In this study, using gain and loss of function approaches, we show that PC4 up-regulation in skeletal muscle in vivo stimulates regeneration and that the mechanism underlying this effect involves the control of MyoD by PC4 through NF-κB.
In fact, in transgenic mice conditionally up-regulating PC4
in adult skeletal muscle, we observed a significantly induced expression of MyoD
as well as that of several satellite cells markers, together with a remarkable increase of adult muscle regeneration following acute chemical damage. Conversely, the deprivation of PC4
in adult satellite cell-derived C2C12 myoblasts produced the opposite effects, i.e.
a dramatic impairment of myoblast fusion and greatly reduced expression of both MyoD
and downstream differentiation markers, accompanied by an up-regulation of G1
-S cyclins, in particular cyclin D1. The induction of cyclin levels, detected in proliferating as well as differentiating conditions, led to a prolongation of the proliferative state of PC4
-deprived myoblasts in differentiation medium. This effect was MyoD-dependent, because it was not observed in PC4
-deprived C3H10T1/2 fibroblasts cultured in similar conditions, unless these cells were rendered myogenic through ectopic MyoD
expression. This conclusion is consistent with the known ability of MyoD to inhibit cell cycle progression through several mechanisms, including induction of the retinoblastoma growth suppressor gene (Rb
) and the cyclin-dependent kinase inhibitor p21
, or its interaction with cyclin-dependent kinase 4 (CDK4) assembled to cyclin D1 (43
). Furthermore, it is worth noting that proliferating MyoD−/−
myoblasts cultured from knock-out mice display increased levels of cyclins D and E that remain high after mitogen withdrawal (60
), which is similar to what we observe in myoblasts expressing decreased MyoD levels as the consequence of PC4
Thus, the functional ablation of PC4 in C2C12 differentiating myoblasts results in delayed exit from the cell cycle and impaired differentiation and fusion as the consequence of MyoD down-regulation. Furthermore, in this study we show that the down-regulation of MyoD by PC4 relies on multiple mechanisms.
We have previously demonstrated that PC4 stimulates the transcriptional activity of MEF2C through displacement of HDAC4 from the MADS domain of MEF2C, because of its ability to bind both HDAC4 and MEF2C (13
). MEF2 factors are known to participate in the autoregulatory loop exerted by MyoD on its own transcription (45
). Our present data indicate that the down-modulation of MyoD levels observed in the absence of PC4
depends, at least in part, on a reduced activity of MEF2C.
Because, however, in the absence of PC4
the most evident decrease of MyoD levels occurred in proliferating myoblasts, i.e.
when MEF2C is not expressed (62
), the deprivation of PC4
also inhibited the levels of MyoD
transcript produced from a transfected expression construct under the control of a heterologous promoter, and a further PC4
-dependent mechanism controlling MyoD
mRNA levels post-transcriptionally is conceivably operating in myoblasts. A key observation made in our study is that the deprivation of PC4
or its overexpression in myoblasts significantly stimulates or inhibits, respectively, the transcriptional activity of NF-κB, revealing that PC4
is a negative regulator of NF-κB. This is a noteworthy finding, because NF-κB is known to function as an inhibitor of skeletal myogenesis through several mechanisms (47
), including suppression of MyoD
mRNA expression at the post-transcriptional level (47
We show that PC4
synergizes with HDAC4 and HDAC3 to inhibit the NF-κB transcriptional activity. It is known that the activity of NF-κB is critically controlled by acetylation. In particular, HDAC3, by deacetylating the p65 subunit of NF-κB, favors its binding to the IκBα repressor, with consequent nuclear export and relocalization into the cytoplasm of the p65-IκBα complex (52
). Thus, we hypothesized that PC4 may facilitate the recruitment of HDAC3 to p65. Indeed, our immunoprecipitation experiments show that PC4 can form trimolecular complexes with HDAC3 and p65. Strong support for the idea that PC4 may favor the recruitment of HDACs to p65, thus promoting deacetylation and hence inactivation of p65, comes from the observations that primary myoblasts overexpressing transgenic PC4
display reduced levels of acetylated p65, although deprivation of PC4 in myoblasts results in an increase of acetylated p65. In further agreement with the model proposed, we also find that overexpression of PC4
causes a parallel decrease of nuclear p65, although the absence of PC4
leads to accumulation of p65 in the nucleus, where p65 exerts its action. Notably, up-regulation of transgenic PC4
in primary myoblasts induces MyoD levels in concomitance with deacetylation of p65 and a decrease of p65 levels in the nucleus, indicating that PC4 can control MyoD through p65 also in vivo
In activated neutrophils, PC4 has been recently shown to associate with NF-κB p65 and HDAC1 (65
). However, we identify here for the first time PC4 as a negative regulator of NF-κB activity, and in skeletal muscle this is relevant because the activation of NF-κB activity has been linked with disease states such as cachexia and various dystrophinopathies, although disruption of the NF-κB pathway inhibits skeletal muscle atrophy (66
). In particular, a recent study has shown that in Duchenne muscular dystrophy mouse models (mdx) and patients, NF-κB signaling is persistently elevated in dystrophic muscles, and its down-regulation results in improved pathology and muscle function in mdx mice, thus implicating NF-κB as a potential therapeutic target for Duchenne muscular dystrophy (69
). Thus, PC4
may behave as a pivotal regulator of muscle differentiation and regeneration, in physiological and pathological condition, acting as an upstream regulator of MyoD levels by corepressing NF-κB activity through HDACs.
It is also noteworthy that in the absence of PC4
Myf5 levels increase, consistently with the notion that inactivation of MyoD
in mice leads to up-regulation of Myf5
). This may only partially compensate for MyoD
down-regulation; in fact, Myf5 appears to substitute for MyoD during embryogenesis but not during regeneration in adult muscle (14
We also provide evidence that PC4
transcription is directly regulated by MyoD; in fact, although we have previously shown that MyoD can transactivate the PC4
promoter, here we report that MyoD is recruited to the promoter region of the endogenous PC4
). Altogether, these findings point to the existence of a positive regulatory loop between MyoD and PC4
, where PC4
links the MyoD activity to that of NF-κB and MEF2C, acting as repressor or enhancer, respectively. Given that in both cases PC4 acts through HDACs, it follows that, depending on the target to which is associated, PC4 can act as coactivator or corepressor.
Interestingly, the activation of Tg PC4
since embryogenesis induced significant increases in the number of satellite cells (i.e.
cells) and myofibers in adult mice, suggesting that PC4
is also relevant during early postnatal muscle growth. In fact, satellite cells are myogenic progenitors set apart during late fetal development to give rise to proliferating myoblasts for postnatal muscle growth, in addition to homeostasis and repair of adult skeletal muscle (36
). Our data also show that PC4 positively regulates the expression of Pax7 in vivo
. It has been suggested that the expression of Pax7
may favor the self-renewal of satellite cells (71
). However, because it has been shown that Pax7 acts genetically upstream of MyoD
), it seems unlikely that the PC4-dependent increase of Pax7
expression is mediated through MyoD
; instead, one possibility is that up-regulation of PC4
during embryonic and postnatal development, by enhancing the MyoD
-dependent process of differentiation, may also trigger the activity and the renewal of the pool of satellite cells.
Considering the dramatic decrease in the number of myogenic cells occurring in muscle degenerative pathologies such as Duchenne dystrophy, our data highlighting the ability of PC4 to potentiate the regenerative process suggest that PC4 might be further investigated as a therapeutic target.