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Akt-mediated phosphorylation controls the activity of the Y-box protein MSY3 in skeletal muscle.

De Angelis L, Balasubramanian S, Berghella L - Skelet Muscle (2015)

Bottom Line: This correlated well with the reduction of phosphorylated active Akt.Knocking down Akt expression increased the amount of dephosphorylated MSY3 and reduced myogenin expression and muscle differentiation.These results support the hypothesis that MSY3 phosphorylation by Akt interferes with MSY3 repression of myogenin circuit activity during muscle development.

View Article: PubMed Central - PubMed

Affiliation: DAHFMO, Unit of Histology and Medical Embryology, University La Sapienza, Via Scarpa 16, Rome, 00161 Italy.

ABSTRACT

Background: The Y-box protein MSY3/Csda represses myogenin transcription in skeletal muscle by binding a highly conserved cis-acting DNA element located just upstream of the myogenin minimal promoter (myogHCE). It is not known how this MSY3 activity is controlled in skeletal muscle. In this study, we provide multiple lines of evidence showing that the post-translational phosphorylation of MSY3 by Akt kinase modulates the MSY3 repression of myogenin.

Methods: Skeletal muscle and myogenic C2C12 cells were used to study the effects of MSY3 phosphorylation in vivo and in vitro on its sub-cellular localization and activity, by blocking the IGF1/PI3K/Akt pathway, by Akt depletion and over-expression, and by mutating potential MSY3 phosphorylation sites.

Results: We observed that, as skeletal muscle progressed from perinatal to postnatal and adult developmental stages, MSY3 protein became gradually dephosphorylated and accumulated in the nucleus. This correlated well with the reduction of phosphorylated active Akt. In C2C12 myogenic cells, blocking the IGF1/PI3K/Akt pathway using LY294002 inhibitor reduced MSY3 phosphorylation levels resulting in its accumulation in the nuclei. Knocking down Akt expression increased the amount of dephosphorylated MSY3 and reduced myogenin expression and muscle differentiation. MSY3 phosphorylation by Akt in vitro impaired its binding at the MyogHCE element, while blocking Akt increased MSY3 binding activity. While Akt over-expression rescued myogenin expression in MSY3 overexpressing myogenic cells, ablation of the Akt substrate, (Ser126 located in the MSY3 cold shock domain) promoted MSY3 accumulation in the nucleus and abolished this rescue. Furthermore, forced expression of Akt in adult skeletal muscle induced MSY3 phosphorylation and myogenin derepression.

Conclusions: These results support the hypothesis that MSY3 phosphorylation by Akt interferes with MSY3 repression of myogenin circuit activity during muscle development. This study highlights a previously undescribed Akt-mediated signaling pathway involved in the repression of myogenin expression in myogenic cells and in mature muscle. Given the significance of myogenin regulation in adult muscle, the Akt/MSY3/myogenin regulatory circuit is a potential therapeutic target to counteract muscle degenerative disease.

No MeSH data available.


Related in: MedlinePlus

Dephosphorylated MSY3 accumulates in C2C12 nuclei. a Expression of MSY3 evaluated by IF with anti-MSY3 Ab, ZONAB, in C2C12 myoblasts in proliferating (30 % confluence) (IF left) and confluent (80 % confluence) growth (IF right) upon LY294002 (LY) treatment. Scale bar = 200 μm. b High magnification image (60×) of MSY3 expression (top) and Hoechst nuclei staining (bottom) in C2C12 myoblasts untreated (−) and upon LY treatment (LY). Scale bar = 60 μm. c N/C ratios of MSY3 signal in C2C12 myoblasts untreated (−) and upon LY treatment (+), evaluated as a mean ± DS for 60–80 cells from a total of five independent experiments. Quantification was performed as described in the “Methods” section. *P <0.05 by Student’s t test. d Densitometry calculations for MSY3 faster (dephosph) and slower (phosph) migration bands in nuclear (N) and cytosolic (C) compartments in untreated (−) and upon LY treatment (+), analyzed on Western blots performed as that shown in Fig S2B. Figure displays results representative from three independent RNAi experiments. *P <0.01; **P <0.001 by Student’s t test
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Fig3: Dephosphorylated MSY3 accumulates in C2C12 nuclei. a Expression of MSY3 evaluated by IF with anti-MSY3 Ab, ZONAB, in C2C12 myoblasts in proliferating (30 % confluence) (IF left) and confluent (80 % confluence) growth (IF right) upon LY294002 (LY) treatment. Scale bar = 200 μm. b High magnification image (60×) of MSY3 expression (top) and Hoechst nuclei staining (bottom) in C2C12 myoblasts untreated (−) and upon LY treatment (LY). Scale bar = 60 μm. c N/C ratios of MSY3 signal in C2C12 myoblasts untreated (−) and upon LY treatment (+), evaluated as a mean ± DS for 60–80 cells from a total of five independent experiments. Quantification was performed as described in the “Methods” section. *P <0.05 by Student’s t test. d Densitometry calculations for MSY3 faster (dephosph) and slower (phosph) migration bands in nuclear (N) and cytosolic (C) compartments in untreated (−) and upon LY treatment (+), analyzed on Western blots performed as that shown in Fig S2B. Figure displays results representative from three independent RNAi experiments. *P <0.01; **P <0.001 by Student’s t test

