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PC4/Tis7/IFRD1 stimulates skeletal muscle regeneration and is involved in myoblast differentiation as a regulator of MyoD and NF-kappaB.

Micheli L, Leonardi L, Conti F, Maresca G, Colazingari S, Mattei E, Lira SA, Farioli-Vecchioli S, Caruso M, Tirone F - J. Biol. Chem. (2010)

Bottom Line: Conversely, we observe that PC4 silencing in myoblasts causes delayed exit from the cell cycle, accompanied by delayed differentiation, and we show that such an effect is MyoD-dependent.On the contrary, PC4 silencing in myoblasts induces the acetylation and nuclear import of p65, in parallel with a decrease of MyoD levels.As a whole, these results indicate that PC4 plays a role in muscle differentiation by controlling the MyoD pathway through multiple mechanisms, and as such, it positively regulates regenerative myogenesis.

View Article: PubMed Central - PubMed

Affiliation: Istituto di Neurobiologia e Medicina Molecolare, Consiglio Nazionale delle Ricerche, Fondazione S Lucia, Via del Fosso di Fiorano 64, 00143 Rome, Italy.

ABSTRACT
In skeletal muscle cells, the PC4 (Tis7/Ifrd1) protein is known to function as a coactivator of MyoD by promoting the transcriptional activity of myocyte enhancer factor 2C (MEF2C). In this study, we show that up-regulation of PC4 in vivo in adult muscle significantly potentiates injury-induced regeneration by enhancing myogenesis. Conversely, we observe that PC4 silencing in myoblasts causes delayed exit from the cell cycle, accompanied by delayed differentiation, and we show that such an effect is MyoD-dependent. We provide evidence revealing a novel mechanism underlying the promyogenic actions of PC4, by which PC4 functions as a negative regulator of NF-κB, known to inhibit MyoD expression post-transcriptionally. In fact, up-regulation of PC4 in primary myoblasts induces the deacetylation, and hence the inactivation and nuclear export of NF-κB p65, in concomitance with induction of MyoD expression. On the contrary, PC4 silencing in myoblasts induces the acetylation and nuclear import of p65, in parallel with a decrease of MyoD levels. We also observe that PC4 potentiates the inhibition of NF-κB transcriptional activity mediated by histone deacetylases and that PC4 is able to form trimolecular complexes with p65 and HDAC3. This suggests that PC4 stimulates deacetylation of p65 by favoring the recruitment of HDAC3 to p65. As a whole, these results indicate that PC4 plays a role in muscle differentiation by controlling the MyoD pathway through multiple mechanisms, and as such, it positively regulates regenerative myogenesis.

