Henri Bernardi

Mechanisms involved in the inhibition of myoblast proliferation and differentiation by myostatin

Muscle growth results from a set of complex processes including myogenic transcription factors expression and activity, cell cycle withdrawal, myoblast fusion in myotubes, and acquisition of an apoptosis-resistant phenotype. Myostatin, a member of the TGFbeta family, described as a strong regulator of myogenesis in vivo Nature 387 (1997), 83; FEBS Lett. 474 (2000), 71 is upregulated during in vitro differentiation Biochem. Biophys. Res. Commun. 280 (2001), 561. To improve characterization of myostatins myogenic influence, we stably transfected vectors expressing myostatin and myostatin antisense in C2C12 myoblasts. Here, we found that myostatin inhibits cell proliferation and differentiation. Our results also indicate that myogenin is an important target of myostatin. In addition, overexpressed but not endogenous myostatin decreases MyoD protein levels and induces changes in its phosphorylation pattern. We also established that myostatin overexpression reduces the frequency of G0/G1-arrested cells during differentiation. Conversely, inhibition of myostatin synthesis leads to enhanced cell cycle withdrawal and consequently stimulates myoblast differentiation. We examined the expression patterns of the pRb, E2F1, p53, and p21 proteins involved in cell cycle withdrawal. We found that myostatin overexpression increases p21 and p53 expression, as it does accumulation of hypophosphorylated Rb. Interestingly, myostatin overexpression strongly reduced low-mitogen-induced apoptosis, whereas antisense expression induced contrary changes. In conclusion, these data show the influence of overexpressed myostatin on myoblast proliferation, differentiation, and apoptosis is extended to endogenous myostatin. Though some differences in overexpression or inhibition of endogenous myostatin were observed, it appears that myogenin and p21 are essential targets of this growth factor.

The role of AMP-activated protein kinase in the coordination of skeletal muscle turnover and energy homeostasis.

The AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase that acts as a sensor of cellular energy status switch regulating several systems including glucose and lipid metabolism. Recently, AMPK has been implicated in the control of skeletal muscle mass by decreasing mTORC1 activity and increasing protein degradation through regulation of ubiquitin-proteasome and autophagy pathways. In this review, we give an overview of the central role of AMPK in the control of skeletal muscle plasticity. We detail particularly its implication in the control of the hypertrophic and atrophic signaling pathways. In the light of these cumulative and attractive results, AMPK appears as a key player in regulating muscle homeostasis and the modulation of its activity may constitute a therapeutic potential in treating muscle wasting syndromes in humans.

Autophagy is essential to support skeletal muscle plasticity in response to endurance exercise.

Physical exercise is a stress that can substantially modulate cellular signaling mechanisms to promote morphological and metabolic adaptations. Skeletal muscle protein and organelle turnover is dependent on two major cellular pathways: Forkhead box class O proteins (FOXO) transcription factors that regulate two main proteolytic systems, the ubiquitin-proteasome, and the autophagy-lysosome systems, including mitochondrial autophagy, and the MTORC1 signaling associated with protein translation and autophagy inhibition. In recent years, it has been well documented that both acute and chronic endurance exercise can affect the autophagy pathway. Importantly, substantial efforts have been made to better understand discrepancies in the literature on its modulation during exercise. A single bout of endurance exercise increases autophagic flux when the duration is long enough, and this response is dependent on nutritional status, since autophagic flux markers and mRNA coding for actors involved in mitophagy are more abundant in the fasted state. In contrast, strength and resistance exercises preferentially raise ubiquitin-proteasome system activity and involve several protein synthesis factors, such as the recently characterized DAGK for mechanistic target of rapamycin activation. In this review, we discuss recent progress on the impact of acute and chronic exercise on cell component turnover systems, with particular focus on autophagy, which until now has been relatively overlooked in skeletal muscle. We especially highlight the most recent studies on the factors that can impact its modulation, including the mode of exercise and the nutritional status, and also discuss the current limitations in the literature to encourage further works on this topic.

