HDAC5 regulates β-catenin expression. A, shown is a schematic of β-catenin promoter luciferase construct and ChIP analysis. Black bar, MEF2 binding site. Outside arrows, primer positions for construct are shown. Inside arrows, primers positions for ChIP analysis are shown. B, C3H10T1/2 cells were treated with scriptaid (1 μm) for 24 h, and β-catenin mRNA content was analyzed. C and D, C3H10T1/2 cells were transfected with either a HDAC5 or GFP vector, and β-catenin mRNA expression (C) and H3K9 acetylation levels and β-catenin protein content (D) were analyzed. E and F, C3H10T1/2 cells were transfected with HDAC5 shRNA or control shRNA and β-catenin mRNA expression (E), and H3K9 acetylation level (AC- H3K9) and β-catenin content were analyzed (F). G, C3H10T1/2 cells were transfected with a β-catenin promoter-luciferase vector or mutated β-catenin promoter, which lacks the MEF2 binding site together with or without co-transfection of the HDAC5 vector. Luciferase activity was analyzed. *, p < 0.05; bars atop with a different letter differ; mean ± S.E.; n = 3; for luciferase assay, n = 6.

AMPK regulates β-catenin expression through phosphorylation of HDAC5. A, C3H10T1/2 cells were treated with AICAR (0.25 mm) or vehicle (PBS) for 3 h, and phosphorylated HDAC5 at Ser-259 and Ser-498 was analyzed by immunoblotting. B, C3H10T1/2 cells were transfected with a vector carrying HDAC5 wild type, HDAC5 S259A mutant, HDAC5 S498A mutant, or GFP control and treated with AICAR (0.25 mm) or vehicle (PBS) for 48 h. β-Catenin content was analyzed by immunoblotting. C, C3H10T1/2 cells were co-transfected with HDAC5 expression vector and the β-catenin promoter luciferase construct followed by AICAR (0.25 mm) or vehicle (PBS) for 24 h. Luciferase activity was determined. D, C3H10T1/2 cells were co-transfected with the β-catenin promoter luciferase construct and the HDAC5 vector or vectors carrying mutated HDAC5 (S259A or S498A) followed by AICAR (0.25 mm) or vehicle (PBS) treatments for 24 h. Luciferase activity was determined. E, C3H10T1/2 cells were transfected with wild-type HDAC5, HDAC5 S259A mutant, and HDAC5 S498A mutant together with AICAR (0.25 mm) or vehicle (PBS) treatment for 6 h, and ChIP was used to analyze the binding of HDAC5 to the MEF2 binding site on the β-catenin promoter. F, C3H10T1/2 cells were transfected with HDAC5 (shown in Fig. 3D) and its two mutants followed by AICAR (0.25 mm) or vehicle (PBS) for 3 h. Subcellular localization of HDAC5 was analyzed by immunocytochemical staining. *, p < 0.05; **, p < 0.01; bars atop with a different letter differ; Mean ± S.E.; n = 3; for luciferase assay, n = 6).

AMP-activated protein kinase regulates β-catenin expression. A and B, C3H10T1/2 cells were treated with AICAR (0.25 mm) or vehicle (PBS), and mRNA (after 6 h) and protein content (after 48 h) were analyzed by RT-PCR and immunoblotting, respectively (ACC, acetyl-CoA carboxylase). C and D, C3H10T1/2 cells were transfected with AMPKα2 WT, K45R, or GFP and assayed as in A and B. E and F, MEFs from AMPKα2 heterozygous (+/−) or knock-out mice (−/−) were isolated from mice of the same pregnancy, and β-catenin content was determined. G, C3H10T1/2 cells were treated with AICAR (0.25 mm) or vehicle (PBS), and nuclear β-catenin content (after 48 h) was analyzed by immunoblotting. *, p < 0.05; **, p < 0.01; bars of the same group atop with a different letter differ; mean ± S.E.; n = 3).

AMPK activation leads HDAC5 relocalization. A, C3H10T1/2 cells were treated with AICAR (0.25 mm) or vehicle (PBS) for 3 h, and co-immunoprecipitation (IP) was conducted using an anti-HDAC5 antibody followed by immunoblotting (IB) with an anti-14-3-3 antibody. B, C3H10T1/2 cells were treated with AICAR (0.25 mm) or vehicle (PBS) for 3 h, and nuclear and cytoplasmic fractions were isolated and analyzed by immunoblotting. C, C3H10T1/2 cells were treated with AICAR (0.25 mm) or vehicle (PBS) for 3 h, and H3K9 acetylation levels were analyzed by immunoblotting. D, C3H10T1/2 cells were transfected with HDAC5, treated with AICAR (0.25 mm) or vehicle (PBS) for 3 h, and immunocytochemical staining was carried out using an anti-tag antibody. *, p < 0.05; mean ± S.E.; n = 3.

Schematic diagram shows the proposed mechanism for AMPK regulation of β-catenin transcription. Without AMPK activation, MEF2 recruits HDAC5 to β-catenin promoter region, leading to deacetylation and inhibition of β-catenin gene expression. On the other hand, activation of AMPK phosphorylates HDAC5 on both Ser-259 and Ser-498 and initiates the interaction between HDAC5 and 14-3-3; this interaction releases HDAC5 from MEF2 and promotes its relocalization from nuclei to cytoplasm, promoting β-catenin gene transcription.