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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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Smart is a novel ubiquitin ligase required for denervation-dependent atrophy.(a) Co-immunoprecipitation experiment showing that SMART is a F-box protein that forms a SCF complex. C2C12 muscle cell lines were transfected with SMART, Skp1, Cul1 and Roc1 expression plasmids. After 24 h, cells were lysed and immunoprecipitation against FLAG-tag or control IgG was performed. Western blots for the different SCF components are shown. (b) RNAi-mediated knockdown of SMART revealed by quantitative RT–PCR (qRT–PCR). Adult TA muscles were transfected with bicistronic expressing vectors that encode either oligo 4 or scramble and GFP. Two weeks later TA muscles were collected, RNA extracted and endogenous SMART, MUSA1, Atrogin1, MuRF1 and Fbxo31 expression were analysed by qRT–PCR, n=4. (c) Inhibition of SMART prevents muscle atrophy in denervated muscles. Adult muscle fibres were co-transfected with bicistronic expressing vectors that encode shRNAs against SMART (oligo 4) or scramble and GFP and denervated. Two weeks later cross-sectional area of transfected fibres, identified by GFP fluorescence, was measured. n=6 muscles for each group. (d) Densitometric quantification of polyubiquitinated proteins in muscle extracts transfected with shRNAi against SMART or scramble. Values are normalized to GAPDH and expressed as fold increase of fed control mice. n=3 muscles for each group. Data are shown as mean±s.e.m. Error bars indicate s.e.m. *P<0.05, **P<0.01 (Student's t-test). con, control; den, denervated; IB, immunoblotting.
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f8: Smart is a novel ubiquitin ligase required for denervation-dependent atrophy.(a) Co-immunoprecipitation experiment showing that SMART is a F-box protein that forms a SCF complex. C2C12 muscle cell lines were transfected with SMART, Skp1, Cul1 and Roc1 expression plasmids. After 24 h, cells were lysed and immunoprecipitation against FLAG-tag or control IgG was performed. Western blots for the different SCF components are shown. (b) RNAi-mediated knockdown of SMART revealed by quantitative RT–PCR (qRT–PCR). Adult TA muscles were transfected with bicistronic expressing vectors that encode either oligo 4 or scramble and GFP. Two weeks later TA muscles were collected, RNA extracted and endogenous SMART, MUSA1, Atrogin1, MuRF1 and Fbxo31 expression were analysed by qRT–PCR, n=4. (c) Inhibition of SMART prevents muscle atrophy in denervated muscles. Adult muscle fibres were co-transfected with bicistronic expressing vectors that encode shRNAs against SMART (oligo 4) or scramble and GFP and denervated. Two weeks later cross-sectional area of transfected fibres, identified by GFP fluorescence, was measured. n=6 muscles for each group. (d) Densitometric quantification of polyubiquitinated proteins in muscle extracts transfected with shRNAi against SMART or scramble. Values are normalized to GAPDH and expressed as fold increase of fed control mice. n=3 muscles for each group. Data are shown as mean±s.e.m. Error bars indicate s.e.m. *P<0.05, **P<0.01 (Student's t-test). con, control; den, denervated; IB, immunoblotting.

Mentions: IP experiments confirmed that SMART forms an SCF complex with Skp1, Cullin1 and Roc1 (Fig. 8a) and therefore belongs to the SCF family of E3 ligases. To confirm the role of SMART in promoting atrophy during denervation, we knocked down SMART in TA in vivo. Four different short hairpin RNAs (shRNAs) have been tested to specifically reduce SMART protein levels (Supplementary Fig. 18a), three of which efficiently knocked down SMART. Next, we transfected oligo 4 into innervated and denervated muscles. These shRNAs did not affect the expression of the other atrophy-related ubiquitin ligases, MUSA, MuRF1 and atrogin1 both at protein and mRNA level (Supplementary Fig. 18b, Fig. 8b). Importantly, SMART inhibition significantly protected denervated muscles from atrophy (Fig. 8c). This sparing is due to the fact that by blocking SMART we greatly reduced protein ubiquitination in denervated muscles (Fig. 8d, Supplementary Fig. 18c). Therefore, we have identified SMART as an additional critical gene whose upregulation is required for atrophy, yet must be carefully controlled to avoid excessive protein breakdown. In conclusion, our findings underline the concept that FoxO members are the master regulatory factor for protein homeostasis during catabolic conditions.


