FIGURE 5.

PEPCK plays a role in cAMP-induced steroidogenic gene expression. A, MA-10 cells were transfected with StAR-Luc (200 ng), 3β-HSD-Luc (200 ng), P450c17-Luc (200 ng), shPEPCK (200 ng), and pSuper vector (200 ng) for 24 h after transfection; MA-10 cells were serum-starved followed by cAMP (100 μm) treatment. Experiments were performed in triplicate, and data are expressed in relative luciferase units (RLU) and as the fold activation relative to the control. Data represent mean ± S.D. of three individual experiments. B, MA-10 cells were transfected with StAR-Luc (200 ng), 3β-HSD-Luc (200 ng), and P450c17-Luc (200 ng) for 12 h after transfection. MA-10 cells were serum-starved and pretreated with 3-MPA (1 and 10 μm) for 2 h, followed by treatment with cAMP (100 μm). Experiments were performed in triplicate, and data are expressed in relative luciferase units and as the fold activation relative to the control. Data represent mean ± S.D. of three individual experiments. C, MA-10 cells were transfected with shPEPCK (200 or 400 ng) for 24 h after transfection, followed by treatment with cAMP (100 μm) for 12 h. Total RNA was isolated for semiquantitative RT-PCR analysis. Data represent mean ± S.D. of three individual experiments. D, RNA was isolated from C samples for real time PCR analysis. PEPCK, StAR, P450scc, and 3β-HSD were evaluated. Data represent mean ± S.D. of three individual experiments. E, MA-10 cells were pretreated with 3-MPA (1, 5, and 10 μm) for 2 h, followed by treatment with cAMP (100 μm) for 12 h. Total RNA was isolated for semiquantitative RT-PCR analysis. Data represent mean ± S.D. of three individual experiments.