Atrogin-1 inhibits Akt-dependent cardiac hypertrophy in mice via ubiquitin-dependent coactivation of Forkhead proteins

Abstract

Cardiac hypertrophy is a major cause of human morbidity and mortality. Although much is known about the pathways that promote hypertrophic responses, mechanisms that antagonize these pathways have not been as clearly defined. Atrogin-1, also known as muscle atrophy F-box, is an F-box protein that inhibits pathologic cardiac hypertrophy by participating in a ubiquitin ligase complex that triggers degradation of calcineurin, a factor involved in promotion of pathologic hypertrophy. Here we demonstrated that atrogin-1 also disrupted Akt-dependent pathways responsible for physiologic cardiac hypertrophy. Our results indicate that atrogin-1 does not affect the activity of Akt itself, but serves as a coactivator for members of the Forkhead family of transcription factors that function downstream of Akt. This coactivator function of atrogin-1 was dependent on its ubiquitin ligase activity and the deposition of polyubiquitin chains on lysine 63 of Foxo1 and Foxo3a. Transgenic mice expressing atrogin-1 in the heart displayed increased Foxo1 ubiquitylation and upregulation of known Forkhead target genes concomitant with suppression of cardiac hypertrophy, while mice lacking atrogin-1 displayed the opposite physiologic phenotype. These experiments define a role for lysine 63-linked ubiquitin chains in transcriptional coactivation and demonstrate that atrogin-1 uses this mechanism to disrupt physiologic cardiac hypertrophic signaling through its effects on Forkhead transcription factors.

Figure 1. Overexpression of atrogin-1 represses insulin- and IGF-1–induced cardiomyocyte hypertrophy and Foxo1 and Foxo3a dephosphorylation.

(A) Cardiomyocytes were infected with Ad-GFP or Ad–atrogin-1–GFP (MOI 10), and cells were stimulated with insulin (20 μg/ml^–1), IGF-1 (20 ng/ml^–1), or PE (100 μM) for 24 h and stained with α-actinin antibody (red). Quantitation of cell surface area was performed on 100 cells per group. *P < 0.001 vs. serum-free control and Ad-GFP. (B) Protein levels of endogenous Akt pathway members were determined in cardiomyocytes expressing atrogin-1 ectopically by IB with the indicated antibodies. (C) Foxo1 and Foxo3a expression and phosphorylation in cytoplasmic and nuclear fractions from cardiomyocytes were assessed by IB using anti-total or –phospho-Foxo1 and -Foxo3a antibodies after the indicated treatments. *P < 0.001 vs. Ad-GFP + insulin or IGF-1; ^#P < 0.01 vs. Ad-GFP + IGF-1. (D) Cardiomyocytes were infected with increasing MOIs of Ad–atrogin-1–GFP and Ad-GFP with IGF-1 (20 μg/ml^–1) stimulation for 24 h. Endogenous Foxo1 and Foxo3a levels were determined by IB with the indicated antibodies. Maximal suppression of Foxo1 and Foxo3a occurred at MOI 30. *P < 0.001 vs. Ad-GFP + IGF-1 at MOI 30. (E) Cardiomyocytes were infected with Ad-siRNA–control or Ad-siRNA–atrogin-1 and treated with insulin (20 μg/ml^–1) or PE (100 μM) for 24 h. (F) Cardiomyocytes treated as indicated were stained with α-actinin antibody. Quantitation of cell surface area was performed on 100 cells per group. Original magnification, ×200. *P < 0.01 vs. Ad-siRNA control + insulin or PE. Scale bars: 50 μm.

Figure 2. Atrogin-1 interacts with Foxo1 and Foxo3a in vivo and in vitro.

