Abstract
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 TRα1 (HepG2-TRα1). 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-TRα1 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-TRα1 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.
Highlights
From the ‡Department of Biochemistry, School of Medicine, Chang-Gung University, Taoyuan, Taiwan 333; §Molecular Medicine Research Center, School of Medicine, Chang-Gung University, Taoyuan, Taiwan 333; ¶Department of Nursing, Chang-Gung University of Science and Technology, Taoyuan, Taiwan 333; ʈFirst Cardiovascular Division, Chang Gung Memorial Hospital, Taoyuan, Taiwan 333; **Medical Research Central, Chang Gung Memorial Hospital, Taoyuan, Taiwan 333
These results demonstrate for the first time that proteins involved in the urokinase plasminogen activator system, including plasminogen activator inhibitor-1 (PAI-1), urokinase plasminogen activator surface receptor (uPAR), and brain-specific serine protease 4 (BSSP4), are augmented in the extra- and intracellular space of T3-treated HepG2-thyroid hormone receptors (TRs)␣1 cells
We applied the stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative proteomic approaches to identify secreted proteins regulated by T3 and study their underlying physiological significance in hepatoma cell lines stably expressing wild-type TR
Summary
Cell Culture—Human hepatoma cells, HepG2, Huh, J7, and Mahlavu were routinely cultured at 37 °C in a humidified atmosphere of 95% air and 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS). For SILAC experiments, human HepG2-TR␣1 cells were maintained in SILAC medium comprising DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% dialyzed fetal bovine serum (Invitrogen), L-lysine and L-arginine (Sigma-Aldrich) or [13C6]-L-lysine, [13C6]-L-arginine (Isotec) added at a concentration of 0.1 g/L for light or heavy stable isotope labeling [28]. Quantitative Reverse Transcription Polymerase Chain Reaction (QRT-PCR)—Total RNA was extracted from T3-treated HepG2-TR␣1 cells using TRIzol reagent, as described previously [38]. HepG2-TR␣1 cells treated with 10 nM T3 for 24 h were cotransfected with 0.6 g DNA/well of pA3TK vector containing the PAI-1 promoter sequence and 0.3 g of SV plasmid, a -galactosidase expression vector (Clontech, Palo Alto, CA), in 24-well plates using the TurboFect in transfection reagent (Fermentas, Glen Burnie, MD) to determine the transcriptional activities of TREs within the PAI-1 promoter. The PAI-1 open reading frame was ligated into pcDNA 3.0 expression vector, and the resulting construct sequenced
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