Figure 2.

CLOCK/BMAL1 regulates both the transactivation and transrepression activities of the GR. A–C) CLOCK/BMAL1 knockdown enhances GR-induced transcriptional activity. HeLa cells were transfected with siRNAs for CLOCK and/or BMAL1 and were treated with 10⁻⁶ M of dexamethasone. Total RNA was harvested, and mRNA levels of glucocorticoid-responsive GILZ (A), CLOCK (B), and BMAL1 (C) were measured with SYBR-Green real-time PCR. Bars represent mean ± se values (n=3) of fold induction over baseline (control siRNA in the absence of dexamethasone) in the presence or absence of 10⁻⁶ M of dexamethasone. *P < 0.01 vs. control with dexamethasone. n.s., not significant. D) CLOCK/BMAL1 overexpression and knockdown respectively suppresses and enhances GR-induced transcriptional activity of the glucocorticoid-responsive G6Pase gene. HepG2 cells were transfected with CLOCK- and BMAL1-expressing plasmids or CLOCK and BMAL1 siRNAs and were treated with 10⁻⁶ M of dexamethasone. Total RNA was harvested, and mRNA levels of G6Pase were measured with SYBR-Green real-time PCR. Bars represent mean ± se values (n=4) of fold induction over baseline (with mock transfection in the absence of dexamethasone) in the presence or absence of 10⁻⁶ M of dexamethasone. *P < 0.01 vs. mock transfection with dexamethasone. E) CLOCK/BMAL1 enhances the repressive effect of GR on NF-κB-induced transcriptional activity. HCT116 cells were transfected with CLOCK- and BMAL1-expressing plasmids together with (κB)₃-Luc and pSV40-β-Gal in the presence or absence of pRShGRα and/or pSRV-RelA (p65) and pRSV-NF-κB I (p50) transfection. Bars represent mean ± se values (n=3) of the luciferase activity normalized for β-galactosidase activity in the presence or absence of 10⁻⁶ M of dexamethasone. *P < 0.01.