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  • Author: Ray-Jade Chen x
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Ray-Jade Chen, His-Chin Wu, Mu-Hsin Chang, Chao-Hung Lai, Yun-Chen Tien, Jin-Ming Hwang, Wu-Hsien Kuo, Fuu-Jen Tsai, Chang-Hai Tsai, Li-Mien Chen, Chih-Yang Huang and Chun-Hsien Chu

This study examines the role of IGF2/mannose 6-phosphate receptor (IGF2R) signaling in the signaling transduction regulation and cell apoptosis in H9c2 cardiomyoblast cells. However, it is difficult to recognize the distinct activation of IGF2 signaling without interfacing with IGFI receptor (IGF1R) after exposure to IGF2. Leu27IGF2, an analog of IGF2 that interacts selectively with the IGF2R, was used to specifically activate IGF2R signaling in this study. DNA fragmentation and TUNEL assay revealed that in contrast to IGF1 treatment preventing angiotensin II and AG1024-induced cell apoptosis, Leu27IGF2 appears to synergistically increase apoptosis in those cells. We further found cell apoptosis induction and an increase in the active form of caspase 3 in the treatment of cells with Leu27IGF2, but not IGF1. To detect the interaction between IGF2R and Gαq using the immunoprecipitation assay, we found that IGF2R could directly interact with Gαq and after treatment with Leu27IGF2 the binding ability of Gαq to IGF2R had increased. This sequentially resulted in the phosphorylation of phospholipase C-β, a key downstream modulator of Gαq, on serine 537. Moreover, disruption of the Gαq protein by small interferon RNA reduced the cell apoptosis induced by Leu27IGF2. Our findings demonstrate that IGF2R activation appears to induce cell apoptosis via Gαq-deriving signaling cascades and its effect is completely different from IGF1R survival signaling.

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Chuan-Chou Tu, V Bharath Kumar, Cecilia Hsuan Day, Wei-Wen Kuo, Su-Peng Yeh, Ray-Jade Chen, Chen-Rong Liao, Hsiao-Yu Chen, Fuu-Jen Tsai, Wen-Jun Wu and Chih-Yang Huang

Previous studies have reported that estrogen receptors (ERs) are expressed in normal human liver, chronic hepatitis, and benign hepatic tumor tissues. However, decreased expression of ERs can be observed in hepatocellular carcinoma (HCC) and the role of ERs in HCC is not fully understood. Thus, the present study aimed to investigate the molecular mechanism induced by the overexpression of ERα (ERα (ESR1)) in Hep3B cells. We first detected the induction of apoptosis in ER-negative Hep3B cells using DNA fragmentation assay and flow cytometry. We found that ERα and ERα plus 17β-estradiol treatment increased apoptosis in Hep3B cells. Additionally, western blotting showed increased expression of active caspase 3 and tumor necrosis factor α (TNFα (TNF)) in ER α-transfected cells. To further understand the importance of SP1-binding sites in the TNF α promoter, ERα-negative Hep3B cells were co-transfected with ER α and a wild-type TNFα plasmid or TNF α with deleted SP1 regions. Deletion of both distant and primal SP1 sites abolished the activity of ERα, and similar results were observed by blocking the expression of SP1 protein using mithramycin (MA). This result indicates that SP1 protein is essential for ERα-activated TNF α promoter activity. Co-immunoprecipitation assay further confirmed the binding interaction between ERα and SP1 in a ligand-dependent manner. In general, we demonstrate that the overexpression of ERα mediates apoptosis in ERα-negative Hep3B cells by the binding of ERα to SP1 protein. Additionally, this ERα–SP1 complex binds to the proximal and distal sites of the TNF α gene promoter and further induces the expression of active caspase 3 in a ligand-dependent manner.

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Mu-Hsin Chang, Wei-Wen Kuo, Ray-Jade Chen, Ming-Chin Lu, Fuu-Jen Tsai, Wu-Hsien Kuo, Ling-Yun Chen, Wen-Jun Wu, Chih-Yang Huang and Chun-Hsien Chu

The IGF-II/mannose 6-phosphate receptor (IGF2R) function in extracellular matrix (ECM) remodeling is known to occur as a result of transforming growth factor-β (TGF-β) activation and plasmin in the proteolytic cleavage level caused by the interaction between latent TGF-β and urokinase plasminogen activator receptor (uPAR) respectively. In one of our previous studies, we found IGF-II and IGF2R dose-dependently correlated with the progression of pathological hypertrophy remodeling following complete abdominal aorta ligation. However, how this IGF2R signaling pathway responds specifically to IGF-II and regulates the myocardial ECM remodeling process is unclear. We found that IGF2R was aberrantly expressed in myocardial infarction scars. The matrix metalloproteinase-9 (MMP-9) zymographic activity was elevated in H9c2 cardiomyoblast cells treated with IGF-II, but not IGF-I. Treatment with Leu27IGF-II, an IGF2R specifically binding IGF-II analog, resulted in significant time-dependent increases in the MMP-9, tissue-type plasminogen activator (tPA), and urokinase plasminogen activator (uPA); and a reduction in the tissue inhibitor of matrix metalloproteinases-2 (TIMP-2) protein expression. Furthermore, IGF2R expression inhibition by siRNA blocked the IGF-II-induced MMP-9 activity. We hypothesize that after IGF-II is bound with IGF2R, the resulting signal disrupts the balance in the MMP-9/TIMP-2 expression level and increases plasminogen activator (PAs) expression involved in the development of myocardial remodeling. If so, IGF2R signaling inhibition may have potential use in the development of therapies preventing heart fibrosis progression.