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)

Smart is a novel ubiquitin ligase required for denervation-dependent atrophy.(a) Co-immunoprecipitation experiment showing that SMART is a F-box protein that forms a SCF complex. C2C12 muscle cell lines were transfected with SMART, Skp1, Cul1 and Roc1 expression plasmids. After 24 h, cells were lysed and immunoprecipitation against FLAG-tag or control IgG was performed. Western blots for the different SCF components are shown. (b) RNAi-mediated knockdown of SMART revealed by quantitative RT–PCR (qRT–PCR). Adult TA muscles were transfected with bicistronic expressing vectors that encode either oligo 4 or scramble and GFP. Two weeks later TA muscles were collected, RNA extracted and endogenous SMART, MUSA1, Atrogin1, MuRF1 and Fbxo31 expression were analysed by qRT–PCR, n=4. (c) Inhibition of SMART prevents muscle atrophy in denervated muscles. Adult muscle fibres were co-transfected with bicistronic expressing vectors that encode shRNAs against SMART (oligo 4) or scramble and GFP and denervated. Two weeks later cross-sectional area of transfected fibres, identified by GFP fluorescence, was measured. n=6 muscles for each group. (d) Densitometric quantification of polyubiquitinated proteins in muscle extracts transfected with shRNAi against SMART or scramble. Values are normalized to GAPDH and expressed as fold increase of fed control mice. n=3 muscles for each group. Data are shown as mean±s.e.m. Error bars indicate s.e.m. *P<0.05, **P<0.01 (Student's t-test). con, control; den, denervated; IB, immunoblotting.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4403316&req=5

f8: Smart is a novel ubiquitin ligase required for denervation-dependent atrophy.(a) Co-immunoprecipitation experiment showing that SMART is a F-box protein that forms a SCF complex. C2C12 muscle cell lines were transfected with SMART, Skp1, Cul1 and Roc1 expression plasmids. After 24 h, cells were lysed and immunoprecipitation against FLAG-tag or control IgG was performed. Western blots for the different SCF components are shown. (b) RNAi-mediated knockdown of SMART revealed by quantitative RT–PCR (qRT–PCR). Adult TA muscles were transfected with bicistronic expressing vectors that encode either oligo 4 or scramble and GFP. Two weeks later TA muscles were collected, RNA extracted and endogenous SMART, MUSA1, Atrogin1, MuRF1 and Fbxo31 expression were analysed by qRT–PCR, n=4. (c) Inhibition of SMART prevents muscle atrophy in denervated muscles. Adult muscle fibres were co-transfected with bicistronic expressing vectors that encode shRNAs against SMART (oligo 4) or scramble and GFP and denervated. Two weeks later cross-sectional area of transfected fibres, identified by GFP fluorescence, was measured. n=6 muscles for each group. (d) Densitometric quantification of polyubiquitinated proteins in muscle extracts transfected with shRNAi against SMART or scramble. Values are normalized to GAPDH and expressed as fold increase of fed control mice. n=3 muscles for each group. Data are shown as mean±s.e.m. Error bars indicate s.e.m. *P<0.05, **P<0.01 (Student's t-test). con, control; den, denervated; IB, immunoblotting.
Mentions: IP experiments confirmed that SMART forms an SCF complex with Skp1, Cullin1 and Roc1 (Fig. 8a) and therefore belongs to the SCF family of E3 ligases. To confirm the role of SMART in promoting atrophy during denervation, we knocked down SMART in TA in vivo. Four different short hairpin RNAs (shRNAs) have been tested to specifically reduce SMART protein levels (Supplementary Fig. 18a), three of which efficiently knocked down SMART. Next, we transfected oligo 4 into innervated and denervated muscles. These shRNAs did not affect the expression of the other atrophy-related ubiquitin ligases, MUSA, MuRF1 and atrogin1 both at protein and mRNA level (Supplementary Fig. 18b, Fig. 8b). Importantly, SMART inhibition significantly protected denervated muscles from atrophy (Fig. 8c). This sparing is due to the fact that by blocking SMART we greatly reduced protein ubiquitination in denervated muscles (Fig. 8d, Supplementary Fig. 18c). Therefore, we have identified SMART as an additional critical gene whose upregulation is required for atrophy, yet must be carefully controlled to avoid excessive protein breakdown. In conclusion, our findings underline the concept that FoxO members are the master regulatory factor for protein homeostasis during catabolic conditions.

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