Phosphoproteomic Study of Human Tubular Epithelial Cell in Response to Transforming Growth Factor-Beta-1-Induced Epithelial-to-Mesenchymal Transition

Abstract

Background: Transforming growth factor-β (TGF-β)-induced epithelial-to-mesenchymal transition (EMT) plays an important role in renal fibrosis and progression of chronic kidney disease (CKD). Phosphorylation of proteins is essential to TGF-β signaling. We applied isobaric tags for relative and absolute quantification (iTRAQ) technology to profile the phosphoproteins in tubular epithelial cells in response to TGF-β-induced EMT in order to further study molecular events. Methods: HK-2 cells were treated with TGF-β1 to induce EMT. The cells were divided into a control group (without TGF-β1 treatment) and a TGF-β1-treated group. Phosphoproteins from two groups were extracted and differentially labeled with iTRAQ reagents and processed by 2D-nano-HPLC-MS/MS. Validating of iTRAQ analysis was performed by western blot. Bioinformatic analysis was performed by on-line databases. Results: By iTRAQ-2D-nano-HPLC-MS/MS, 38 differentially expressed phosphoproteins were identified which included 19 up-regulated phosphoproteins and 19 down-regulated phosphoproteins. Western blot confirmed up-regulation of phosphorylated moesin and HSP90α. Bioinformatic analysis suggested that the majority of proteins were located in the nucleus and endoplasmic reticulum lumen. The phosphoproteins were categorized into 17 molecular function classifications. Nucleic acid binding protein, cytoskeletal protein and chaperone were the major categories of molecular function. A biological network was built to analyze interaction between up-regulated proteins. Conclusion: We demonstrate a TGF-β1-mediated post-transcriptional regulation of EMT in tubular epithelial cells. Phosphorylation of moesin and HSP90α might play a role in TGF-β-induced EMT.