Transforming growth factor (TGF)-beta is a pro-oncogenic cytokine that induces the epithelial-mesenchymal transition (EMT), a crucial event in tumor progression. During TGF-beta-mediated EMT in NMuMG mouse mammary epithelial cells, we observed sustained increases in reactive oxygen species (ROS) levels in the cytoplasm and mitochondria with a concomitant decrease in mitochondrial membrane potential and intracellular glutathione levels. In pseudo rho 0 cells, whose respiratory chain function was impaired, the increase in intracellular ROS levels was abrogated, suggesting an important role of mitochondrial activity as a trigger for TGF-beta-stimulated ROS generation. In line with this, TGF-beta-mediated expression of the EMT marker fibronectin was inhibited not only by chemicals that interfere with ROS signaling but also by exogenously expressed mitochondrial thioredoxin (TXN2) independent of Smad signaling. Of note, TGF-beta-mediated induction of HMGA2, a central mediator of EMT and metastatic progression, was similarly impaired by TXN2 expression, revealing a novel mechanism involving a thiol oxidation reaction in mitochondria, which regulates TGF-beta-mediated gene expression associated with EMT. (C) 2013 Elsevier Inc. All rights reserved.