Figure 8.

Effect of wortmannin on GnRH-iduced gonadotropin subunit promoter activities and PI3K-responsive regions in αGSU-promoter. αT3-1 cells were transiently transfected with αGSU-Luc (A) and LβT2 cells were transiently transfected with LHβ-Luc (B) and FSHβ-Luc (C) promoter constructs before being pretreated for 1 h with wortmannin (10nM) followed by addition of GnRH (100nM) for 6 h. Cells were harvested and assayed for an increase in reporter gene expression expressed as relative light units (RLU), after normalization of transfection efficiency with an internal control. One-way ANOVA determined that ***p-val<0.001; **p-val<0.01 and *p-val<0.05 were significantly different between treatment groups. D. Subconfluent αT3-1 cells were transfected with 5′-deletion mutants of h-846αGSU-Luc for 8 h. Cells were washed several times and incubated for 36 h. Cells were preincubated with or without wortmannin (10nM) followed by GnRH treatment (100nM) for 6 h. αGSU-Luc activity was determined as described in Materials and Methods. PGBE, pituitary glycoprotein basal element; αBE, α basal elements; GSE, glycoprotein specific element; URE, upstream response element; CRE, cAMP response element; JRE, junctional response element; TSS, transcription start site.