TY - JOUR
T1 - Stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative proteomics study of a thyroid hormone-regulated secretome in human hepatoma cells
AU - Chen, G. Yi
AU - Chi, Lang Ming
AU - Chi, Hsiang Cheng
AU - Tsai, Ming Ming
AU - Tsai, Chung Ying
AU - Tseng, Yi Hsin
AU - Lin, Yang Hsiang
AU - Chen, Wei Jan
AU - Huang, Ya Hui
AU - Lin, Kwang Huei
PY - 2012/4
Y1 - 2012/4
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=84859807729&partnerID=8YFLogxK
U2 - 10.1074/mcp.M111.011270
DO - 10.1074/mcp.M111.011270
M3 - 文章
C2 - 22171322
AN - SCOPUS:84859807729
SN - 1535-9476
VL - 11
JO - Molecular and Cellular Proteomics
JF - Molecular and Cellular Proteomics
IS - 4
ER -