SMILE decreases Pgc-1α gene expression via inhibition of CREB/CRTC2.
A, Ad-GFP and Ad-SMILE were infected with primary hepatocytes and treated with forskolin. Expression of Pgc-1α was analyzed by quantitative PCR. B, AML12 cells were cotransfected with Pgc-1 promoter and SMILE expression vector. The cells were treated with vehicle or forskolin 24 h after the transfection. C, AML12 cells were cotransfected with Pgc-1–Luc, CRTC2, and SMILE expression vector. D, AML12 cells were cotransfected with WT Pgc-1–Luc, CRE site-mutated Pgc-1–Luc, CREB, CRTC2, and SMILE expression vector. E, AML12 cells were cotransfected with CRE-Luc, CREB, PKA, CRTC2, and SMILE. F, AML12 cells were cotransfected with CRE-Luc, PKA, S171A (constitutive active CRTC2), SMILE (long form), and ZF (short form of SMILE) expression vector. Error bars show mean ± S.D. n.s., not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.005 by two-tailed Student's t test. n.s. means not significant. Results are representative of three independently performed experiments.