Up-regulation of gonadotropin alpha-subunit gene by phosphatidylinositol 3-kinase inhibitors in clonal gonadotroph cells.

OBJECTIVE: Phosphatidylinositol-3 kinase (PI3-kinase) has been known to play an important role in cell survival and proliferation by activation of its downstream target, Akt/protein kinase B (PKB). In this present study, we investigated the effects of PI3-kinase inhibitors on gonadotropin alpha-subunit gene expression in pituitary gonadotrophs.

METHODS: Alpha T3-1 cells, a pituitary gonadotroph cell line, were used in this study. alphaT3-1 cells were transfected with alpha-subunit promoter region-linked luciferase vector, and stimulated with GnRH in the presence or absence of two different PI3-kinase inhibitors, LY 294002 and wortmannin. Dose response effects of these inhibitors were also examined. Extracellular signal-regulated kinase (ERK) phosphorylation were determined by western blotting analysis.

RESULTS: Treatment of alphaT3-1 cells with PI3-kinase inhibitor, LY 294002, significantly increased alpha-subunit gene expression up to 6.89 +/- 0.26-fold, and showed additive effect with gonadotropin-releasing hormone (GnRH). The increasing effect of LY 294002 on alpha-subunit gene expression was observed at the concentration more than 1 microM. The experiment using another PI3-kinase inhibitor, wortmannin, showed similar effects, where wortmannin alone increased alpha-subunit gene expression by dose dependent manner and showed additive effect with GnRH. The inhibitor of PKB failed to modulate basal activity of alpha-subunit promoter as well as GnRH-induced promoter activities. Western blotting analysis using phosphorylated form specific antibody for ERK demonstrated that both LY 294002 and wortmannin increased ERK phosphorylation.

CONCLUSION: These results suggested that PI3-kinase inhibitor, LY 294002 and wortmannin increased gonadotropin alpha-subunit gene expression related with ERK activation.