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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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MyoD is recruited to the PC4 gene promoter. A, ChIP analysis of MyoD binding to the mouse PC4 promoter in proliferating (GM) or differentiating (DM) C2C12 myoblasts. The scheme above the graph illustrates the first exon of the mouse PC4/Tis7 gene and the promoter region analyzed, located 780 nt before the transcription start. The amounts of PC4 promoter region retrieved from immunoprecipitates obtained with anti-MyoD antibody (black columns) or with normal rabbit serum (gray columns) are expressed as percentage of the amounts of promoter region from input cell lysates. The same amount of chromatin was immunoprecipitated in ChIPs with a-MyoD or normal rabbit serum. B, ChIP analysis of MyoD binding to the NeuroD1 promoter in C2C12 myoblasts, performed as negative control. C, PC4 mRNA levels, detected by real time PCR in duplicate cultures of myoblasts analyzed by ChIP, expressed as fold values relative to the mRNA amount present in GM cultures. A–C, the average ± S.E. values are from three experiments.
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Figure 10: MyoD is recruited to the PC4 gene promoter. A, ChIP analysis of MyoD binding to the mouse PC4 promoter in proliferating (GM) or differentiating (DM) C2C12 myoblasts. The scheme above the graph illustrates the first exon of the mouse PC4/Tis7 gene and the promoter region analyzed, located 780 nt before the transcription start. The amounts of PC4 promoter region retrieved from immunoprecipitates obtained with anti-MyoD antibody (black columns) or with normal rabbit serum (gray columns) are expressed as percentage of the amounts of promoter region from input cell lysates. The same amount of chromatin was immunoprecipitated in ChIPs with a-MyoD or normal rabbit serum. B, ChIP analysis of MyoD binding to the NeuroD1 promoter in C2C12 myoblasts, performed as negative control. C, PC4 mRNA levels, detected by real time PCR in duplicate cultures of myoblasts analyzed by ChIP, expressed as fold values relative to the mRNA amount present in GM cultures. A–C, the average ± S.E. values are from three experiments.

Mentions: In a previous study, we have shown that the expression of MyoD in fibroblasts mediates the induction of PC4 mRNA expression during the ensuing process of myogenic differentiation by stimulating the activity of the PC4 gene promoter through a mechanism independent of MyoD binding to E-box motifs (13). We were now interested to define whether MyoD is nonetheless recruited to the PC4 promoter. To this end we performed ChIP experiments in C2C12 myoblasts using an anti-MyoD antibody and amplifying a fragment of the mouse PC4 gene (Tis7) promoter region. We observed that the MyoD protein was significantly recruited to the PC4/Tis7 promoter, whereas the binding of MyoD to the NeuroD1 negative control promoter was not above background levels (Fig. 10, A and B). This indicates that MyoD directly regulates PC4 mRNA expression during differentiation. Notably, however, the MyoD protein was found to associate to the PC4/Tis7 promoter not only during differentiation but also in proliferating myoblasts, i.e. in GM, where MyoD is transcriptionally inactive and PC4 is expressed at levels similar to those of differentiating myoblasts (Fig. 10C). Indeed, as we have previously shown, PC4 mRNA expression is positively regulated in proliferating conditions by serum growth factor-dependent mechanisms (13). The constitutive binding of MyoD to the PC4 promoter suggests that PC4 may belong to the category of genes directly activated by MyoD during differentiation through a feed-forward mechanism (58).


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)

MyoD is recruited to the PC4 gene promoter. A, ChIP analysis of MyoD binding to the mouse PC4 promoter in proliferating (GM) or differentiating (DM) C2C12 myoblasts. The scheme above the graph illustrates the first exon of the mouse PC4/Tis7 gene and the promoter region analyzed, located 780 nt before the transcription start. The amounts of PC4 promoter region retrieved from immunoprecipitates obtained with anti-MyoD antibody (black columns) or with normal rabbit serum (gray columns) are expressed as percentage of the amounts of promoter region from input cell lysates. The same amount of chromatin was immunoprecipitated in ChIPs with a-MyoD or normal rabbit serum. B, ChIP analysis of MyoD binding to the NeuroD1 promoter in C2C12 myoblasts, performed as negative control. C, PC4 mRNA levels, detected by real time PCR in duplicate cultures of myoblasts analyzed by ChIP, expressed as fold values relative to the mRNA amount present in GM cultures. A–C, the average ± S.E. values are from three experiments.
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Figure 10: MyoD is recruited to the PC4 gene promoter. A, ChIP analysis of MyoD binding to the mouse PC4 promoter in proliferating (GM) or differentiating (DM) C2C12 myoblasts. The scheme above the graph illustrates the first exon of the mouse PC4/Tis7 gene and the promoter region analyzed, located 780 nt before the transcription start. The amounts of PC4 promoter region retrieved from immunoprecipitates obtained with anti-MyoD antibody (black columns) or with normal rabbit serum (gray columns) are expressed as percentage of the amounts of promoter region from input cell lysates. The same amount of chromatin was immunoprecipitated in ChIPs with a-MyoD or normal rabbit serum. B, ChIP analysis of MyoD binding to the NeuroD1 promoter in C2C12 myoblasts, performed as negative control. C, PC4 mRNA levels, detected by real time PCR in duplicate cultures of myoblasts analyzed by ChIP, expressed as fold values relative to the mRNA amount present in GM cultures. A–C, the average ± S.E. values are from three experiments.
Mentions: In a previous study, we have shown that the expression of MyoD in fibroblasts mediates the induction of PC4 mRNA expression during the ensuing process of myogenic differentiation by stimulating the activity of the PC4 gene promoter through a mechanism independent of MyoD binding to E-box motifs (13). We were now interested to define whether MyoD is nonetheless recruited to the PC4 promoter. To this end we performed ChIP experiments in C2C12 myoblasts using an anti-MyoD antibody and amplifying a fragment of the mouse PC4 gene (Tis7) promoter region. We observed that the MyoD protein was significantly recruited to the PC4/Tis7 promoter, whereas the binding of MyoD to the NeuroD1 negative control promoter was not above background levels (Fig. 10, A and B). This indicates that MyoD directly regulates PC4 mRNA expression during differentiation. Notably, however, the MyoD protein was found to associate to the PC4/Tis7 promoter not only during differentiation but also in proliferating myoblasts, i.e. in GM, where MyoD is transcriptionally inactive and PC4 is expressed at levels similar to those of differentiating myoblasts (Fig. 10C). Indeed, as we have previously shown, PC4 mRNA expression is positively regulated in proliferating conditions by serum growth factor-dependent mechanisms (13). The constitutive binding of MyoD to the PC4 promoter suggests that PC4 may belong to the category of genes directly activated by MyoD during differentiation through a feed-forward mechanism (58).

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