Stimulatory effect of interleukin-1beta on growth hormone gene expression and growth hormone release from rat GH3 cells

Our previous studies demonstrated that interferon gamma increases the human (h) growth hormone (GH) gene promoter activity in rat pituitary GH3 cells, and its regulatory mechanism may be different from the classical GH-releasing hormone-induced regulatory mechanism. Interleukin-1beta (IL-1beta) is thought to induce the release of GH by pituitary cells, but whether or not and by which mechanisms IL-1beta regulates GH synthesis remains unclear. The purpose of our study was thus to investigate the effect of IL-1beta on the hGH gene expression in GH3 rat pituitary tumor cells using stable transfection of the hGH promoter fused to a luciferase reporter gene. Our results showed that IL-1beta (10-10(4) U/ml) increased GH secretion and synthesis and that 10(2) to 10(4) U/ml IL-1beta promoted the luciferase expression in stable GH3 cells, with a maximal action of 1.61 times over that of controls. Among inhibitors of intracellular signaling transduction pathways, mitogen-activated protein kinase kinase (MAPKK/MEK) inhibitor PD98059 (40 microM) and p38 MAPK inhibitor SB203580 (5 microM)blocked completely the stimulatory effect of IL-1beta, and the phosphoinositide 3-kinase inhibitor LY294002 (10 microM) blocked partially the induction of IL-1beta. Western blot analysis demonstrated that IL-1beta increased the activation of phosphorylated MEK and p38 MAPK in GH3 cells. Neither overexpression of Pit-1 nor inhibiting Pit-1 expression affected IL-1beta induction of hGH promoter activity. To identify the DNA sequence that mediated the effect of IL-1beta, six deletion constructs of hGH promoter were created. The stimulatory effect of IL-1beta was abolished following deletion of the -196- to -132-bp fragment. In conclusion, our data show that IL-1beta promotes GH secretion and synthesis by rat pituitary GH3 cells. The stimulatory effect of IL-1beta on the hGH gene promoter appears to require the activation of MEK, p38 MAPK, and phosphoinositide 3-kinase and a fragment of promoter sequence that spans the -196- to -132-bp fragment of the gene, but is unrelated to the Pit-1 protein.