Mentions: Although MSY3 is a nucleic acid binding protein, in epithelial cells, it is sequestered in the cytosol by a tight junction-associated protein, ZO-1, which controls its accumulation and activity as a transcription factor in the nuclei [25]. This suggests that sub-cellular localization of MSY3 is a possible mechanism for regulating MSY3 functions. Phosphorylation by Akt is a requirement for initiating sub-cellular trafficking of many Akt targets, including the Y-box proteins [29, 36]. To investigate the role of Akt in MSY3 intracellular distribution in myogenic cells, we treated proliferating (GM) and differentiated C2C12 cells (DM) with the Akt inhibitor LY and detected MSY3 protein localization by IF with ZONAB. Fluorescence images of untreated C2C12 cells showed that MSY3 is not localized in the nuclei and is dispersed in the cytoplasm, both in myoblasts and myotubes, with some exceptions of selective nuclear localization (0.1 %, white arrow in Additional file 1: Figure S5A). To examine MSY3 localization in more detail, we quantified the extent of fluorescence observed in the cytoplasm and nucleus from confocal sections and calculated the nuclear/cytoplasmic (N/C) ratio for 60–80 cells across five independent experiments (Fig. 3c). This analysis showed that, although a high variability exists in the N/C ratio across different experiments in untreated cells, MSY3 preferentially localized in the cytosol or was equivalently distributed between nucleus and cytosol. Additionally, MSY3 localization is independent from cell confluence, since similar N/C ratios were observed in proliferating cells at 30 and 80 % of confluency (Fig. 3c). After LY treatment, we observed a robust MSY3 accumulation in the nucleus both in proliferating and differentiated cells (Fig. 3a, c and Additional file 1: Figure S5A). A higher magnification (60×) of the IF shows that in a low percentage of the nuclei MSY3 protein is completely depleted (Fig. 3b). When C2C12 cells were treated with LY in DM, myogenesis was inhibited and mono-nucleated cells with MSY3 localized in the nuclei were observed. Although, in some cases, we observed small myotubes with diffuse MSY3 staining (in nucleus and cytoplasm) (green arrow in Additional file 1: Figure S5A). We analyzed the distribution of phosphorylated and dephosphorylated MSY3 in the nuclear (N) and (C) cytoplasmic compartments in untreated and LY treated C2C12 myoblasts by Western blot. In concordance with the IF observations, densitometry measurements showed that phosphorylated and dephosphorylated forms of MSY3 protein were distributed equally between the nuclei and cytoplasm in untreated myoblasts and upon LY treatment the nuclei were enriched for the dephosphorylated form (Fig. 3d and Additional file 1: Figure S5B). These results demonstrate that Akt phosphorylation inhibited by PI3K/Akt pharmacological blockade induces a robust accumulation of dephosphorylated MSY3 in the nuclei of myogenic cells. In the nucleus, dephosphorylated MSY3 acts as a transcriptional repressor of differentiation promoting genes, such as myogenin.Fig. 3


Akt-mediated phosphorylation controls the activity of the Y-box protein MSY3 in skeletal muscle.

