The Mechanism
of Interferonγ Increases Growth Hormone
Expression in Rat Pituitary GH3 Cells
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 )
Abstract The method
of luciferase reporter gene was used to investigate the effect of interferon-γ
(IFN-γ) on the activity of human growth hormone (hGH) gene promoter in rat
pituitary GH3 cells, to elucidate the post-receptor signal
transduction pathway and the key promoter sequence which mediated the action of
IFN-γ. The luciferase expression plasmid pGL3-484-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 GH3 cells. The changes of luciferase expression
in the GH3 cells were determined, after treatment with IFN-γ or
IFN-γ plus inhibitors of intracellular signaling pathways, to observe the
effect of IFN-γ and these inhibitors on the activity of hGH gene promoter. The
various deletion constructs of Luc reporter: pGL3-380-Luc,
pGL3-250-Luc, pGL3-132-Luc
and pGL3-66-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 GH3 cells, then the changes of luciferase
expression in the GH3 cells were assayed, after treatment with
IFN-γ, to find out the key sequence which mediated the action of IFN-γ. Our
results showed that IFN-γ (105 u/L, 106 u/L) could
increase luciferase expression in GH3cells transfected with pGL3-484-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 μmol/L) could completely blocked the stimulatory effect of IFN-γ on hGH gene promoter activity. Pit-1 overexpression or inhibited Pit-1 expression, both had no effect on IFN-γ-induced hGH gene promoter activity. When various deletion constructs of >Luc
reporter were transfected into GH3 cells, only pGL3380-Luc
and pGL3250-Luc still responded to IFN-γ. In
conclusion, IFN-γ could increase the activity of hGH gene promoter in rat
pituitary GH3 cells. This stimulatory effect of IFN-γ 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.
Key words interferon-γ; hGH gene promoter; GH3
cell line; luciferase reporter gene
*Corresponding author: Tel,
86-10-65295075; e-mail, [email protected]