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PC4 represses the NF-κB transactivation function. A, C2C12 cells were plated in duplicate 35-mm culture dishes (7 × 104 cells) and cotransfected the next day with the NF-κB-LUC reporter (0. 1 μg) and either the pSCT-PC4 expression construct or the empty pSCT vector (0.5 μg). Cells were either maintained in GM or switched to DM 24 h after transfection and harvested 48 h later. TNF (10 ng/ml) was added 6 h before harvesting, where indicated. Luciferase activity from cell extracts is expressed as fold induction relative to the activity of the GM control sample (transfected with the empty vector and not treated with TNF). Bars represent the average fold induction ± S.E. determined from five independent experiments, each performed in duplicate. *, p < 0.05 (Student's t test). B, C2C12 cells transfected as in A were analyzed for PC4 protein expression by Western blot. C, C2C12 cells infected with the RS/2 retrovirus expressing shRNA to PC4 or the control retrovirus (RS) were plated in duplicate 35-mm culture dishes (7 × 104 cells) and transfected the next day with the NF-κB-LUC reporter (0.1 μg). Cells were either maintained in GM or switched to DM 24 h after transfection and harvested 48 h later; where indicated, TNF (10 ng/ml) was added 6 h before harvesting. Luciferase activity is expressed as fold induction relative to the activity of the GM RS-infected control sample not treated with TNF. Bars represent the average fold induction ± S.E. determined from five independent experiments performed in duplicate. *, p < 0.05; **, p < 0.01 (Student's t test).
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Figure 8: PC4 represses the NF-κB transactivation function. A, C2C12 cells were plated in duplicate 35-mm culture dishes (7 × 104 cells) and cotransfected the next day with the NF-κB-LUC reporter (0. 1 μg) and either the pSCT-PC4 expression construct or the empty pSCT vector (0.5 μg). Cells were either maintained in GM or switched to DM 24 h after transfection and harvested 48 h later. TNF (10 ng/ml) was added 6 h before harvesting, where indicated. Luciferase activity from cell extracts is expressed as fold induction relative to the activity of the GM control sample (transfected with the empty vector and not treated with TNF). Bars represent the average fold induction ± S.E. determined from five independent experiments, each performed in duplicate. *, p < 0.05 (Student's t test). B, C2C12 cells transfected as in A were analyzed for PC4 protein expression by Western blot. C, C2C12 cells infected with the RS/2 retrovirus expressing shRNA to PC4 or the control retrovirus (RS) were plated in duplicate 35-mm culture dishes (7 × 104 cells) and transfected the next day with the NF-κB-LUC reporter (0.1 μg). Cells were either maintained in GM or switched to DM 24 h after transfection and harvested 48 h later; where indicated, TNF (10 ng/ml) was added 6 h before harvesting. Luciferase activity is expressed as fold induction relative to the activity of the GM RS-infected control sample not treated with TNF. Bars represent the average fold induction ± S.E. determined from five independent experiments performed in duplicate. *, p < 0.05; **, p < 0.01 (Student's t test).

Mentions: The above results indicated that the PC4 silencing inhibited the levels of MyoD transcript in C2C12 cells (Fig. 5), as well as the MyoD levels produced from a transfected expression construct under the control of a heterologous promoter (Fig. 7, D and E). This suggested that PC4, besides regulating MyoD transcription through MEF2C, might be implicated also in a second mechanism controlling MyoD mRNA accumulation. Noteworthy, it has been shown that NF-κB can suppress MyoD expression at the post-transcriptional level through a destabilization element in the MyoD transcript (47). This observation raised the intriguing hypothesis that PC4 could have an impact on MyoD through NF-κB. Thus, we investigated a possible functional interaction between PC4 and NF-κB by analyzing in C2C12 cells the effect of an overexpression or deprivation of PC4 on the transactivation potential of NF-κB. To this aim, a luciferase reporter gene fused to two tandem repeats of the κB site (2×NF-κB-LUC (48)) was transfected in C2C12 myoblasts with or without an expression vector for PC4, and its activity was measured in proliferating conditions (GM) or after 48 h in differentiating conditions (DM; Fig. 8A). We observed that the overexpression of PC4 significantly inhibited the endogenous activity of NF-κB, both in proliferating and in differentiating myoblasts (p = 0.02 for both conditions; Fig. 8A, left). Moreover, PC4 overexpression was also able to inhibit significantly the tumor necrosis factor α (TNF)-induced activity of NF-κB in proliferating but not in differentiating conditions (p = 0.04 and p = 0.11, respectively; Fig. 8A, right). The expression of equal levels of PC4 protein produced under the corresponding conditions (i.e. GM and DM) by the transfected PC4 expression construct was checked by Western blot (Fig. 8B). Conversely, C2C12 myoblasts where the expression of PC4 had been silenced by shRNA presented a significant stimulation of NF-κB activity both in proliferating and differentiating conditions (p = 0.02 and p = 0.01, respectively; Fig. 8C, left), also following stimulation by TNF (p = 0.049 in GM; p = 0.005 in DM; Fig. 8C, right). These findings reveal that PC4 can function as a negative regulator of NF-κB transcriptional activity.


