Kanamaru Yutaka, Nakao Atsuhito, Tanaka Yuichi, Inagaki Yutaka, Ushio Hiroko, Shirato Isao, Horikoshi Satoshi, Okumura Ko, Ogawa Hideoki, Tomino Yasuhiko
Division of Nephrology, Department of Internal Medicine, Juntendo University School of Medicine, Tokyo, Japan.
Nephron Exp Nephrol. 2003;95(1):e36-42. doi: 10.1159/000073022.
Transforming growth factor beta 1 (TGF-beta1) induces alpha2(I) collagen gene (COL1A2) expression in mesangial cells through physical and functional cooperation of Smad proteins and Sp1. A transcriptional coactivator, p300, is also suggested to play an important role in TGF-beta1/Smad signal transduction. However, the role of p300 in TGF-beta1/Smad-pathway-mediated transcriptional activation of the COL1A2 gene in mesangial cells is still obscure.
Endogenous p300 expression and its modulation by TGF-beta1 were evaluated by Western blotting and immunofluorescence. The physical interaction of p300 with Smad2/3 was examined by immunoprecipitation followed by Western blotting. The functional role of p300 in TGF-beta1/Smad-pathway-mediated COL1A2 transcription was investigated in cotransfection experiments using a COL1A2 promoter-luciferase reporter gene construct and p300 expression plasmids.
TGF-beta1 induced COL1A2 gene expression in cultured mouse mesangial cells which was blocked by overexpression of inhibitory Smad7. In addition, TGF-beta1-induced nuclear export of endogenous Smad7 was observed in mouse mesangial cells. Endogenous p300 was expressed in the nucleus of the cells. TGF-beta1 induced interaction of endogenous p300 with Smad2/3, and a dominant negative construct of p300 inhibited the TGF-beta1-induced COL1A2 expression in cultured mouse mesangial cells.
p300 may be involved in TGF-beta1/Smad-pathway-mediated type I collagen gene transcription in mouse mesangial cells. Our findings would reveal a molecular basis of TGF-beta1-induced type I collagen gene transcription in mouse mesangial cells.
转化生长因子β1（TGF-β1）通过Smad蛋白与Sp1的物理和功能协作诱导系膜细胞中α2（I）胶原基因（COL1A2）表达。转录共激活因子p300也被认为在TGF-β1/Smad信号转导中起重要作用。然而，p300在系膜细胞中TGF-β1/Smad通路介导的COL1A2基因转录激活中的作用仍不清楚。
通过蛋白质免疫印迹法和免疫荧光评估内源性p300表达及其受TGF-β1的调节。通过免疫沉淀后蛋白质免疫印迹法检测p300与Smad2/3的物理相互作用。在使用COL1A2启动子-荧光素酶报告基因构建体和p300表达质粒的共转染实验中研究p300在TGF-β1/Smad通路介导的COL1A2转录中的功能作用。
TGF-β1诱导培养的小鼠系膜细胞中COL1A2基因表达，而抑制性Smad7的过表达可阻断该表达。此外，在小鼠系膜细胞中观察到TGF-β1诱导的内源性Smad7核输出。内源性p300在细胞的细胞核中表达。TGF-β1诱导内源性p300与Smad2/3相互作用，p300的显性负性构建体抑制培养的小鼠系膜细胞中TGF-β1诱导的COL1A2表达。
p300可能参与小鼠系膜细胞中TGF-β1/Smad通路介导的I型胶原基因转录。我们的研究结果将揭示小鼠系膜细胞中TGF-β1诱导的I型胶原基因转录的分子基础。
