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Regulation of autophagy and the ubiquitin-proteasome system by the FoxO transcriptional network during muscle atrophy.

Milan G, Romanello V, Pescatore F, Armani A, Paik JH, Frasson L, Seydel A, Zhao J, Abraham R, Goldberg AL, Blaauw B, DePinho RA, Sandri M - Nat Commun (2015)

Bottom Line: Notably, in the setting of low nutrient signalling, we demonstrate that FoxOs are required for Akt activity but not for mTOR signalling.FoxOs control several stress-response pathways such as the unfolded protein response, ROS detoxification, DNA repair and translation.Finally, we identify FoxO-dependent ubiquitin ligases including MUSA1 and a previously uncharacterised ligase termed SMART (Specific of Muscle Atrophy and Regulated by Transcription).

View Article: PubMed Central - PubMed

Affiliation: Venetian Institute of Molecular Medicine, via Orus 2, 35129 Padova, Italy.

ABSTRACT
Stresses like low nutrients, systemic inflammation, cancer or infections provoke a catabolic state characterized by enhanced muscle proteolysis and amino acid release to sustain liver gluconeogenesis and tissue protein synthesis. These conditions activate the family of Forkhead Box (Fox) O transcription factors. Here we report that muscle-specific deletion of FoxO members protects from muscle loss as a result of the role of FoxOs in the induction of autophagy-lysosome and ubiquitin-proteasome systems. Notably, in the setting of low nutrient signalling, we demonstrate that FoxOs are required for Akt activity but not for mTOR signalling. FoxOs control several stress-response pathways such as the unfolded protein response, ROS detoxification, DNA repair and translation. Finally, we identify FoxO-dependent ubiquitin ligases including MUSA1 and a previously uncharacterised ligase termed SMART (Specific of Muscle Atrophy and Regulated by Transcription). Our findings underscore the central function of FoxOs in coordinating a variety of stress-response genes during catabolic conditions.

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Deletion of FoxOs prevents muscle loss and weakness during fasting.(a) Frequency histograms of cross-sectional areas (μm2) of FoxO1,3,4f/f (black bars) and FoxO1,3,4−/− (magenta bars) fibres in fed (upper panel) and fasted (lower panel) conditions, n=4, each group. (b) Force measurements preformed in vivo on gastrocnemius showed that FoxO1,3,4−/− muscles preserve maximal tetanic force after fasting; n=6 muscles in each group. (c) Force/frequency curve of starved gastrocnemius muscle underlines the important protection achieved by the absence of FoxOs; n=6 muscles in each group. (d) Immunoblot of protein extracts from gastrocnemius muscles. Phosphorylation of AKT and S6 is reduced in fed and starved FoxO1,3,4−/− muscles when compared with controls. Data are representative of three independent experiments. (e) Immunoblot analysis of p62 and LC3 in homogenates of gastrocnemius muscles from fed and starved FoxO1,3,4−/− or controls. Fasting did not induce LC3 lipidation and p62 upregulation in FoxO-deficient muscles. Data are representative of three independent experiments. (f) Quantification of GFP–LC3-positive vesicles in FoxO1,3,4f/f and FoxO1,3,4−/− TA muscles; n=4 muscles in each group (g) Autophagy flux is not increased in FoxO-deficient TA muscles. Inhibition of autophagy–lysosome fusion by colchicine treatment induces accumulation of LC3II band in starved control but not in starved FoxO1,3,4−/− muscles. Upper panel: immunoblot analysis of gastrocnemius homogenates. Lower panel: quantification of LC3 lipidation. n=4 muscles in each group (h) The scheme shows the overlap between FoxO-dependent genes, identified by gene expression profiling of fed (n=4) and fasted (n=4) muscles from FoxO1,3,4f/f and FoxO1,3,4−/− and atrophy-related genes or atrogenes. The data in the graphs are shown as mean±s.e.m. Error bars indicate s.e.m. *P<0.05, **P<0.01 (Student's t-test).
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f2: Deletion of FoxOs prevents muscle loss and weakness during fasting.(a) Frequency histograms of cross-sectional areas (μm2) of FoxO1,3,4f/f (black bars) and FoxO1,3,4−/− (magenta bars) fibres in fed (upper panel) and fasted (lower panel) conditions, n=4, each group. (b) Force measurements preformed in vivo on gastrocnemius showed that FoxO1,3,4−/− muscles preserve maximal tetanic force after fasting; n=6 muscles in each group. (c) Force/frequency curve of starved gastrocnemius muscle underlines the important protection achieved by the absence of FoxOs; n=6 muscles in each group. (d) Immunoblot of protein extracts from gastrocnemius muscles. Phosphorylation of AKT and S6 is reduced in fed and starved FoxO1,3,4−/− muscles when compared with controls. Data are representative of three independent experiments. (e) Immunoblot analysis of p62 and LC3 in homogenates of gastrocnemius muscles from fed and starved FoxO1,3,4−/− or controls. Fasting did not induce LC3 lipidation and p62 upregulation in FoxO-deficient muscles. Data are representative of three independent experiments. (f) Quantification of GFP–LC3-positive vesicles in FoxO1,3,4f/f and FoxO1,3,4−/− TA muscles; n=4 muscles in each group (g) Autophagy flux is not increased in FoxO-deficient TA muscles. Inhibition of autophagy–lysosome fusion by colchicine treatment induces accumulation of LC3II band in starved control but not in starved FoxO1,3,4−/− muscles. Upper panel: immunoblot analysis of gastrocnemius homogenates. Lower panel: quantification of LC3 lipidation. n=4 muscles in each group (h) The scheme shows the overlap between FoxO-dependent genes, identified by gene expression profiling of fed (n=4) and fasted (n=4) muscles from FoxO1,3,4f/f and FoxO1,3,4−/− and atrophy-related genes or atrogenes. The data in the graphs are shown as mean±s.e.m. Error bars indicate s.e.m. *P<0.05, **P<0.01 (Student's t-test).

