Abstract
TGF-β1 is a key fibrotic factor mediating epithelial mesenchymal transition (EMT) of epithelial cells through various signaling pathways. However, roles of proteolytic cleavage and endogenous peptide dynamics in TGF-β1-induced EMT remain unknown. We therefore performed quantitative peptidomics of TGF-β1-induced EMT in renal tubular epithelial cells. The acquired mesenchymal characteristics were confirmed, including morphological change (from cobblestone-like to fibroblast-like), decreased epithelial marker (ZO-1), and increased mesenchymal marker (vimentin). Quantitative peptidomics using stable isotope labeling revealed significantly altered levels of 70 unique endogenous peptides (derived from internal and C-terminal parts of 39 unique precursor proteins) after EMT induction. Interestingly, the majority of these peptides were derived from non-short-lived proteins, and analysis of P1 position revealed predominance of hydrophobic residues, suggesting that these endogenous peptides were generated mainly from proteasome cleavage. This hypothesis was confirmed by treating the cells with MG132 (a proteasome inhibitor), which provided almost identical endogenous peptide pattern as of the TGF-β1-treated cells. Moreover, validation assay showed marked reduction of proteasome peptidase activity in both TGF-β1-treated and MG132-treated cells. This is the first peptidome dataset that provides several novel aspects of mechanisms for TGF-β1-induced EMT. Our data also suggest that TGF-β1 exerts inhibitory effect against proteasome activity during EMT induction.