Characterization of the RAS/RAF/ERK Signal Cascade as a Novel Regulating Factor in Alpha-Amanitin-Induced Cytotoxicity in Huh-7 Cells

The well-known hepatotoxicity mechanism resulting from alpha-amanitin (alpha-AMA) exposure arises from RNA polymerase II (RNAP II) inhibition. RNAP II inhibition occurs through the dysregulation of mRNA synthesis. However, the signaling pathways in hepatocytes that arise from alpha-AMA have not yet been fully elucidated. Here, we identified that the RAS/RAF/ERK signaling pathway was activated through quantitative phosphoproteomic and molecular biological analyses in Huh-7 cells. Bioinformatics analysis showed that alpha-AMA exposure increased protein phosphorylation in a time-dependent alpha-AMA exposure. In addition, phosphorylation increased not only the components of the ERK signaling pathway but also U2AF65 and SPF45, known splicing factors. Therefore, we propose a novel mechanism of alpha-AMA as follows. The RAS/RAF/ERK signaling pathway involved in aberrant splicing events is activated by alpha-AMA exposure followed by aberrant splicing events leading to cell death in Huh-7 cells.