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. 2012 Oct 16;287(50):41875–41887. doi: 10.1074/jbc.M112.421552

FIGURE 4.

FIGURE 4.

AMPK negatively regulates LRH-1-mediated steroidogenic gene promoter activities and gene expression. A, MA-10 cells were transfected with StAR-Luc (200 ng), P450c17-Luc (200 ng), and 3β-HSD-Luc (200 ng), respectively. Twenty four hours after the transfection, MA-10 cells were cultured in 0.5% charcoal-stripped horse serum and glucose-free medium supplemented with 20 mmol/liter sodium lactate, 1 mmol/liter sodium pyruvate, and 15 mmol/liter HEPES, followed by cAMP treatment at the indicated concentrations for 12 h. Experiments were performed in triplicate, and data are expressed in relative luciferase units (RLU) and as the fold activation relative to the control, representing mean ± S.D. of three individual experiments. B, MA-10 cells were transfected with several deletion constructs of StAR-Luc (200 ng) with or without CA-AMPK and treated with 8-Br-cAMP as indicated for 24 h. Experiments were performed in triplicate, and data are expressed in relative luciferase units (RLU) and as the fold activation relative to the control, representing mean ± S.D. of three individual experiments. *, p < 0.001 compared with untreated control and cAMP treated cells. C, HeLa cells were transfected with Sft4-Luc (200 ng), LRH-1 (200 ng), and CA-AMPK (100 or 200 ng) and were then treated with AICAR (100 or 400 μm). Experiments were performed in triplicate, and data are expressed in RLU and as the fold activation relative to the control. Data represent mean ± S.D. of three individual experiments. D, MA-10 cells were transfected with StAR-Luc (200 ng), P450c17-Luc (200 ng), and LRH-1 (200 ng) as indicated, with or without AICAR (400 μm). Experiments were performed in triplicate, and data are expressed in RLU and as the fold activation relative to the control. Data represent mean ± S.D. of three individual experiments. E and F, MA-10 cells were infected with the LRH-1 adenovirus (30 m.o.i.) for 24 h followed by AICAR (100 and 400 μm) treatment for 12 h or adenovirus CA-AMPK overexpression (10 or 30 m.o.i.). Total RNA was isolated for semiquantitative RT-PCR analysis. Data represent mean ± S.D. of three individual experiments. G, MA-10 cells were transfected with Sft4-Luc (200 ng), LRH-1 (200 ng), and shPEPCK (200 ng) and treated with 3-MPA (10 or 20 μm) or 8-Br-cAMP (100 μm). Experiments were performed in triplicate, and data are expressed in RLU and as the fold activation relative to the control. Data represent mean ± S.D. of three individual experiments.