Mixed lineage kinase 3 (MLK3)-activated p38 MAP kinase mediates transforming growth factor-β-induced apoptosis in hepatoma cells

Although transforming growth factor beta1 (TGF-beta1) acts via the Smad signaling pathway to initiate de novo gene transcription, the TGF-beta1-induced MAPK kinase activation that is involved in the regulation of apoptosis is less well understood. Even though the p38 MAP kinase and c-Jun NH2-terminal kinases (JNKs) are involved in TGF-beta1- induced cell death in hepatoma cells, the upstream mediators of these kinases remain to be defined. We show here that the members of the mixed lineage kinase (MLK) family ( including MLK1, MLK2, MLK3, and dual leucine zipper-bearing kinase (DLK)) are expressed in FaO rat hepatoma cells and are likely to act between p38 and TGF-beta receptor kinase in death signaling. TGF-beta1 treatment leads to an increase in MLK3 activity. Overexpression of MLK3 enhances TGF-beta1-induced apoptotic death in FaO cells and Hep3B human hepatoma cells, whereas expression of the dominant-negative forms of MLK3 suppresses cell death induced by TGF-beta1. The dominant-negative forms of MLK1 and -2 also suppress TGF-beta1-induced cell death. In MLK3-overexpressing cells, ERK, JNKs, and p38 MAP kinases were further activated in response to TGF-beta1 compared with the control cells. In contrast, overexpression of the dominant-negative MLK3 resulted in suppression of TGF-beta1-induced MAP kinase activation and TGF-beta1-induced caspase-3 activation. We also show that only the inhibition of the p38 pathway suppressed TGF-beta1-induced apoptosis. These observations support a role for MLKs in the TGF-beta1-induced cell death mechanism.