关键词: 蛋白磷酸酶2A, 去磷酸化, 细胞外基质合成, EMT, Smad3中间连接区

Abstract:

【Abstract】 Objective: To observed the effect of PP2Ac on accumulation of extracellular matrix and EMT by PP2Ac overexpression plasmid and PP2Ac siRNA transfected with Human kidney proximal tubular epithelial cell line(HK-2) cells, and to explore the role of PP2Ac in tubulointersitial firosis. Methodology: 1. HK-2 cells were incubated and randomly divided into 3 groups including TGF-β1 group(treated with TGF-β1 5ng/ml for 24 h or 1 h), plasmid transfection plus TGF-β1 stimulation group（treated with TGF-β1 5ng/ml for 24 h or 1 h after HK-2 cells transfected with PP2Ac over expression or SiRNA plasmid）and control group (only FBS-free DMEM/F12) RT-PCR and Western blot were used to detect the mRNA and protein expression of PP2Ac, fibronectin, collagen I, α-SMA and E-cadherin. The expression of Smad3 and Smad3-L Ser204, and protein of Smad3-L Ser208 in nucleus were tested by Western blot. Results 1. RT-PCR and Western blot showed that the expression of PP2Ac was significantly increased in HK-2 cells stimulated with TGF-β1. Furthermore, fibronectin, collagen I and α-SMA expression was also increased while the E-cadherin expression was decreased. Compared with TGF-β1 group, PP2Ac expression was increased significantly by incubation with TGF-β1 after transfection of PP2Ac over expression plasmid. Moreover, the expression of fibronectin, collagen I and α-SMA was upregulated while E-cadherin was downregulated. Howeover, after transfected with the PP2Ac siRNA plasmid, fibronectin, collagen I and α-SMA expression was reduced, but E-cadherin was increased. 2. After HK-2 cells stimulated with TGF-β1 for 1 h, the expression of Smad3L Ser204 and protein of Smad3-L Ser208 in nucleus was elevated, both of which were furtherly downregulated when HK-2 cells were transfected with PP2Ac over expression plasmid. While they were upregulated when HK-2 cells were transfected with PP2Ac SiRNA plasmid compared with that in TGF-β1 stimulation group. Conclusion: PP2Ac promoted the accumulation of extracellular matrix and epithelial–mesenchymal transition (EMT) in HK2 cells, which could contribute to regulate the dephosphorylation of Smad3 linker region.

Key words: protein phosphatase 2A, accumulation of extracellular matrix , dephosphorylation, epithelial–mesenchymal transition (EMT), Smad3 link region