c-Jun N-Terminal Kinase 1 Promotes Transforming Growth Factor-β1-Induced Epithelial-to-Mesenchymal Transition via Control of Linker Phosphorylation and Transcriptional Activity of Smad3

Transforming growth factor (TGF)-beta 1 is a key mediator of lung remodeling and fibrosis. Epithelial cells are both a source of and can respond to TGF-beta 1 with epithelial-to-mesenchymal transition (EMT). We recently determined that TGF-beta 1-induced EMT in lung epithelial cells requires the presence of c-Jun N-terminal kinase (JNK) 1. Because TGF-beta 1 signals via Smad complexes, the goal of the present study was to determine the impact of JNK1 on phosphorylation of Smad3 and Smad3-dependent transcriptional responses in lung epithelial cells. Evaluation of JNK1-deficient lung epithelial cells demonstrated that TGF-beta 1-induced terminal phosphorylation of Smad3 was similar, whereas phosphorylation of mitogen-activated protein kinase sites in the linker regions of Smad3 was diminished, in JNK1-deficient cells compared with wild-type cells. In comparison to wild-type Smad3, expression of a mutant Smad3 in which linker mitogen-activated protein kinase sites were ablatedc aused a marked attenuation in JNK1 or TGF-beta 1-induced Smad-binding element transcriptional activity, and expression of plasminogen activator inhibitor-1, fibronectin-1, high-mobility group A2, CArG box-binding factor-A, and fibroblast-specific protein-1, genes critical in the process of EMT. JNK1 enhanced the interaction between Smad3 and Smad4, which depended on linker phosphorylation of Smad3. Conversely, Smad3 with phosphomimetic mutations in the linker domain further enhanced EMT-related genes and proteins, even in the absence of JNK1. Finally, we demonstrated a TGF-beta 1-induced interaction between Smad3 and JNK1. Collectively, these results demonstrate that Smad3 phosphorylation in the linker region and Smad transcriptional activity are directly or indirectly controlled by JNK1, and provide a putative mechanism whereby JNK1 promotes TGF-beta 1-induced EMT.