Mentions: To further characterize the role of FoxO1,3,4 in skeletal muscles, we then analysed the phenotype of FoxO1,3,4−/− mice under conditions of muscle wasting. Initially, we used fasting as a model of muscle loss since it is an established condition that induces nuclear translocation of FoxO members and their binding to target promoters2814 (Supplementary Fig. 2) and we compared FoxO1,3,4 muscles with controls. Importantly, FoxO1,3,4 knockout mice were completely spared from muscle loss after fasting (Fig. 2a, Supplementary Fig. 3). To understand whether sparing of muscle mass is also functionally relevant, we measured muscle force in living animals. While control fasted animals became significantly weaker than fed ones, FoxO1,3,4−/− gastrocnemius muscles did not loose strength after fasting (Fig. 2b). Importantly the comparison of force/frequency curve of fasted wild-type versus fasted FoxO1,3,4 muscle underlined the important protection achieved by the absence of FoxO family when nutrients are low or absent (Fig. 2c). These findings confirm that the absence of FoxO members prevents atrophy and profound weakening. To explain this profound effect on sparing force and muscle mass, we monitored the level of the contractile protein, myosin, when nutrients are removed. As expected, fasting induced an important reduction of myosin content in controls, while FoxO1,3,4 knockout were completely protected (Supplementary Fig. 4a). The maintenance of myosins in knockout is consequent to inhibition of protein ubiquitination (Supplementary Fig. 4b-c).


Regulation of autophagy and the ubiquitin-proteasome system by the FoxO transcriptional network during muscle atrophy.

Milan G, Romanello V, Pescatore F, Armani A, Paik JH, Frasson L, Seydel A, Zhao J, Abraham R, Goldberg AL, Blaauw B, DePinho RA, Sandri M - Nat Commun (2015)

