METTL3 regulates rifampicin-induced CYP3A4 expression by activating PXR translation and nuclear import and stabilizing CYP3A4 mRNA.

The pregnane X receptor (PXR) activator rifampicin (RIF) plays a critical role in drug-drug interactions (DDIs) by inducing cytochrome P450 (CYP) 3A4 expression. Increasing evidence indicates that n6-methyladenosine (m6A) modification is involved in the regulation of CYP basal expression. Here, we showed its effect on RIF-induced CYP3A4 expression. This study found that m6A levels and methyltransferase-like 3 (METTL3) expression were upregulated in HepG2 and LS174T cells treated with RIF, as well as in mice treated with pregnenolone-16α-carbonitrile (PCN, a typical PXR activator in mice), associated with the expression of CYP3A4 and Cyp3a11 (mouse homolog of human CYP3A4). Specifically, knockdown or overexpression of METTL3 downregulated and upregulated the basal and RIF-induced expression of CYP3A4, respectively. Similar results were obtained in a mouse model with liver-specific knockdown of Mettl3 (homolog of human METTL3) treated with PCN, indicating the involvement of m6A methylation in RIF-induced CYP3A4 expression. Mechanistically, METTL3 promotes PXR nuclear translocation and protein translation, while potentially affecting CYP3A4 mRNA stability through its binding to CYP3A4 mRNA (enhanced by RIF), thereby contributing to RIF-induced upregulation of CYP3A4 expression. Functionally, we observed that METTL3 knockdown or treatment with the METTL3 inhibitor STM2457 reduced the cytotoxicity induced by RIF combined with ritonavir. In conclusion, this study identified a novel mechanism of m6A modification in the regulation of RIF-induced CYP3A4 expression, providing valuable insights into CYP3A4-mediated DDIs.