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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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Generation of a conditional bitransgenic mouse expressing PC4 in skeletal muscle. A, transgene CMV-βactin-rtTA contains the CMV enhancer/chicken β-actin promoter, followed by the doxycycline rtTA and the rabbit β-globin polyadenylation site. The TRE-PC4 transgene contains TRE fused downstream to the minimal CMV promoter, the PC4 ORF, and the SV40 late polyadenylation site. The rtTA protein binds the TRE and activates transcription of the PC4 transgene in the presence of doxycycline. B, analysis of the PC4 protein expressed in skeletal muscle by the three lines, B, G, and H, of PC4 bitransgenic mice, activated or not with doxycycline supplied in the drinking water since P30. C, tissue expression analysis of the PC4 protein by Western blot. D, PC4 mRNA by real time PCR in line G of bitransgenic mice treated or not with doxycycline (Doxy). PC4 mRNA fold expression was calculated relative to the level of expression in untreated mice. Tg PC4 mice used for experiments were 2 months old and isogenic, being the progeny of at least six generations of interbreeding. Skm, skeletal muscle; H, heart; B, brain; L, liver; K, kidney; S, spleen; TA, tibialis anterior; EDL, extensor digitorum longum; Dia, diaphragm.
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Figure 1: Generation of a conditional bitransgenic mouse expressing PC4 in skeletal muscle. A, transgene CMV-βactin-rtTA contains the CMV enhancer/chicken β-actin promoter, followed by the doxycycline rtTA and the rabbit β-globin polyadenylation site. The TRE-PC4 transgene contains TRE fused downstream to the minimal CMV promoter, the PC4 ORF, and the SV40 late polyadenylation site. The rtTA protein binds the TRE and activates transcription of the PC4 transgene in the presence of doxycycline. B, analysis of the PC4 protein expressed in skeletal muscle by the three lines, B, G, and H, of PC4 bitransgenic mice, activated or not with doxycycline supplied in the drinking water since P30. C, tissue expression analysis of the PC4 protein by Western blot. D, PC4 mRNA by real time PCR in line G of bitransgenic mice treated or not with doxycycline (Doxy). PC4 mRNA fold expression was calculated relative to the level of expression in untreated mice. Tg PC4 mice used for experiments were 2 months old and isogenic, being the progeny of at least six generations of interbreeding. Skm, skeletal muscle; H, heart; B, brain; L, liver; K, kidney; S, spleen; TA, tibialis anterior; EDL, extensor digitorum longum; Dia, diaphragm.

Mentions: To activate the expression of the PC4 transgene, the Tg TRE-PC4 B, G, and H lines were crossed with a second transgenic mouse, i.e. CMV-βactin-rtTA, carrying the reverse tetracycline transcriptional activator (rtTA) under control of the CMV enhancer/chicken β-actin promoter, whose expression is mainly restricted to skeletal muscle and heart (18). In this way, we obtained three lineages (named B, G, and H) of the bitransgenic mouse CMV-βactin-rtTA/TRE-PC4 (hereafter referred to as Tg PC4 for brevity), in which doxycycline (a tetracycline analog), administered to mice at the desired time, triggers the production of the transcription transactivator rtTA, which in turn induces the expression of exogenous PC4 by binding to the TRE within the TRE-PC4 transgene (Fig. 1A).


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)

Generation of a conditional bitransgenic mouse expressing PC4 in skeletal muscle. A, transgene CMV-βactin-rtTA contains the CMV enhancer/chicken β-actin promoter, followed by the doxycycline rtTA and the rabbit β-globin polyadenylation site. The TRE-PC4 transgene contains TRE fused downstream to the minimal CMV promoter, the PC4 ORF, and the SV40 late polyadenylation site. The rtTA protein binds the TRE and activates transcription of the PC4 transgene in the presence of doxycycline. B, analysis of the PC4 protein expressed in skeletal muscle by the three lines, B, G, and H, of PC4 bitransgenic mice, activated or not with doxycycline supplied in the drinking water since P30. C, tissue expression analysis of the PC4 protein by Western blot. D, PC4 mRNA by real time PCR in line G of bitransgenic mice treated or not with doxycycline (Doxy). PC4 mRNA fold expression was calculated relative to the level of expression in untreated mice. Tg PC4 mice used for experiments were 2 months old and isogenic, being the progeny of at least six generations of interbreeding. Skm, skeletal muscle; H, heart; B, brain; L, liver; K, kidney; S, spleen; TA, tibialis anterior; EDL, extensor digitorum longum; Dia, diaphragm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3037682&req=5

Figure 1: Generation of a conditional bitransgenic mouse expressing PC4 in skeletal muscle. A, transgene CMV-βactin-rtTA contains the CMV enhancer/chicken β-actin promoter, followed by the doxycycline rtTA and the rabbit β-globin polyadenylation site. The TRE-PC4 transgene contains TRE fused downstream to the minimal CMV promoter, the PC4 ORF, and the SV40 late polyadenylation site. The rtTA protein binds the TRE and activates transcription of the PC4 transgene in the presence of doxycycline. B, analysis of the PC4 protein expressed in skeletal muscle by the three lines, B, G, and H, of PC4 bitransgenic mice, activated or not with doxycycline supplied in the drinking water since P30. C, tissue expression analysis of the PC4 protein by Western blot. D, PC4 mRNA by real time PCR in line G of bitransgenic mice treated or not with doxycycline (Doxy). PC4 mRNA fold expression was calculated relative to the level of expression in untreated mice. Tg PC4 mice used for experiments were 2 months old and isogenic, being the progeny of at least six generations of interbreeding. Skm, skeletal muscle; H, heart; B, brain; L, liver; K, kidney; S, spleen; TA, tibialis anterior; EDL, extensor digitorum longum; Dia, diaphragm.
Mentions: To activate the expression of the PC4 transgene, the Tg TRE-PC4 B, G, and H lines were crossed with a second transgenic mouse, i.e. CMV-βactin-rtTA, carrying the reverse tetracycline transcriptional activator (rtTA) under control of the CMV enhancer/chicken β-actin promoter, whose expression is mainly restricted to skeletal muscle and heart (18). In this way, we obtained three lineages (named B, G, and H) of the bitransgenic mouse CMV-βactin-rtTA/TRE-PC4 (hereafter referred to as Tg PC4 for brevity), in which doxycycline (a tetracycline analog), administered to mice at the desired time, triggers the production of the transcription transactivator rtTA, which in turn induces the expression of exogenous PC4 by binding to the TRE within the TRE-PC4 transgene (Fig. 1A).

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