PC4/Tis7/IFRD1 Stimulates Skeletal Muscle Regeneration and Is Involved in Myoblast Differentiation as a Regulator of MyoD and NF-κB

Laura Micheli,Luca Leonardi,Filippo Conti,Giovanna Maresca,Sandra Colazingari,Elisabetta Mattei,Sergio A Lira,Stefano Farioli-Vecchioli,Maurizia Caruso,Felice Tirone

doi:10.1074/jbc.m110.162842

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.

Highlights

Skeletal myoblast differentiation is a multistep process characterized by permanent exit from the cell cycle, maturation into mononucleated myocytes, and fusion in multinucleated myotubes
To activate the expression of the PC4 transgene, the Tg tetracyclineresponsive element (TRE)-PC4 B, G, and H lines were crossed with a second transgenic mouse, i.e. CMV-␤actin-regulated transactivator (rtTA), carrying the reverse tetracycline transcriptional activator under control of the CMV enhancer/chicken ␤-actin promoter, whose expression is mainly restricted to skeletal muscle and heart [18]
We obtained three lineages of the bitransgenic mouse CMV-␤actin-rtTA/TRE-PC4, in which doxycycline, 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)

Summary

Introduction

Skeletal myoblast differentiation is a multistep process characterized by permanent exit from the cell cycle, maturation into mononucleated myocytes, and fusion in multinucleated myotubes This differentiation program is controlled by the basic helix-loop-helix family of myogenic regulatory fac-. No indication about the underlying molecular mechanism(s) was obtained from the knock-out experiments In this regard, we have recently found that PC4 (as we refer to both the mouse and rat gene) cooperates with MyoD at inducing the transcriptional activity of MEF2C by counteracting the inhibition exerted by histone deacetylase 4 (HDAC4) on MEF2C. We have recently found that PC4 (as we refer to both the mouse and rat gene) cooperates with MyoD at inducing the transcriptional activity of MEF2C by counteracting the inhibition exerted by histone deacetylase 4 (HDAC4) on MEF2C This relies on the ability of PC4 to bind selectively MEF2C, inhibiting its interaction with HDAC4 [13]. PC4 is expressed in vivo in adult skeletal muscle, it is barely detectable during embryonic development [15], which suggests a prevalent role for PC4 in MyoD-dependent postdevelopmental myogenesis

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Results

Conclusion