De Angelis L, Balasubramanian S, Berghella L - Skelet Muscle (2015)

Dephosphorylated MSY3 accumulates in C2C12 nuclei. a Expression of MSY3 evaluated by IF with anti-MSY3 Ab, ZONAB, in C2C12 myoblasts in proliferating (30 % confluence) (IF left) and confluent (80 % confluence) growth (IF right) upon LY294002 (LY) treatment. Scale bar = 200 μm. b High magnification image (60×) of MSY3 expression (top) and Hoechst nuclei staining (bottom) in C2C12 myoblasts untreated (−) and upon LY treatment (LY). Scale bar = 60 μm. c N/C ratios of MSY3 signal in C2C12 myoblasts untreated (−) and upon LY treatment (+), evaluated as a mean ± DS for 60–80 cells from a total of five independent experiments. Quantification was performed as described in the “Methods” section. *P <0.05 by Student’s t test. d Densitometry calculations for MSY3 faster (dephosph) and slower (phosph) migration bands in nuclear (N) and cytosolic (C) compartments in untreated (−) and upon LY treatment (+), analyzed on Western blots performed as that shown in Fig S2B. Figure displays results representative from three independent RNAi experiments. *P <0.01; **P <0.001 by Student’s t test
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Related In: Results  -  Collection

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Fig3: Dephosphorylated MSY3 accumulates in C2C12 nuclei. a Expression of MSY3 evaluated by IF with anti-MSY3 Ab, ZONAB, in C2C12 myoblasts in proliferating (30 % confluence) (IF left) and confluent (80 % confluence) growth (IF right) upon LY294002 (LY) treatment. Scale bar = 200 μm. b High magnification image (60×) of MSY3 expression (top) and Hoechst nuclei staining (bottom) in C2C12 myoblasts untreated (−) and upon LY treatment (LY). Scale bar = 60 μm. c N/C ratios of MSY3 signal in C2C12 myoblasts untreated (−) and upon LY treatment (+), evaluated as a mean ± DS for 60–80 cells from a total of five independent experiments. Quantification was performed as described in the “Methods” section. *P <0.05 by Student’s t test. d Densitometry calculations for MSY3 faster (dephosph) and slower (phosph) migration bands in nuclear (N) and cytosolic (C) compartments in untreated (−) and upon LY treatment (+), analyzed on Western blots performed as that shown in Fig S2B. Figure displays results representative from three independent RNAi experiments. *P <0.01; **P <0.001 by Student’s t test
Mentions: Although MSY3 is a nucleic acid binding protein, in epithelial cells, it is sequestered in the cytosol by a tight junction-associated protein, ZO-1, which controls its accumulation and activity as a transcription factor in the nuclei [25]. This suggests that sub-cellular localization of MSY3 is a possible mechanism for regulating MSY3 functions. Phosphorylation by Akt is a requirement for initiating sub-cellular trafficking of many Akt targets, including the Y-box proteins [29, 36]. To investigate the role of Akt in MSY3 intracellular distribution in myogenic cells, we treated proliferating (GM) and differentiated C2C12 cells (DM) with the Akt inhibitor LY and detected MSY3 protein localization by IF with ZONAB. Fluorescence images of untreated C2C12 cells showed that MSY3 is not localized in the nuclei and is dispersed in the cytoplasm, both in myoblasts and myotubes, with some exceptions of selective nuclear localization (0.1 %, white arrow in Additional file 1: Figure S5A). To examine MSY3 localization in more detail, we quantified the extent of fluorescence observed in the cytoplasm and nucleus from confocal sections and calculated the nuclear/cytoplasmic (N/C) ratio for 60–80 cells across five independent experiments (Fig. 3c). This analysis showed that, although a high variability exists in the N/C ratio across different experiments in untreated cells, MSY3 preferentially localized in the cytosol or was equivalently distributed between nucleus and cytosol. Additionally, MSY3 localization is independent from cell confluence, since similar N/C ratios were observed in proliferating cells at 30 and 80 % of confluency (Fig. 3c). After LY treatment, we observed a robust MSY3 accumulation in the nucleus both in proliferating and differentiated cells (Fig. 3a, c and Additional file 1: Figure S5A). A higher magnification (60×) of the IF shows that in a low percentage of the nuclei MSY3 protein is completely depleted (Fig. 3b). When C2C12 cells were treated with LY in DM, myogenesis was inhibited and mono-nucleated cells with MSY3 localized in the nuclei were observed. Although, in some cases, we observed small myotubes with diffuse MSY3 staining (in nucleus and cytoplasm) (green arrow in Additional file 1: Figure S5A). We analyzed the distribution of phosphorylated and dephosphorylated MSY3 in the nuclear (N) and (C) cytoplasmic compartments in untreated and LY treated C2C12 myoblasts by Western blot. In concordance with the IF observations, densitometry measurements showed that phosphorylated and dephosphorylated forms of MSY3 protein were distributed equally between the nuclei and cytoplasm in untreated myoblasts and upon LY treatment the nuclei were enriched for the dephosphorylated form (Fig. 3d and Additional file 1: Figure S5B). These results demonstrate that Akt phosphorylation inhibited by PI3K/Akt pharmacological blockade induces a robust accumulation of dephosphorylated MSY3 in the nuclei of myogenic cells. In the nucleus, dephosphorylated MSY3 acts as a transcriptional repressor of differentiation promoting genes, such as myogenin.Fig. 3

