[The mechanism of interferon-gamma increases growth hormone expression in rat pituitary GH(3) cells].
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.
Department of Endocrinology, Peking Union Medical College Hospital, Chinese Academy Medical Science Peking Union Medical College, Beijing 100730, China. dengjy@csc.pumch.ac.cn
Feng-Ying
FY
Gong
Yi-Fan
YF
Shi
Jie-Ying
JY
Deng
D000818:Animals; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics; D015870:Gene Expression / Q000187:drug effects*; D017930:Genes, Reporter; D019382:Human Growth Hormone / Q000235:genetics*; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology*; D008156:Luciferases / Q000235:genetics; D020935:MAP Kinase Signaling System; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors; D010902:Pituitary Gland / Q000166:cytology; D011401:Promoter Regions, Genetic; D051381:Rats; D050817:Transcription Factor Pit-1; D014157:Transcription Factors / Q000235:genetics
D004268:DNA-Binding Proteins; C494187:POU1F1 protein, human; C494186:Pou1f1 protein, rat; D050817:Transcription Factor Pit-1; D014157:Transcription Factors; D019382:Human Growth Hormone; D007371:Interferon-gamma; D008156:Luciferases; D020928:Mitogen-Activated Protein Kinases