Syndecan-3 and Notch cooperate in regulating adult myogenesis

Syndecan-3 is required for Notch processing by ADAM17/TACe and therefore regulates proliferation and viability of muscle satellite cells

Regulation of Pax7 protein levels by caspase-3 and proteasome activity in differentiating myoblasts

The transcription factor Pax7 negatively regulates the activity of the muscle regulatory transcription factor MyoD, preventing muscle precursor cells from undergoing terminal differentiation. In this context, the ratio between Pax7 and MyoD protein levels is thought to be critical in allowing myogenesis to proceed or to maintain the undifferentiated muscle precursor state. We have previously shown that Pax7 is subject to rapid down regulation in differentiating myoblasts, via a proteasome-dependent pathway. Here we present evidence indicating that Pax7 is also subject to caspase-3-dependent regulation. Furthermore, simultaneous inhibition of caspase-3 and proteasome activity induced further accumulation of Pax7 protein in differentiating myoblasts. These results suggest that at early stages of muscle differentiation, Pax7 levels are regulated by at least two independent mechanisms involving caspase-3 and proteasome activity

NEDD4-1 deficiency impairs satellite cell function during skeletal muscle regeneration

Abstract Background Satellite cells are tissue-specific stem cells primarily responsible for the regenerative capacity of skeletal muscle. Satellite cell function and maintenance are regulated by extrinsic and intrinsic mechanisms, including the ubiquitin–proteasome system, which is key for maintaining protein homeostasis. In this context, it has been shown that ubiquitin-ligase NEDD4-1 targets the transcription factor PAX7 for proteasome-dependent degradation, promoting muscle differentiation in vitro. Nonetheless, whether NEDD4-1 is required for satellite cell function in regenerating muscle remains to be determined. Results Using conditional gene ablation, we show that NEDD4-1 loss, specifically in the satellite cell population, impairs muscle regeneration resulting in a significant reduction of whole-muscle size. At the cellular level, NEDD4-1-null muscle progenitors exhibit a significant decrease in the ability to proliferate and differentiate, contributing to the formation of myofibers with reduced diameter. Conclusions These results indicate that NEDD4-1 expression is critical for proper muscle regeneration in vivo and suggest that it may control satellite cell function at multiple levels

Ck2-Dependent Phosphorylation Is Required to Maintain Pax7 Protein Levels in Proliferating Muscle Progenitors.

Skeletal muscle regeneration and long term maintenance is directly link to the balance between self-renewal and differentiation of resident adult stem cells known as satellite cells. In turn, satellite cell fate is influenced by a functional interaction between the transcription factor Pax7 and members of the MyoD family of muscle regulatory factors. Thus, changes in the Pax7-to-MyoD protein ratio may act as a molecular rheostat fine-tuning acquisition of lineage identity while preventing precocious terminal differentiation. Pax7 is expressed in quiescent and proliferating satellite cells, while its levels decrease sharply in differentiating progenitors Pax7 is maintained in cells (re)acquiring quiescence. While the mechanisms regulating Pax7 levels based on differentiation status are not well understood, we have recently described that Pax7 levels are directly regulated by the ubiquitin-ligase Nedd4, thus promoting proteasome-dependent Pax7 degradation in differentiating satellite cells. Here we show that Pax7 levels are maintained in proliferating muscle progenitors by a mechanism involving casein kinase 2-dependent Pax7 phosphorylation at S201. Point mutations preventing S201 phosphorylation or casein kinase 2 inhibition result in decreased Pax7 protein in proliferating muscle progenitors. Accordingly, this correlates directly with increased Pax7 ubiquitination. Finally, Pax7 down regulation induced by casein kinase 2 inhibition results in precocious myogenic induction, indicating early commitment to terminal differentiation. These observations highlight the critical role of post translational regulation of Pax7 as a molecular switch controlling muscle progenitor fate

CK2 inhibition in proliferating myoblasts increases Pax7 ubiquitination and its proteasome-dependent degradation.