PC4/Tis7/IFRD1 stimulates skeletal muscle regeneration and is involved in myoblast differentiation as a regulator of MyoD and NF-kappaB.

Micheli L, Leonardi L, Conti F, Maresca G, Colazingari S, Mattei E, Lira SA, Farioli-Vecchioli S, Caruso M, Tirone F - J. Biol. Chem. (2010)

PC4 represses the NF-κB transactivation function. A, C2C12 cells were plated in duplicate 35-mm culture dishes (7 × 104 cells) and cotransfected the next day with the NF-κB-LUC reporter (0. 1 μg) and either the pSCT-PC4 expression construct or the empty pSCT vector (0.5 μg). Cells were either maintained in GM or switched to DM 24 h after transfection and harvested 48 h later. TNF (10 ng/ml) was added 6 h before harvesting, where indicated. Luciferase activity from cell extracts is expressed as fold induction relative to the activity of the GM control sample (transfected with the empty vector and not treated with TNF). Bars represent the average fold induction ± S.E. determined from five independent experiments, each performed in duplicate. *, p < 0.05 (Student's t test). B, C2C12 cells transfected as in A were analyzed for PC4 protein expression by Western blot. C, C2C12 cells infected with the RS/2 retrovirus expressing shRNA to PC4 or the control retrovirus (RS) were plated in duplicate 35-mm culture dishes (7 × 104 cells) and transfected the next day with the NF-κB-LUC reporter (0.1 μg). Cells were either maintained in GM or switched to DM 24 h after transfection and harvested 48 h later; where indicated, TNF (10 ng/ml) was added 6 h before harvesting. Luciferase activity is expressed as fold induction relative to the activity of the GM RS-infected control sample not treated with TNF. Bars represent the average fold induction ± S.E. determined from five independent experiments performed in duplicate. *, p < 0.05; **, p < 0.01 (Student's t test).
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Figure 8: PC4 represses the NF-κB transactivation function. A, C2C12 cells were plated in duplicate 35-mm culture dishes (7 × 104 cells) and cotransfected the next day with the NF-κB-LUC reporter (0. 1 μg) and either the pSCT-PC4 expression construct or the empty pSCT vector (0.5 μg). Cells were either maintained in GM or switched to DM 24 h after transfection and harvested 48 h later. TNF (10 ng/ml) was added 6 h before harvesting, where indicated. Luciferase activity from cell extracts is expressed as fold induction relative to the activity of the GM control sample (transfected with the empty vector and not treated with TNF). Bars represent the average fold induction ± S.E. determined from five independent experiments, each performed in duplicate. *, p < 0.05 (Student's t test). B, C2C12 cells transfected as in A were analyzed for PC4 protein expression by Western blot. C, C2C12 cells infected with the RS/2 retrovirus expressing shRNA to PC4 or the control retrovirus (RS) were plated in duplicate 35-mm culture dishes (7 × 104 cells) and transfected the next day with the NF-κB-LUC reporter (0.1 μg). Cells were either maintained in GM or switched to DM 24 h after transfection and harvested 48 h later; where indicated, TNF (10 ng/ml) was added 6 h before harvesting. Luciferase activity is expressed as fold induction relative to the activity of the GM RS-infected control sample not treated with TNF. Bars represent the average fold induction ± S.E. determined from five independent experiments performed in duplicate. *, p < 0.05; **, p < 0.01 (Student's t test).
Mentions: The above results indicated that the PC4 silencing inhibited the levels of MyoD transcript in C2C12 cells (Fig. 5), as well as the MyoD levels produced from a transfected expression construct under the control of a heterologous promoter (Fig. 7, D and E). This suggested that PC4, besides regulating MyoD transcription through MEF2C, might be implicated also in a second mechanism controlling MyoD mRNA accumulation. Noteworthy, it has been shown that NF-κB can suppress MyoD expression at the post-transcriptional level through a destabilization element in the MyoD transcript (47). This observation raised the intriguing hypothesis that PC4 could have an impact on MyoD through NF-κB. Thus, we investigated a possible functional interaction between PC4 and NF-κB by analyzing in C2C12 cells the effect of an overexpression or deprivation of PC4 on the transactivation potential of NF-κB. To this aim, a luciferase reporter gene fused to two tandem repeats of the κB site (2×NF-κB-LUC (48)) was transfected in C2C12 myoblasts with or without an expression vector for PC4, and its activity was measured in proliferating conditions (GM) or after 48 h in differentiating conditions (DM; Fig. 8A). We observed that the overexpression of PC4 significantly inhibited the endogenous activity of NF-κB, both in proliferating and in differentiating myoblasts (p = 0.02 for both conditions; Fig. 8A, left). Moreover, PC4 overexpression was also able to inhibit significantly the tumor necrosis factor α (TNF)-induced activity of NF-κB in proliferating but not in differentiating conditions (p = 0.04 and p = 0.11, respectively; Fig. 8A, right). The expression of equal levels of PC4 protein produced under the corresponding conditions (i.e. GM and DM) by the transfected PC4 expression construct was checked by Western blot (Fig. 8B). Conversely, C2C12 myoblasts where the expression of PC4 had been silenced by shRNA presented a significant stimulation of NF-κB activity both in proliferating and differentiating conditions (p = 0.02 and p = 0.01, respectively; Fig. 8C, left), also following stimulation by TNF (p = 0.049 in GM; p = 0.005 in DM; Fig. 8C, right). These findings reveal that PC4 can function as a negative regulator of NF-κB transcriptional activity.