Bottom Line: This correlated well with the reduction of phosphorylated active Akt.Knocking down Akt expression increased the amount of dephosphorylated MSY3 and reduced myogenin expression and muscle differentiation.These results support the hypothesis that MSY3 phosphorylation by Akt interferes with MSY3 repression of myogenin circuit activity during muscle development.

View Article: PubMed Central - PubMed

Affiliation: DAHFMO, Unit of Histology and Medical Embryology, University La Sapienza, Via Scarpa 16, Rome, 00161 Italy.

ABSTRACT

Background: The Y-box protein MSY3/Csda represses myogenin transcription in skeletal muscle by binding a highly conserved cis-acting DNA element located just upstream of the myogenin minimal promoter (myogHCE). It is not known how this MSY3 activity is controlled in skeletal muscle. In this study, we provide multiple lines of evidence showing that the post-translational phosphorylation of MSY3 by Akt kinase modulates the MSY3 repression of myogenin.

Methods: Skeletal muscle and myogenic C2C12 cells were used to study the effects of MSY3 phosphorylation in vivo and in vitro on its sub-cellular localization and activity, by blocking the IGF1/PI3K/Akt pathway, by Akt depletion and over-expression, and by mutating potential MSY3 phosphorylation sites.

Results: We observed that, as skeletal muscle progressed from perinatal to postnatal and adult developmental stages, MSY3 protein became gradually dephosphorylated and accumulated in the nucleus. This correlated well with the reduction of phosphorylated active Akt. In C2C12 myogenic cells, blocking the IGF1/PI3K/Akt pathway using LY294002 inhibitor reduced MSY3 phosphorylation levels resulting in its accumulation in the nuclei. Knocking down Akt expression increased the amount of dephosphorylated MSY3 and reduced myogenin expression and muscle differentiation. MSY3 phosphorylation by Akt in vitro impaired its binding at the MyogHCE element, while blocking Akt increased MSY3 binding activity. While Akt over-expression rescued myogenin expression in MSY3 overexpressing myogenic cells, ablation of the Akt substrate, (Ser126 located in the MSY3 cold shock domain) promoted MSY3 accumulation in the nucleus and abolished this rescue. Furthermore, forced expression of Akt in adult skeletal muscle induced MSY3 phosphorylation and myogenin derepression.

Conclusions: These results support the hypothesis that MSY3 phosphorylation by Akt interferes with MSY3 repression of myogenin circuit activity during muscle development. This study highlights a previously undescribed Akt-mediated signaling pathway involved in the repression of myogenin expression in myogenic cells and in mature muscle. Given the significance of myogenin regulation in adult muscle, the Akt/MSY3/myogenin regulatory circuit is a potential therapeutic target to counteract muscle degenerative disease.

No MeSH data available.


Related in: MedlinePlus