(A) We transfected 293 cells with the indicated plasmids. Equal amounts of protein lysates were immunoprecipitated with anti-HA or anti-Myc antibody and analyzed by IB with the indicated antibodies. WCE, whole cell extract. (B) The ability of Foxo1 and Foxo3a expressed in 293 cells to be retained by GST or a GST–atrogin-1 fusion protein was analyzed by IB after GST pulldown. (C) Endogenous protein interactions were examined in cardiomyocyte cell lysates immunoprecipitated with rabbit IgG or anti–atrogin-1 antibody and analyzed by IB with antibodies to detect Foxo1, Foxo3a, and atrogin-1. (D) Domains of atrogin-1 involved in binding to Foxo1 and Foxo3a expressed in 293 cells were mapped with GST pulldown assays. GST–atrogin-1 proteins were affinity purified and analyzed by SDS-PAGE and Coomassie blue staining. The ability of the truncated atrogin-1 fusion proteins to bind to Foxo1 and Foxo3a from whole-cell lysates was determined by blotting with antibodies against Flag and HA, respectively. (E) Deletion constructs of atrogin-1 in D that bind to Foxo1 and Foxo3a. NLS, nuclear localization sequence; PDZ, PDZ-binding domain. (F) Interaction domains of Foxo1 required for binding to atrogin-1 were mapped by co-IP. We transfected 293 cells with the indicated Flag-Foxo1 and HA–atrogin-1 plasmids. Equal amounts of lysates were prepared for IP with a Flag antibody and immunoblotted with antibodies for HA or Flag. (G) Deletion constructs of Foxo1 in F that bind to atrogin-1. FK, Forkhead domain; NES, nuclear export sequence; LxxLL, nuclear receptor–interacting domain or LxxLL motif; TAD, transactivation domain.

Figure 3. Atrogin-1 induces Foxo1 and Foxo3a nuclear localization and increased transcriptional activity.

(A) Cardiomyocytes were transiently transfected with expression vectors encoding HA-Foxo3a or Myc–atrogin-1. At 24 h after transfection, cells were treated without or with insulin for 12 h and immunostained with antibodies against HA (green) and Myc (red). Staining was assessed by confocal microscopy; representative images are shown. Scale bars: 50 μm. (B) Cardiomyocytes (100 random cells per condition) expressing the indicated proteins were scored for Foxo1 or Foxo3a localization. Data are mean ± SEM of 2 independent experiments. Original magnification, ×200. (C) To assess the role of atrogin-1 on Foxo3a-dependent transcription, cardiomyocytes were transfected with luciferase expression plasmids driven by the indicated promoters and vectors expressing Foxo3a or Foxo3aA3 (a constitutively active form) and atrogin-1. Data were normalized by cotransfection with a plasmid expressing β-gal. Cells were harvested 24 h later for measurement of luciferase activities. Results are expressed relative to the level of expression with the reporter gene alone and representative of 3 independent experiments. Error bars indicate SEM. (D) Cardiomyocytes were transfected with a luciferase reporter driven by the p27^kip1 promoter along with Foxo1 or Foxo3a and plasmids expressing siRNA–atrogin-1 or siRNA-control. Luciferase activity was measured 24 h after transfection. *P < 0.01 vs. control siRNA.

Figure 4. Atrogin-1 induces lysine 63–dependent ubiquitylation of Foxo3a.

(A) Cultured 293 cells were transfected with the indicated plasmids, and pulse-chase analysis was performed. Extracts were subjected to IP using either anti-Flag or anti-HA antibodies. Immunoprecipitates were analyzed by SDS–PAGE followed by autoradiography. (B) The indicated plasmids were cotransfected, and 293 cells were treated for 4 h with DMSO or MG132 (20 μM). The expression levels of the respective proteins were analyzed by IB with anti-Flag, anti-HA, or anti-Myc antibodies. (C) We transfected 293 cells with vectors expressing HA-ubiquitin (HA-Ub), HA-Foxo3a, atrogin-1, or atrogin-1 ΔF-box mutant lacking the E3-ligase activity. Cell extracts were immunoprecipitated with Foxo3a antibody and analyzed by IB with the indicated antibodies. An aliquot of the cell extracts was subjected to direct IB analysis using anti-Foxo3a or anti-Myc antibodies. (D) In vitro ubiquitylation reactions were performed with purified ubiquitin, E1, the E2 UBC13, GST-Foxo3a, and the SCF^atrogin-1 complex. Reactions were resolved by SDS-PAGE followed by IB with anti-ubiquitin antibody. (E) We cotransfected 293 cells with the indicated plasmids. At 24 h after transfection, ubiquitin-conjugated proteins were prepared for IP with anti-Foxo3a. Immunoprecipitates were subject to SDS–PAGE followed by IB with anti-ubiquitin, anti-Foxo3a, or anti-Myc antibodies. An aliquot of the cell extracts was subjected to direct IB analysis using anti-Foxo3a or anti-Myc antibodies.

