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活化素抑制大鼠垂体GH3细胞中人生长激素基因启动子的活性

龚凤英, 邓洁英, 朱惠娟, 潘慧

中国医学科学院&； 北京协和医院内分泌科，卫生部内分泌重点实验室，北京 100730

摘要

本研究旨在探讨活化素(activin)对大鼠垂体GH3 细胞中人生长激素(hGH)基因启动子活性的影响及其可能的调节机制。采用荧光素酶报告基因方法。首先建立含hGH 基因启动子(-484~+30 bp)和荧光素酶融合基因的稳定转染GH3 细胞株，然后加入活化素或同时加入活化素与相关信号转导途径的激动剂，通过检测细胞培养液和细胞裂解液中GH 的含量，以及GH3 细胞内荧光素酶的变化，反映活化素对GH 分泌、合成和hGH 基因启动子活性的影响。将含不同长度hGH 基因启动子序列的荧光素酶表达质粒分别转染GH3 细胞，观察它们对活化素的反应，寻找活化素影响hGH 基因启动子活性的关键DNA序列。结果表明，活化素(5, 50 nmol/L)能抑制大鼠垂体GH3 细胞中GH 的分泌和合成，活化素(5, 50 nmol/L)还能够抑制GH3 细胞中hGH 基因启动子的活性，使之仅达对照组的77% 和69%；在胞内信号转导激动剂中，丝裂原活化蛋白激酶激酶(MAPKK/MEK)特异性激动剂C6 ceramide (1 μmol/L)完全取消了活化素对hGH 基因启动子活性的抑制作用；活化素发挥抑制作用所需要的hGH 基因启动子关键序列位于-132~-66 bp 之间。上述研究表明，活化素能抑制大鼠垂体GH3 中hGH基因启动子的活性，它可能是通过抑制细胞内依赖MAPK 的信号转导途径来完成的，同时hGH 启动子上-132~-66 bp 的序列在其中发挥重要的作用。

关键词： 活化素; 生长激素; 启动子; GH3 细胞; 丝裂原活化蛋白激酶激酶

分类号：R322.5+4

[Activin inhibits the promoter activity of human growth hormone gene in rat pituitary GH3 cells.] [Ariticle in Chinese]

GONG Feng-Ying, DENG Jie-Ying, ZHU Hui-Juan, PAN Hui

Department of Endocrinology, Key Laboratory of Endocrinology of Ministry of Health, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences &； Peking Union Medical College, Beijing 100730, China

Abstract

The present study was aimed at investigating the effect of activin on the activity of human growth hormone (hGH) genepromoter in rat pituitary GH3 cells and the underlying molecular mechanism. The method of luciferase reporter gene was used. Wefirstly established a stable GH3 cell line which contains hGH gene promoter (-484 to 30 bp) and luciferase reporter gene by transfectingpGL3-484-Luc2 luciferase expression plasmid into GH3 cells using Lipofectamine transfection reagent. After treating these cells withactivin or activin plus various signaling transduction activators, the concentration of GH in the medium and lysate of GH3 cells andluciferase activities in GH3 cells were measured. The results showed that activin (5 nmol/L, 50 nmol/L) decreased the secretion andsynthesis of GH. The amounts of GH content in GH3 lysate and medium treated with 50 nmol/L activin were 82% and 59% of thecontrol, respectively. Furthermore, activin (5, 50 nmol/L) reduced the luciferase expression in stable GH3 cells, with the expressionbeing 77% and 69% of the control (P<0.001). Among the activators of intracellular signaling transduction pathways, mitogen-activatedprotein kinases kinase (MAPKK/MEK) activators C6 ceramide (1 μmol/L) abolished completely the inhibitory effect of activin.Western blot analysis further confirmed the inhibition of phosphorylated MEK in GH3 cells. The inhibitory effect of activin wasabrogated following the deletion of the fragment from -132 to -66 bp within the hGH gene promoter. These results indicate that activindecreases the activity of hGH gene promoter in rat pituitary GH3 cells. The intracellular MEK dependent signaling pathway and thepromoter sequence that spans the -132 to -66 bp fragment of hGH gene are involved in the inhibitory effect of activin.

Key words: activin; growth hormone; promoter; GH3 cells; MAPKK/MEK

收稿日期：2009-10-23　　录用日期：2010-01-06

通讯作者：龚凤英　　E-mail: fygong5074@yahoo.com.cn

引用本文：

龚凤英, 邓洁英, 朱惠娟, 潘慧. 活化素抑制大鼠垂体GH3细胞中人生长激素基因启动子的活性[J]. 生理学报 2010; 62 (1): 49-54.

GONG Feng-Ying, DENG Jie-Ying, ZHU Hui-Juan, PAN Hui. [Activin inhibits the promoter activity of human growth hormone gene in rat pituitary GH3 cells.] [Ariticle in Chinese] . Acta Physiol Sin 2010; 62 (1): 49-54 (in Chinese with English abstract).