中华皮肤科杂志 ›› 2009, Vol. 42 ›› Issue (7): 463-466.

• 论著 • 上一篇    下一篇

内皮素对黑素瘤A375细胞转化生长因子-β1及磷酸化Smad 3蛋白表达的调节

黄巍1,方险峰2,陶娟2,林能兴3,陈宏翔4,刘业强5,李延6,杨井2,李艳秋7,涂亚庭3,黄长征2   

  1. 1. 武汉市皮肤病防治研究所
    2. 华中科技大学同济医学院附属协和医院皮肤科
    3. 武汉华中科技大学同济医学院附属协和医院皮肤科
    4. 华中科技大学同济医学院附属协和医院
    5. 华中科技大学同济医学院协和医院
    6. 武汉华中科技大学同济医学院附属同济医院皮肤科
    7. 湖北省武汉市协和医院皮肤科
  • 收稿日期:2008-07-01 修回日期:2008-07-20 出版日期:2009-07-15 发布日期:2009-07-08
  • 通讯作者: 黄巍
  • 基金资助:

    30671891;国家自然科学基金资助项目（编号）

Regulatory effect of endothelin on the expression of transformation growth factor-beta 1 and phosphorylated Smad 3 in A375 cells in vitro

  • Received:2008-07-01 Revised:2008-07-20 Online:2009-07-15 Published:2009-07-08

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摘要：

目的 探讨内皮素家族（ET）对恶性黑素瘤（MM）A375细胞转化生长因子beta1（TGF-β1）表达的差异性调节及对TGF-β信号通路蛋白Smad 3磷酸化水平的影响。方法 ELISA、RT-PCR技术测定不同浓度ET-1、ET-3（0、0.1、1、10、100 nmol/L）及其与内皮素受体阻断剂BQ123、BQ788联合干预下MM细胞TGF-β1在蛋白质和mRNA水平的表达。Western印迹法检测联合干预下A375细胞Smad 3蛋白磷酸化的表达。结果 ET-1对A375细胞（105细胞）TGF-β1蛋白表达的影响呈浓度依赖性上调，100 nmol/L浓度下达1289.38 ± 89.42 ng/L（P ＜ 0.05）；ET-3的作用与此相反，100 nmol/L浓度下表达仅为85.09 ± 9.37 ng/L（P ＜ 0.05）。BQ123明显阻断ET-1上调TGF-β1的效应（P ＜ 0.05），BQ788对此差异无统计学意义（P ＞ 0.05）；联合干预下，在TGF-β1 mRNA水平也表现出相同的趋势。Western印迹法检测磷酸化Smad 3蛋白（P-Smad 3）表达显示，ET-1显著上调P-Smad 3的表达（P ＜ 0.05），BQ123干预下其表达水平接近对照组（P ＞ 0.05），BQ788不能阻断其对P-Smad 3表达的上调（P ＞ 0.05）；ET-3则明显抑制P-Smad 3的表达（P ＜ 0.05）。结论 ET-1可诱导MM A375细胞TGF-β1的高表达，ET-3有相反的作用。ET受体-A介导ET-1 诱导的TGF-β通路信号蛋白Smad 3的磷酸化。

关键词: 恶性黑素瘤;内皮素;内皮素受体;转化生长因子beta1;磷酸化Smad3

Abstract:

Objective To investigate the effects of endothelin-1 （ET-1） and endothelin-3 （ET-3） on the expression of transformation growth factor-beta 1 （TGF-β1） and phosphorylation of Smad 3 in malignant melanoma cell line, A375. Methods Cultured A375 cells were classified into 5 groups, i.e. control group （no stimulation）, ET-1 group （stimulated with ET-1）, ET-1 + BQ123 group （treated with ET-1 and BQ123）, ET-1 + BQ788 group （treated with ET-1 and BQ788）, ET-3 group （stimulated with ET-3）, to receive different stimulation. The working concentrations were 0, 0.1, 1, 10, 100 nmol/L for ET-1 and ET-3, 10 μmol/L for BQ123 and BQ788. After another 12- and 24-hour culture, ELISA, RT-PCR and Western blot were used to detect the expression of TGF-β1 protein and mRNA as well as phosphorylated Smad 3 （P-Smad 3）. Results The expression of TGF-β1 in A375 cells was up-regulated by ET-1, but down-regulated by ET-3, and both of the effects were in a concentration-dependent manner. Under the stimulation with ET-1 and ET-3 of 100 nmol/L, the level of TGF-β1 reached 1289.38 ± 89.42 ng/L per 105 cells and 85.09 ± 9.37 ng/L per 105 cells, respectively, significantly different from that in unstimulated cells （both P < 0.05）. BQ123 significantly blocked the up-regulatory effect of ET-1 on the expression TGF-β1 protein（P < 0.05）, but BQ788 had no significant influence on the effect, so was the case with TGF-β1 mRNA. Western blot revealed that ET-1 significantly elevated the expression of P-Smad 3 in A375 cells （P < 0.05）, and the elevation was significantly inhibited by BQ123, but not by BQ788. The expression of P-Smad 3 was statistically decreased by ET-3 in A375 cells （P < 0.05）. Conclusions The expression of TGF-β1 could be enhanced by ET-1, but suppressed by ET-3. It is likely that endothelin receptor A mediates the phosphorylation of Smad 3 induced by ET-1.