities of p Cp GL-CYP3A4-EP unmethylation, p Cp GL-CYP3A4-EP unmethylation+PSG5-PXR, p Cp GLCYP3A4-EP methylation, p Cp GL-CYP3A4-EP methylation+PSG5-PXR, were88.57±6.06, 164.95±21.66,29.75±4.43 and 31.55±7.91, respectively. In the p Cp GL-CYP3A4-EP unmethylation groups, the PSG5-PXR group was 1.88 folds than the control group; In the p Cp GL-CYP3A4-EP methylation groups, the PSG5-PXR group was 1.05 folds than the control group. So, there was a significant difference between plasmid methylation groups and unmethylation groups.The results showed that, the methylation of CYP3A4 enhancer had significantly effect on the PXR-mediated CYP3A4 transcriptional activity.5. The effect of Cp G methylation in the distinct regulatory elements of the CYP3A4 on the PXR-mediated CYP3A4 transcriptional activityConclusions:1. The low expression of CYP3A4 is significantly increased following treatment with 5-Aza-2-d C and shown concentration and time dependence in Hep G2 cells.2. the methylation of CYP3A4 promoter has no significantly effect on the PXRmediated CYP3A4 transcriptional activity in Hep G2 cells.3. CpG methylation in the distinct regulatory elements of the CYP3A4 has significantly effect on the PXR-mediated CYP3A4 transcriptional activity in Hep G2 cells.