Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC)-based Quantitative Proteomics Study of a Thyroid Hormone-regulated Secretome in Human Hepatoma Cells

The thyroid hormone, 3, 3',5-triiodo-L-thyronine (T-3), regulates cell growth, development, differentiation, and metabolism via interactions with thyroid hormone receptors (TRs). However, the secreted proteins that are regulated by T-3 are yet to be characterized. In this study, we used the quantitative proteomic approach of stable isotope labeling with amino acids in cell culture coupled with nano-liquid chromatography-tandem MS performed on a LTQ-Orbitrap instrument to identify and characterize the T-3-regulated proteins secreted in human hepatocellular carcinoma cell lines overexpressing TR alpha 1 (HepG2-TR alpha 1). In total, 1742 and 1714 proteins were identified and quantified, respectively, in three independent experiments. Among these, 61 up-regulated twofold and 11 down-regulated twofold proteins were identified. Eight proteins displaying increased expression and one with decreased expression in conditioned media were validated using Western blotting. Real-time quantitative RT-PCR further disclosed induction of plasminogen activator inhibitor-1 (PAI-1), a T-3 target, in a time-course and dose-dependent manner. Serial deletions of the PAI-1 promoter region and subsequent chromatin immunoprecipitation assays revealed that the thyroid hormone response element on the promoter is localized at positions -327/-312. PAI-1 overexpression enhanced tumor growth and migration in a manner similar to what was seen when T-3 induced PAI-1 expression in J7-TR alpha 1 cells, both in vitro and in vivo. An in vitro neutralizing assay further supported a crucial role of secreted PAI-1 in T-3/TR-regulated cell migration. To our knowledge, these results demonstrate for the first time that proteins involved in the urokinase plasminogen activator system, including PAI-1, uPAR, and BSSP4, are augmented in the extra-and intracellular space of T-3-treated HepG2-TR alpha 1 cells. The T-3-regulated secretome generated in the current study may provide an opportunity to establish the mechanisms underlying T-3-associated tumor progression and prognosis. Molecular & Cellular Proteomics 11: 10.1074/mcp.M111.011270, 1-19, 2012.