Figure 5. Lysine 63–linked ubiquitin chains are required for transcriptional coactivation of Foxo3a by atrogin-1.

(A) Cultured cardiomyocytes were transfected with vectors expressing HA-Foxo3a, Myc–atrogin-1, and/or atrogin-1 ΔF-box together with the p27^kip1 luciferase reporter and β-gal constructs. At 24 h after transfection, cells were lysed and luciferase activity was measured. Lysates were immunoblotted with anti-HA and Myc. *P < 0.01 vs. atrogin-1 WT. (B) We transfected 293 cells with the indicated plasmids. Equal amounts of protein lysates were immunoprecipitated with anti-Myc or anti-HA antibodies to detect atrogin-1 and Foxo3a, respectively, followed by IB to detect protein-protein interactions. (C) Reporter assays were performed using the p27^kip1 promoter in cardiomyocytes cotransfected with plasmids expressing Foxo3a and/or atrogin-1, along with vectors expressing WT ubiquitin or mutant ubiquitins containing mutations of lysine 48 (K48R) or lysine 63 (K63R). At 24 h after transfection, cells were lysed and luciferase activity was measured. Lysates were immunoblotted with anti-HA and Myc. *P < 0.001 vs. WT ubiquitin. Results are expressed relative to the level of expression with the reporter gene alone and representative of 3 independent experiments. Error bars indicate SEM.

Figure 6. Enhanced Forkhead protein ubiquitylation and activity and suppressed IGF-1/GH-dependent cardiac hypertrophy in cardiac-specific atrogin-1 Tg mice.

(A) Cardiac hypertrophy was induced in 8-wk-old mice with IGF-1/GH injections. Atrogin-1 Tg and WT mice were sacrificed 14 d later; their hearts were freshly isolated; and levels of the indicated total and phospho-proteins were determined by IB. Results were normalized to relative expression levels of phospho-Foxo1, Foxo3a, and Akt (n = 6). *P < 0.001 vs. WT. (B) Equal amounts of lysates from WT and Tg hearts were immunoprecipitated with Foxo3a antibody and analyzed by IB with antibodies against ubiquitin or Foxo3a to detect ubiquitylated forms of Foxo3a in vivo before and after IGF-1/GH treatment. (C) RT-PCR was performed to measure the expression of known Foxo1 and Foxo3a target genes in WT and Tg mouse hearts before and after IGF-1/GH treatment. Total RNA was isolated from mouse hearts, and expression of transcripts for Bim, p27^Kip1, GADD45, SOD2, and GAPDH was determined and normalized to GAPDH (n = 6). *P < 0.001 vs. WT. A representative analysis is shown. (D) M-mode echocardiographic analysis of hearts from Tg and WT mice after 2 wk IGF-1/GH injection. (E) Representative macroscopic histologic analysis of H&E-stained hearts from indicated mice after 2 wk IGF-1/GH injection. Histologic sections were also stained with wheat germ agglutinin–TRITC conjugate to determine cell size. Scale bars: 1 mm (top); 50 μm (bottom). (F) Cardiomyocyte size from WT and Tg hearts after IGF-1/GH treatment.

Figure 7. Lack of atrogin-1 expression results in exaggerated cardiac hypertrophy in response to voluntary running exercise.

(A) Representative macroscopic histologic analysis of H&E-stained mouse hearts after 3 wk voluntary wheel exercise. Scale bar: 1 mm. Histologic sections were also stained with wheat germ agglutinin-TRITC to determine cell size. Scale bars: 1 mm (top); 50 μm (bottom). (B) Quantitation of cardiomyocyte size from WT and Atrogin-1^–/– heart after 3 wk voluntary wheel exercise and sham controls. (C) M-mode echocardiographic analysis of hearts from WT and Atrogin-1^–/– mice after 3 wk voluntary wheel exercise.