Control of Foxo1 gene expression by co-activator P300

Abstract

FOXO1 is an important downstream mediator of the insulin signaling pathway. In the fed state, elevated insulin phosphorylates FOXO1 via AKT, leading to its nuclear exclusion and degradation. A reduction in nuclear FOXO1 levels then leads to suppression of hepatic glucose production. However, the mechanism leading to expression of Foxo1 gene in the fasted state is less clear. We found that Foxo1 mRNA and FOXO1 protein levels of Foxo1 were increased significantly in the liver of mice after 16 h of fasting. Furthermore, dibutyrl cAMP stimulated the expression of Foxo1 at both mRNA and protein level in hepatocytes. Because cAMP-PKA regulates hepatic glucose production through cAMP-response element-binding protein co-activators, we depleted these co-activators using adenoviral shRNAs. Interestingly, only depletion of co-activator P300 resulted in the decrease of Foxo1 mRNA and FOXO1 protein levels. In addition, inhibition of histone acetyltransferase activity of P300 significantly decreased hepatic Foxo1 mRNA and FOXO1 protein levels in fasted mice, as well as fasting blood glucose levels. By characterization of Foxo1 gene promoter, P300 regulates the Foxo1 gene expression through the binding to tandem cAMP-response element sites in the proximal promoter region of Foxo1 gene.

Keywords: CREB; Foxo; Gluconeogenesis; Histone Acetylase; P300.

FIGURE 1.

Fasting induces Foxo1 gene expression. a, Foxo1 mRNA levels compared in the liver of mice sacrificed at fed or fasted (16 h) states (n = 5). Real-time qPCR was used to measure gene expression (normalized to 36B4 expression levels). Asterisk (*) signifies that groups with same treatment are significantly different (p < 0.05). Error bars, S.E. b, phosphorylation status of CREB, AKT and total AKT, CREB, and Foxo1 protein levels in the liver from fed and 16-h fasted mice are shown (n = 5). c, fasting led to the early induction of Foxo1 expression. The Foxo1 protein levels in the liver of mice sacrificed are shown at the indicated fasting time points. Each lane represents sample pooled from two mice.

FIGURE 2.

Activation of cAMP-PKA pathway leads to the expression of Foxo1 gene. a and b, 16 h after the seeding, Hepa1–6 cells were subjected to serum starvation in FBS-free DMEM for 3 h, then 1 mm Bt-cAMP was added and cells were incubated for 5h before harvesting. b, Foxo1 mRNA levels were measured by using real-time qPCR, and data were normalized to 36B4 expression levels) (n = 3). Asterisk (*) signifies that groups with same treatment are significantly different (p < 0.05). Error bars, S.D. c, Hepa1–6 cells were treated with actinomycin D (2.5 μg/ml) for 30 min in FBS-free DMEM prior to the addition of 1 mm Bt-cAMP (6 h).

FIGURE 3.

Co-activator P300 mediates the Foxo1 gene expression. a and b, 48 h after the addition of adenoviral shRNAs to deplete CBP, P300, and CRTC2, Hepa1–6 cells were then treated with 0.2 mm Bt-cAMP for 6 h. Immunoblot analyses were conducted to determine the protein levels using indicated antibodies. c, 48 h after adenoviral shRNAs mediated depletion of CBP or P300, primary hepatocytes were subjected to serum starvation for 3 h. After washing with PBS, 0.2 mm Bt-cAMP was added in glucose production medium. d, in Hepa1–6 cells, 48 h after the addition of adenoviral shRNAs of scrambled, CREB, CBP, and P300, growth medium was changed to FBS-free DMEM and incubated for 3 h, then 1 mm Bt-cAMP was added and incubated for 5 h before harvesting. Real-time qPCR was used to measure gene expression (n = 3), and data were normalized to 36B4 expression levels. Each lane represents sample pooled from three treatments. Asterisk (*) signifies that groups with same treatment are significantly different (p < 0.05). Error bars, S.D.

FIGURE 4.

Depletion of P300 decreased Foxo1 mRNA levels in the liver. Foxo1 mRNA levels in the liver of mice with adenoviral shRNAs mediated depletion of P300 (a) or CBP (b), and sacrificed after 16 h fasting (n = 3). Asterisk (*) signifies that groups with the same treatment are significantly different (p < 0.05). Error bars, S.D.

FIGURE 5.

Identification of the functional regions in Foxo1 gene promoter. a, schematic shows reporter constructs tested. Nucleotides are numbered relative to the transcription start site (bp +1). 20 ng of reporter constructs was transfected into Hepa1–6 cells together with 400 ng of control RSV-cat or PKA expression plasmids. Reporter activities were measured 48 h after the transfection. b, three CRE site were identified in the proximal promoter region of the Foxo1 gene. c, three CREs were individually deleted in the context of the proximal promoter construct (−125 bp) and tested again. Hepa1–6 cells were transfected with 20 ng of each construct together with 200 ng of control RSV-cat or PKA expression plasmids. d, 20 ng of the proximal promoter construct and its mutants and 200 ng of PKA expression plasmid were co-transfected into Hepa1–6 cells together with 400 ng of control pcDNA or P300 expression vector. Reporter activities were measured 48 h after the transfection. Asterisk (*) signifies that groups with same treatment are significantly different (p < 0.05). Error bars, S.D.

FIGURE 6.

P300 binds to the proximal promoter region of the Foxo1 gene. a, PKA stimulated the binding of CREB and p300 to CREs in the proximal promoter of the Foxo1 gene. 48 h after the transfection of PKA expression plasmid, Hepa1–6 cells were fixed with formaldehyde followed by immunoprecipitation with CREB and P300 antibodies. The DNA in the immunoprecipitates was amplified using primers encompassing 150 bp including the CREs in the promoter of the mouse Foxo1 gene (n = 3). b, Hepa1–6 cells were transfected with PKA or together with HA-tagged P300 expression plasmid, followed by the fixation and immunoprecipitation using anti-HA tag-specific antibody. The binding of HA-tagged P300 was determined by real-time PCR (n = 3). Asterisk (*) signifies that groups with same treatment are significantly different (p < 0.05). Error bars, S.D.

FIGURE 7.

P300 binds to the proximal promoter region of the Foxo1 gene. a, 48 h after the addition of adenoviral shRNAs, Hepa1–6 cells were grown in FBS-free DMEM supplemented with 10 μm control C37 or HAT inhibitor C646 for 3 h, followed by the addition of 0.2 mm Bt-cAMP and incubated for 6 h. b, Hepa1–6 cells were transfected with 20 ng of promoter constructs, 400 ng of PKA, and 500 ng of P300 expression plasmids. 24 h later, 20 μm C37 or C646 was added, and cells were incubated for another 24 h. c, in primary hepatocytes, 20 μm C37 or C646 was added to FBS-free medium during serum starvation. After washing with PBS, 0.2 mm Bt-cAMP together with 20 μm C37 or C646 was added in glucose production medium for another 3 h. d–f, administration of HAT inhibitor significantly decreased fasting blood glucose levels (d), and hepatic Foxo1 mRNA levels (e), as well as the protein levels (f) of mice (n = 3∼4). Asterisk (*) signifies that groups with same treatment are significantly different (p < 0.05). Error bars, S.D.

FIGURE 8.

Proposed model for the regulation of Foxo1 gene by P300.