Bottom Line: Conversely, we observe that PC4 silencing in myoblasts causes delayed exit from the cell cycle, accompanied by delayed differentiation, and we show that such an effect is MyoD-dependent.On the contrary, PC4 silencing in myoblasts induces the acetylation and nuclear import of p65, in parallel with a decrease of MyoD levels.As a whole, these results indicate that PC4 plays a role in muscle differentiation by controlling the MyoD pathway through multiple mechanisms, and as such, it positively regulates regenerative myogenesis.

View Article: PubMed Central - PubMed

Affiliation: Istituto di Neurobiologia e Medicina Molecolare, Consiglio Nazionale delle Ricerche, Fondazione S Lucia, Via del Fosso di Fiorano 64, 00143 Rome, Italy.

ABSTRACT
In skeletal muscle cells, the PC4 (Tis7/Ifrd1) protein is known to function as a coactivator of MyoD by promoting the transcriptional activity of myocyte enhancer factor 2C (MEF2C). In this study, we show that up-regulation of PC4 in vivo in adult muscle significantly potentiates injury-induced regeneration by enhancing myogenesis. Conversely, we observe that PC4 silencing in myoblasts causes delayed exit from the cell cycle, accompanied by delayed differentiation, and we show that such an effect is MyoD-dependent. We provide evidence revealing a novel mechanism underlying the promyogenic actions of PC4, by which PC4 functions as a negative regulator of NF-κB, known to inhibit MyoD expression post-transcriptionally. In fact, up-regulation of PC4 in primary myoblasts induces the deacetylation, and hence the inactivation and nuclear export of NF-κB p65, in concomitance with induction of MyoD expression. On the contrary, PC4 silencing in myoblasts induces the acetylation and nuclear import of p65, in parallel with a decrease of MyoD levels. We also observe that PC4 potentiates the inhibition of NF-κB transcriptional activity mediated by histone deacetylases and that PC4 is able to form trimolecular complexes with p65 and HDAC3. This suggests that PC4 stimulates deacetylation of p65 by favoring the recruitment of HDAC3 to p65. As a whole, these results indicate that PC4 plays a role in muscle differentiation by controlling the MyoD pathway through multiple mechanisms, and as such, it positively regulates regenerative myogenesis.

Show MeSH
Related in: MedlinePlus