<p>(A) TBB-induced Pax7 decline is prevented by concomitant proteasome inhibition. Proliferating C2C12 cells were treated as indicated with TBB 125 μM and/or 1 μM of the proteasome inhibitor epoxomicin (Epo) for 6 hours and analyzed by Western blotting. GAPDH was used as loading control and anti-phospho-CK2 substrate antibody was used as a control of TBB treatment. Right panel shows quantification of protein levels (Pax7/GAPDH) normalized to control (DMSO); mean±SEM, n = 3; ANOVA, * p<0.05. (B) TBB increases Pax7 ubiquitination in proliferating myoblasts. C2C12 cells were transfected with Pax7 and myc-6xHis-ubiquitin, treated with DMSO or 100 μM TBB for 12 hours and 12.5 μM MG132 was added for the last 6 hours before cell lysis. Denaturing Ni-NTA pull-down, followed by Western Blot shows higher levels of ubiquitinated Pax7 in TBB treated cells. Inputs = 10% of cell extracts prior to Ni-NTA pull-down. (C) Disruption of Pax7 phosphorylation by CK2, increases its ubiquitination <i>in vivo</i>. C2C12 myoblasts were transfected with the specified constructs for bimolecular fluorescence complementation (BiFC) and treated with 25 μM MG132 and 125 μM TBB or DMSO as indicated, for 6 hours before fixation. Interaction of bFos-VC and bJun-VN was included as a positive control for BiFC. Transfection of VC and VN was used as a negative control, exhibiting non-specific diffuse fluorescence signal. Arrows indicate positive BiFC and arrowheads shows transfected cells (mRFP positive cells) without complementation. Quantification of BiFC positive cells from total mRFP positive population mean±SEM, n = 3; ANOVA, * p<0.05.</p

CK2 regulates Pax7 stability in proliferating myoblasts.

<p>(A) C3H10T1/2 cells transfected with myc-Pax7-WT or mutants were treated with DMSO or 100 μM of CK2 inhibitor (TBB) for 6 hours prior to lysis and Western Blot analysis. GFP was used as transfection/loading control. Right panels show quantification of fold reduction in myc-Pax7 levels (myc/GFP) for each treatment compared to vehicle (DMSO); mean±SEM, n = 5 (upper), n = 4 (lower). Pax7-DS and Pax7-DD phospho-mimetics exhibit enhanced stability upon CK2 inhibition compared to other phospho-mutants. Proliferating C2C12 cells were incubated with DMSO, TBB (B) or TBCA (C) at the indicated concentration for 6 hours. Endogenous Pax7 levels were analyzed by Western Blot using GAPDH as loading control. Anti-phospho-CK2 substrate antibody was used as a control of TBB treatment. (B)-(C), Lower panels show quantification of Pax7/GAPDH ratio in relative units; mean±SEM, n = 4; ANOVA, * p<0.05. Pax7 protein levels are significantly reduced with 125 μM TBB for 6 hours in proliferating C2C12 cells. (D) (left panel) qPCR analysis determining relative Pax7 mRNA expression upon CK2 inhibition as performed in (B). mean±SEM, n = 3. (Right panel) RT-PCR analysis of Pax7 mRNA expression upon CK2 inhibition in adult primary myoblasts (n = 3). (E) Proliferating C2C12 cells were incubated with DMSO or TBB for 12, 24, 48 and 72 hours, adding fresh doses every 24 hours. Lower panel shows quantification of fold reduction in Pax7 levels (Pax7/GAPDH) for each treatment compared to vehicle (DMSO); mean±SEM, n = 3.</p

Pax7 is phosphorylated by casein kinase II (CK2) in proliferating myoblasts.

