Hui Ding1,2, Jinjun Chen1, Jingping Qin1, Ruhua Chen3, Zili Yi4. 1. College of Bioscience & Biotechnology, Hunan Agricultural University, Changsha, 410128, Hunan, China. 2. Department of Pulmonary and Critical Care Medicine, Yixing People Hospital, Affiliated to Jiangsu University, Yixing, 214200, Jiangsu, China. 3. Department of Pulmonary and Critical Care Medicine, Yixing People Hospital, Affiliated to Jiangsu University, Yixing, 214200, Jiangsu, China. staff252@yxph.com. 4. College of Bioscience & Biotechnology, Hunan Agricultural University, Changsha, 410128, Hunan, China. 923262842@qq.com.
Abstract
BACKGROUND: Although the morbidity and mortality rates associated with idiopathic pulmonary fibrosis (IPF) are high, there is still lack of powerful and precise therapeutic options for IPF. OBJECT: Through in vitro model, this study sought to determine whether binding of acetylated CCAAT/enhancer binding protein β (C/EBPβ) to alpha-smooth muscle actin (α-SMA) promoter could affect the activity of the latter as well as assess if it is essential for epithelial-to-mesenchymal transition (EMT) and extracellular matrix deposition in IPF. METHODS: The expression of EMT and C/EBPβ in A549 cells treated with transforming growth factor-beta (TGF-β) as pulmonary fibrotic model was detected by western blotting and qPCR. Collagen-I expression using ELISA was performed. The luciferase activity was used to examine the activity of C/EBPβ. Knockdown of C/EBPβ was performed by siRNA. We also investigated the effect of deacetylation of C/EBPβ on EMT using sirtuin 1 (SIRT1). The binding ability of C/EBPβ with α-SMA promoter was affirmed via chromatin immunoprecipitation (ChIP) and electrophoresis mobility shift assay (EMSA). The relationship between α-SMA and acetylated C/EBPβ was determined with co-immunoprecipitation (Co-IP). SiRNA-mediated knockdown of C/EBPβ in A549 cells attenuated TGF-β1-induced myofibroblast differentiation and ECM deposition. The extent of association between acetylated C/EBPβ and α-SMA promoter was dynamically monitored. RESULTS: It was confirmed that deacetylation of C/EBPβ in A549 cells successfully ameliorated TGF-β1-induced EMT, as shown by reduction in α-SMA expression and excessive collagen-I accumulation. CONCLUSION: The EMT and fibrotic effect of TGF-β1 is dependent on acetylated C/EBPβ-mediated regulation of α-SMA gene activity. Thus, C/EBPβ acetylation may play a central role in pulmonary fibrosis.
BACKGROUND: Although the morbidity and mortality rates associated with idiopathic pulmonary fibrosis (IPF) are high, there is still lack of powerful and precise therapeutic options for IPF. OBJECT: Through in vitro model, this study sought to determine whether binding of acetylated CCAAT/enhancer binding protein β (C/EBPβ) to alpha-smooth muscle actin (α-SMA) promoter could affect the activity of the latter as well as assess if it is essential for epithelial-to-mesenchymal transition (EMT) and extracellular matrix deposition in IPF. METHODS: The expression of EMT and C/EBPβ in A549 cells treated with transforming growth factor-beta (TGF-β) as pulmonary fibrotic model was detected by western blotting and qPCR. Collagen-I expression using ELISA was performed. The luciferase activity was used to examine the activity of C/EBPβ. Knockdown of C/EBPβ was performed by siRNA. We also investigated the effect of deacetylation of C/EBPβ on EMT using sirtuin 1 (SIRT1). The binding ability of C/EBPβ with α-SMA promoter was affirmed via chromatin immunoprecipitation (ChIP) and electrophoresis mobility shift assay (EMSA). The relationship between α-SMA and acetylated C/EBPβ was determined with co-immunoprecipitation (Co-IP). SiRNA-mediated knockdown of C/EBPβ in A549 cells attenuated TGF-β1-induced myofibroblast differentiation and ECM deposition. The extent of association between acetylated C/EBPβ and α-SMA promoter was dynamically monitored. RESULTS: It was confirmed that deacetylation of C/EBPβ in A549 cells successfully ameliorated TGF-β1-induced EMT, as shown by reduction in α-SMA expression and excessive collagen-I accumulation. CONCLUSION: The EMT and fibrotic effect of TGF-β1 is dependent on acetylated C/EBPβ-mediated regulation of α-SMA gene activity. Thus, C/EBPβ acetylation may play a central role in pulmonary fibrosis.
Authors: Marjolein E Blaauboer; Claire L Emson; Lars Verschuren; Marjan van Erk; Scott M Turner; Vincent Everts; Roeland Hanemaaijer; Reinout Stoop Journal: Matrix Biol Date: 2013-05-03 Impact factor: 11.583
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