Temporal Quantitative Proteomics by iTRAQ 2D-LC-MS/MS and Corresponding mRNA Expression Analysis Identify Post-Transcriptional Modulation of Actin-Cytoskeleton Regulators During TGF-β-Induced Epithelial-Mesenchymal Transition
journal contributionposted on 02.01.2009, 00:00 by Venkateshwar G. Keshamouni, Pratik Jagtap, George Michailidis, John R. Strahler, Rork Kuick, Ajaya Kumar Reka, Panagiotis Papoulias, Rashmi Krishnapuram, Anjaiah Srirangam, Theodore J. Standiford, Philip C. Andrews, Gilbert S. Omenn
To gain insights into how TGF-β regulates epithelial-mesenchymal transition (EMT), we assessed the time course of proteins and mRNAs during EMT by multiplex iTRAQ labeling and 2D-LC-MS/MS, and by hybridization, respectively. Temporal iTRAQ analysis identified 66 proteins as differentially expressed during EMT, including newly associated proteins calpain, fascin and macrophage-migration inhibitory factor (MIF). Comparing protein and mRNA expression overtime showed that all the 14 up-regulated proteins involved in the actin-cytoskeleton remodeling were accompanied by increases in corresponding mRNA expression. Interestingly, siRNA mediated knockdown of cofilin1 potentiated TGF-β-induced EMT. Further analysis of cofilin1 and β-actin revealed an increase in their mRNA stability in response to TGF-β, contributing to the observed increase in mRNA and protein expression. These results are the first demonstration of post-transcriptional regulation of cytoskeletal remodelling and a key role for cofilin1 during TGF-β-induced EMT.