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 and Peking Union Medical College, Beijing 100730, China.
Sheng Li Xue Bao. 2010 Feb 25;62(1):49-54.
The present study was aimed at investigating the effect of activin on the activity of human growth hormone (hGH) gene promoter in rat pituitary GH3 cells and the underlying molecular mechanism. The method of luciferase reporter gene was used. We firstly established a stable GH3 cell line which contains hGH gene promoter (-484 to 30 bp) and luciferase reporter gene by transfecting pGL3-484-Luc2 luciferase expression plasmid into GH3 cells using Lipofectamine transfection reagent. After treating these cells with activin or activin plus various signaling transduction activators, the concentration of GH in the medium and lysate of GH3 cells and luciferase activities in GH3 cells were measured. The results showed that activin (5 nmol/L, 50 nmol/L) decreased the secretion and synthesis of GH. The amounts of GH content in GH3 lysate and medium treated with 50 nmol/L activin were 82% and 59% of the control, respectively. Furthermore, activin (5, 50 nmol/L) reduced the luciferase expression in stable GH3 cells, with the expression being 77% and 69% of the control (P<0.001). Among the activators of intracellular signaling transduction pathways, mitogen-activated protein kinases kinase (MAPKK/MEK) activators C(6) ceramide (1 micromol/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 was abrogated following the deletion of the fragment from -132 to -66 bp within the hGH gene promoter. These results indicate that activin decreases the activity of hGH gene promoter in rat pituitary GH3 cells. The intracellular MEK dependent signaling pathway and the promoter sequence that spans the -132 to -66 bp fragment of hGH gene are involved in the inhibitory effect of activin.
本研究旨在探讨激活素对大鼠垂体GH3细胞中人生长激素（hGH）基因启动子活性的影响及其潜在的分子机制。采用荧光素酶报告基因法。我们首先使用脂质体转染试剂将pGL3 - 484 - Luc2荧光素酶表达质粒转染到GH3细胞中，建立了一个稳定的GH3细胞系，该细胞系包含hGH基因启动子（-484至30 bp）和荧光素酶报告基因。在用激活素或激活素加各种信号转导激活剂处理这些细胞后，测量了GH3细胞培养基和裂解物中GH的浓度以及GH3细胞中的荧光素酶活性。结果表明，激活素（5 nmol/L，50 nmol/L）降低了GH的分泌和合成。用50 nmol/L激活素处理的GH3裂解物和培养基中GH含量分别为对照的82%和59%。此外，激活素（5、50 nmol/L）降低了稳定的GH3细胞中荧光素酶的表达，其表达量分别为对照的77%和69%（P<0.001）。在细胞内信号转导途径的激活剂中，丝裂原活化蛋白激酶激酶（MAPKK/MEK）激活剂C(6)神经酰胺（1 μmol/L）完全消除了激活素的抑制作用。蛋白质免疫印迹分析进一步证实了GH3细胞中磷酸化MEK的抑制。在hGH基因启动子内缺失-132至-66 bp片段后，激活素的抑制作用被消除。这些结果表明，激活素降低了大鼠垂体GH3细胞中hGH基因启动子的活性。细胞内MEK依赖性信号通路以及跨越hGH基因-132至-66 bp片段的启动子序列参与了激活素的抑制作用。
