Gong Feng-Ying, Shi Yi-Fan, Deng Jie-Ying
Department of Endocrinology, Peking Union Medical College Hospital, Chinese Academy Medical Science Peking Union Medical College, Beijing 100730, China.
Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai). 2002 Sep;34(5):619-24.
The method of luciferase reporter gene was used to investigate the effect of interferon-gamma (IFN-gamma) on the activity of human growth hormone (hGH) gene promoter in rat pituitary GH(3) cells, to elucidate the post-receptor signal transduction pathway and the key promoter sequence which mediated the action of IFN-gamma. The luciferase expression plasmid pGL(3)-484-Luc containing hGH gene promoter (-484-2 bp) and luciferase reporter gene were transfected alone or cotransfected with pituitary specific transcription factor Pit-1 expression plasmid (PcDNA-Pit-1-cDNA) or Pit-1 antisense oligonucleotide (Pit-1 OND) into rat GH(3) cells. The changes of luciferase expression in the GH(3) cells were determined, after treatment with IFN-gamma or IFN-gamma plus inhibitors of intracellular signaling pathways, to observe the effect of IFN-gamma and these inhibitors on the activity of hGH gene promoter. The various deletion constructs of Luc reporter: pGL(3)-380-Luc, pGL(3)-250-Luc, pGL(3)-132-Luc and pGL(3)-66-Luc, which contained the -380-2 bp, -250-2 bp, -132-2 bp and -66-2 bp sequences of hGH gene promoter, respectively, were transfected into GH(3) cells, then the changes of luciferase expression in the GH(3) cells were assayed, after treatment with IFN-gamma, to find out the key sequence which mediated the action of IFN-gamma. Our results showed that IFN-gamma (10(5) u/L, 10(6) u/L) could increase luciferase expression in GH(3)cells transfected with pGL(3)-484-Luc alone, the maximal action being 131% of the control (P<0.001). Among the inhibitors of intracellular signaling transduction pathways, only mitogen activated protein kinases (MAPK) inhibitor PD98059 (40 micromol/L) could completely blocked the stimulatory effect of IFN-gamma on hGH gene promoter activity. Pit-1 overexpression or inhibited Pit-1 expression, both had no effect on IFN-gamma-induced hGH gene promoter activity. When various deletion constructs of Luc reporter were transfected into GH(3) cells, only pGL(3)380-Luc and pGL(3)250-Luc still responded to IFN-gamma. In conclusion, IFN-gamma could increase the activity of hGH gene promoter in rat pituitary GH(3) cells. This stimulatory effect of IFN-gamma may be associated with the intracellular MAPK signaling pathway and with -252--132 bp sequence in hGH gene promoter, but had no relationship with pituitary specific transcription factor Pit-1.
采用荧光素酶报告基因方法，研究γ-干扰素（IFN-γ）对大鼠垂体GH(3)细胞中人生长激素（hGH）基因启动子活性的影响，以阐明受体后信号转导途径及介导IFN-γ作用的关键启动子序列。将含hGH基因启动子（-4842 bp）的荧光素酶表达质粒pGL(3)-484-Luc及荧光素酶报告基因单独转染，或与垂体特异性转录因子Pit-1表达质粒（PcDNA-Pit-1-cDNA）或Pit-1反义寡核苷酸（Pit-1 OND）共转染至大鼠GH(3)细胞。用IFN-γ或IFN-γ加细胞内信号通路抑制剂处理后，测定GH(3)细胞中荧光素酶表达的变化，以观察IFN-γ及这些抑制剂对hGH基因启动子活性的影响。将分别含hGH基因启动子-3802 bp、-2502 bp、-1322 bp和-662 bp序列的各种Luc报告基因缺失构建体：pGL(3)-380-Luc、pGL(3)-250-Luc、pGL(3)-132-Luc和pGL(3)-66-Luc转染至GH(3)细胞，用IFN-γ处理后，测定GH(3)细胞中荧光素酶表达的变化，以找出介导IFN-γ作用的关键序列。结果显示，IFN-γ（10^5 u/L、10^6 u/L）可使单独转染pGL(3)-484-Luc的GH(3)细胞中荧光素酶表达增加，最大作用为对照的131%（P<0.001）。在细胞内信号转导途径抑制剂中，只有丝裂原活化蛋白激酶（MAPK）抑制剂PD98059（40 μmol/L）可完全阻断IFN-γ对hGH基因启动子活性的刺激作用。Pit-1过表达或抑制Pit-1表达，均对IFN-γ诱导的hGH基因启动子活性无影响。将各种Luc报告基因缺失构建体转染至GH(3)细胞时，只有pGL(3)380-Luc和pGL(3)250-Luc仍对IFN-γ有反应。综上所述，IFN-γ可增加大鼠垂体GH(3)细胞中hGH基因启动子的活性。IFN-γ的这种刺激作用可能与细胞内MAPK信号通路及hGH基因启动子中的-252-~132 bp序列有关，但与垂体特异性转录因子Pit-1无关。