<p>(A) Schematic representation of <i>in silico</i> analysis of mouse Pax7 sequence, depicting Pax7 domains and aminoacids with the highest probabilities to be phosphorylated by serine/threonine kinases. (B) Mass spectrometry analysis of myc-Pax7 expressed in proliferating C2C12 myoblasts, shows three Pax7 phospho-peptides, indicated by a 79.9663 increase in mass. Pax7 serine 201 and serine 205 are located in two consecutive consensus sequences for CK2 phosphorylation (*S/T-x-x-D/E/pS). (C) Upper panel shows schematic representation of Pax7 point mutations (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154919#sec002" target="_blank">Materials and Methods</a>) at the indicated sites. Lower panel shows <i>in vitro</i> phosphorylation assays using purified WT GST-Pax7 protein (WT) or GST-Pax7 mutants (AS, SA, AA, respectively), in presence or absence of purified CK2. Only WT and SA are detected with anti-phospho-CK2 substrate antibody (anti-pCK2-sub) by Western Blot, indicating that serine 201 is directly phosphorylated by CK2.</p

CK2 inhibition in proliferating cells resulted in accelerated expression of differentiation markers.

<p>24 h after isolation, mouse primary myoblasts were incubated with 100 μM TBB or DMSO for 6 hours before fixation. Indirect immunofluorescence for Pax7, Syndecan-4 (A, B), MyoD (A), Myogenin (B) was performed as indicated. TBB treatment results in a significant increase in the percentage of cells expressing reduced Pax7 levels, while expressing high levels of myogenin (arrows). (C) Quantification of cell subpopulations present in (B), shows a ~10-fold increase in the percentage of Pax7(-)/ myogenin (+) cells (i.e. differentiating cells) with a concomitant decrease in the percentage of proliferating Pax7(+)/myogenin(-) sub population. mean±SEM, n = 3. (D) qPCR analysis determining relative Pax7 mRNA expression upon CK2 inhibition (sample pool obtained from one experiment performed as in A and B).</p

Disruption of Pax7 phosphorylation results in lower Pax7 protein levels.

<p>(A) Pax7 localization is not affected by S201/S205 phosphorylation. Immunofluorescence analysis of C3H10T1/2 cells transfected with myc-Pax7-WT, myc-Pax7-AS, myc-Pax7-SA or myc-Pax7-AA shows nuclear localization (DAPI) of all Pax7 variants (myc). Scale bar = 10μm. (B) the ability of each Pax7 mutant to repress MyoD-dependent myogenic conversion of C3H10T1/2 cells, was evaluated by immunofluorescence. All Pax7 mutants repressed myotube formation resembling the effect of Pax7-WT. Panel depicts MyoD and myc-Pax7 expression in transfected mRFP(+) cells. Quantification of three representative experiments, evaluating myotube formation by fusion index (i.e. myotube associated nuclei/ total MyoD+ or myc+ nuclei; considering ≥3 nuclei in mRFP+ cells as a myotube) is shown. Lower panel: the percentage of MyHC associated MyoD(+) nuclei was also inhibited upon Pax7 and Pax7 phospho-mutants co-expression. Bar = 10μm. (C) Transcriptional activity of Pax7-WT or Pax7 phospho-mutants, was evaluated using the <i>6xPRS9-luc</i> reporter gene in C3H10T1/2 cells [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154919#pone.0154919.ref020" target="_blank">20</a>]. CMV-LacZ expression vector was co-transfected and β-galactosidase activity was used to normalize luciferase activity. The graph represents the relative luciferase activity (RLA) (luciferase/β-galactosidase) / Pax7-WT activity ratio; mean±SEM, n = 3. No significant differences between Pax7-WT and mutants activity was observed. (D) Phosphorylation status of S201/S205 affects Pax7 protein levels. Upper panels: Western Blot analysis of myc-tagged Pax7 point mutants expression in C3H10T1/2 cells. GFP was used as transfection/loading control. Lower panels show quantification of myc-Pax7/GFP ratio normalized to Pax7-WT; mean±SEM, n = 3 (left), n = 4 (right); ANOVA, * p<0.05. Pax7-AA protein levels are significantly lower than Pax7-WT. Pax7-DS and Pax7-DD phospho-mimetic mutants exhibit higher expression levels compared to Pax7-WT.</p