Deletion of FoxOs prevents muscle loss and weakness during fasting.(a) Frequency histograms of cross-sectional areas (μm2) of FoxO1,3,4f/f (black bars) and FoxO1,3,4−/− (magenta bars) fibres in fed (upper panel) and fasted (lower panel) conditions, n=4, each group. (b) Force measurements preformed in vivo on gastrocnemius showed that FoxO1,3,4−/− muscles preserve maximal tetanic force after fasting; n=6 muscles in each group. (c) Force/frequency curve of starved gastrocnemius muscle underlines the important protection achieved by the absence of FoxOs; n=6 muscles in each group. (d) Immunoblot of protein extracts from gastrocnemius muscles. Phosphorylation of AKT and S6 is reduced in fed and starved FoxO1,3,4−/− muscles when compared with controls. Data are representative of three independent experiments. (e) Immunoblot analysis of p62 and LC3 in homogenates of gastrocnemius muscles from fed and starved FoxO1,3,4−/− or controls. Fasting did not induce LC3 lipidation and p62 upregulation in FoxO-deficient muscles. Data are representative of three independent experiments. (f) Quantification of GFP–LC3-positive vesicles in FoxO1,3,4f/f and FoxO1,3,4−/− TA muscles; n=4 muscles in each group (g) Autophagy flux is not increased in FoxO-deficient TA muscles. Inhibition of autophagy–lysosome fusion by colchicine treatment induces accumulation of LC3II band in starved control but not in starved FoxO1,3,4−/− muscles. Upper panel: immunoblot analysis of gastrocnemius homogenates. Lower panel: quantification of LC3 lipidation. n=4 muscles in each group (h) The scheme shows the overlap between FoxO-dependent genes, identified by gene expression profiling of fed (n=4) and fasted (n=4) muscles from FoxO1,3,4f/f and FoxO1,3,4−/− and atrophy-related genes or atrogenes. The data in the graphs are shown as mean±s.e.m. Error bars indicate s.e.m. *P<0.05, **P<0.01 (Student's t-test).
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f2: Deletion of FoxOs prevents muscle loss and weakness during fasting.(a) Frequency histograms of cross-sectional areas (μm2) of FoxO1,3,4f/f (black bars) and FoxO1,3,4−/− (magenta bars) fibres in fed (upper panel) and fasted (lower panel) conditions, n=4, each group. (b) Force measurements preformed in vivo on gastrocnemius showed that FoxO1,3,4−/− muscles preserve maximal tetanic force after fasting; n=6 muscles in each group. (c) Force/frequency curve of starved gastrocnemius muscle underlines the important protection achieved by the absence of FoxOs; n=6 muscles in each group. (d) Immunoblot of protein extracts from gastrocnemius muscles. Phosphorylation of AKT and S6 is reduced in fed and starved FoxO1,3,4−/− muscles when compared with controls. Data are representative of three independent experiments. (e) Immunoblot analysis of p62 and LC3 in homogenates of gastrocnemius muscles from fed and starved FoxO1,3,4−/− or controls. Fasting did not induce LC3 lipidation and p62 upregulation in FoxO-deficient muscles. Data are representative of three independent experiments. (f) Quantification of GFP–LC3-positive vesicles in FoxO1,3,4f/f and FoxO1,3,4−/− TA muscles; n=4 muscles in each group (g) Autophagy flux is not increased in FoxO-deficient TA muscles. Inhibition of autophagy–lysosome fusion by colchicine treatment induces accumulation of LC3II band in starved control but not in starved FoxO1,3,4−/− muscles. Upper panel: immunoblot analysis of gastrocnemius homogenates. Lower panel: quantification of LC3 lipidation. n=4 muscles in each group (h) The scheme shows the overlap between FoxO-dependent genes, identified by gene expression profiling of fed (n=4) and fasted (n=4) muscles from FoxO1,3,4f/f and FoxO1,3,4−/− and atrophy-related genes or atrogenes. The data in the graphs are shown as mean±s.e.m. Error bars indicate s.e.m. *P<0.05, **P<0.01 (Student's t-test).
Mentions: To further characterize the role of FoxO1,3,4 in skeletal muscles, we then analysed the phenotype of FoxO1,3,4−/− mice under conditions of muscle wasting. Initially, we used fasting as a model of muscle loss since it is an established condition that induces nuclear translocation of FoxO members and their binding to target promoters2814 (Supplementary Fig. 2) and we compared FoxO1,3,4 muscles with controls. Importantly, FoxO1,3,4 knockout mice were completely spared from muscle loss after fasting (Fig. 2a, Supplementary Fig. 3). To understand whether sparing of muscle mass is also functionally relevant, we measured muscle force in living animals. While control fasted animals became significantly weaker than fed ones, FoxO1,3,4−/− gastrocnemius muscles did not loose strength after fasting (Fig. 2b). Importantly the comparison of force/frequency curve of fasted wild-type versus fasted FoxO1,3,4 muscle underlined the important protection achieved by the absence of FoxO family when nutrients are low or absent (Fig. 2c). These findings confirm that the absence of FoxO members prevents atrophy and profound weakening. To explain this profound effect on sparing force and muscle mass, we monitored the level of the contractile protein, myosin, when nutrients are removed. As expected, fasting induced an important reduction of myosin content in controls, while FoxO1,3,4 knockout were completely protected (Supplementary Fig. 4a). The maintenance of myosins in knockout is consequent to inhibition of protein ubiquitination (Supplementary Fig. 4b-c).

Bottom Line: Notably, in the setting of low nutrient signalling, we demonstrate that FoxOs are required for Akt activity but not for mTOR signalling.FoxOs control several stress-response pathways such as the unfolded protein response, ROS detoxification, DNA repair and translation.Finally, we identify FoxO-dependent ubiquitin ligases including MUSA1 and a previously uncharacterised ligase termed SMART (Specific of Muscle Atrophy and Regulated by Transcription).

View Article: PubMed Central - PubMed

Affiliation: Venetian Institute of Molecular Medicine, via Orus 2, 35129 Padova, Italy.

ABSTRACT
Stresses like low nutrients, systemic inflammation, cancer or infections provoke a catabolic state characterized by enhanced muscle proteolysis and amino acid release to sustain liver gluconeogenesis and tissue protein synthesis. These conditions activate the family of Forkhead Box (Fox) O transcription factors. Here we report that muscle-specific deletion of FoxO members protects from muscle loss as a result of the role of FoxOs in the induction of autophagy-lysosome and ubiquitin-proteasome systems. Notably, in the setting of low nutrient signalling, we demonstrate that FoxOs are required for Akt activity but not for mTOR signalling. FoxOs control several stress-response pathways such as the unfolded protein response, ROS detoxification, DNA repair and translation. Finally, we identify FoxO-dependent ubiquitin ligases including MUSA1 and a previously uncharacterised ligase termed SMART (Specific of Muscle Atrophy and Regulated by Transcription). Our findings underscore the central function of FoxOs in coordinating a variety of stress-response genes during catabolic conditions.

Show MeSH
Related in: MedlinePlus