Fig. 4. Effect of PG on GnRHR Regulation.
A, Effect of PG on GnRH-induced GnRHR promoter activity. LβT2 cells were transiently transfected with control pGL3 or GnRHR-Luc, pretreated for 30 min with PGE2, iloprost or PGF2α (500 nm) followed by addition of GnRH (100 nm) and continuous PG treatment for 4 h. Luciferase activity was measured, corrected for transfection efficiency and expressed as relative light units (RLU×103). Basal levels of pGL3 and GnRHR-Luc were not detectable (ND) after PG treatment, but there was no effect of PG treatment on the cotransfected control CMV-β-galactosidase plasmid. Fold stimulation was calculated relative to GnRH treated and a representative experiment of the three performed, each done in triplicate is shown. B, Effect of PG on GnRH binding. LβT2 cells were pretreated for 30 min with PGE2, iloprost or PGF2α (500 nm) followed by a competitive binding assay for GnRH in the presence and absence of PGE2, PGF2α or iloprost (500 nm) as detailed in Materials and Methods. A representative experiment of the three performed is shown. C and D, Effect of the phospholipase C inhibitor, U73122 on GnRH-induced GnRHR promoter activity and phosphoinositide turnover. C, LβT2 cells were transiently transfected with GnRHR-Luc, pretreated with U73122 for 30 min, followed by a further addition of GnRH (100 nm for 4 h). Luciferase activity was measured, corrected for transfection efficiency and expressed as relative light units (RLU×103). D, LβT2 cells were pretreated with U73122 for 30 min, followed by stimulation with GnRH (100 nm for 30 min) and total inositol phosphate (InsP) production was determined. A representative experiment of the three preformed, each done in triplicate is shown. E, Effect of PG on GnRH-induced phosphoinositide turnover. LβT2 cells were pretreated for 30 min with PGE2, iloprost or PGF2α (500 nm) followed by a 30-min incubation with increasing doses of GnRH in the presence and absence of PGE2, PGF2α or iloprost (500 nm), and total inositol phosphates (InsP) production was determined as detailed in Materials and Methods. A representative experiment of the three preformed, each done in triplicate is shown. One-way ANOVA determined that ***, P < 0.001; **, P < 0.01; and *, P < 0.05 were significantly different between treatment groups.