Inhibition of myoblast differentiation by Sfrp1 and Sfrp2

Secreted Frizzled-related proteins (Sfrps) are extracellular regulators of Wnt signalling and play important roles in developmental and oncogenic processes. They are known to be upregulated in regenerating muscle and in myoblast cultures but their function is unknown. Here, we show that the addition of recombinant Sfrp1 or Sfrp2 to C2C12 cell line cultures or to primary cultures of satellite cells results in the inhibition of myotube formation with no significant effect on the cell cycle or apoptosis. Even though at confluence, treated and untreated cultures are identical in appearance, analyses have shown that, for maximum effect, the cells have to be treated while they are proliferating. Furthermore, removal of Sfrp from the culture medium during differentiation restores normal myotube formation. We conclude that Sfrp1 and Sfrp2 act to prevent myoblasts from entering the terminal differentiation process.

Inhibition of autocrine secretion of myostatin enhances terminal differentiation in human rhabdomyosarcoma cells

Rhabdomyosarcomas (RMSs) are one of the most common solid tumor of childhood. Rhabdomyosarcoma (RMS) cells fail to both complete the skeletal muscle differentiation program and irreversibly exit the cell cycle as a consequence of an active repression exerted on the muscle-promoting factor MyoD. Myostatin is a negative regulator of normal muscle growth, we have thus studied its possible role in RMS cells. Here, we present evidence that overexpression of myostatin is a common feature of RMS since both subtypes of RMS (embryonal RD and alveolar Rh30 cells) express high levels of myostatin when compared to nontumoral skeletal muscle cells. Interestingly, we found that inactivation of myostatin through overexpression of antisense myostatin or of follistatin (a myostatin antagonist) constructs enhanced differentiation of RD cells. In addition, RD and Rh30 cells treated with blocking antimyostatin antibodies progress into the myogenic terminal differentiation program. Finally, our results suggest that high levels of myostatin could impair MyoD function in RMS cells. These results show that an autocrine myostatin loop contributes to maintain RMS cells in an undifferentiating stage and suggest that new therapeutic approaches could be exploited for the treatment of RMS based on inactivation of myostatin protein.

Wnt4 activates the canonical β-catenin pathway and regulates negatively myostatin: functional implication in myogenesis

Expression of Wnt proteins is known to be important for developmental processes such as embryonic pattern formation and determination of cell fate. Previous studies have shown that Wn4 was involved in the myogenic fate of somites, in the myogenic proliferation, and differentiation of skeletal muscle. However, the function of this factor in adult muscle homeostasis remains not well understood. Here, we focus on the roles of Wnt4 during C2C12 myoblasts and satellite cells differentiation. We analyzed its myogenic activity, its mechanism of action, and its interaction with the anti-myogenic factor myostatin during differentiation. Established expression profiles indicate clearly that both types of cells express a few Wnts, and among these, only Wnt4 was not or barely detected during proliferation and was strongly induced during differentiation. As attested by myogenic factors expression pattern analysis and fusion index determination, overexpression of Wnt4 protein caused a strong increase in satellite cells and C2C12 myoblast differentiation leading to hypertrophic myotubes. By contrast, exposure of satellite and C2C12 cells to small interfering RNA against Wnt4 strongly diminished this process, confirming the myogenic activity of Wnt4. Moreover, we reported that Wnt4, which is usually described as a noncanonical Wnt, activates the canonical β-catenin pathway during myogenic differentiation in both cell types and that this factor regulates negatively the expression of myostatin and the regulating pathways associated with myostatin. Interestingly, we found that recombinant myostatin was sufficient to antagonize the differentiation-promoting activities of Wnt4. Reciprocally, we also found that the genetic deletion of myostatin renders the satellite cells refractory to the hypertrophic effect of Wnt4. These results suggest that the Wnt4-induced decrease of myostatin plays a functional role during hypertrophy. We propose that Wnt4 protein may be a key factor that regulates the extent of differentiation in satellite and C2C12 cells.

Characterization of a null mutation in ace‐1, the gene encoding class A acetylcholinesterase in the nematode Caenorhabditis elegans

Two genes (ace‐1 and ace‐2) encode two major classes (A and B) of acetylcholinesterase (AChE) in the nematode Caenorhabditis elegans. A null mutation in ace‐1 (allele p1000) suppresses all acetylcholinesterase activity of class A. We have identified an opal mutation TG (W99)→TG (Stop) as the only alteration in the mutated gene. This leads to a truncated protein (98 instead of 620 amino acids) with no enzymatic activity. The mutation also reduces the level of ace‐1 transcripts to only 10% of that in wild‐type animals. This most likely results from a destabilization of mRNA containing the nonsense message. In contrast, compensation of class B by class A AChE in the null mutant strain ace‐2 takes place with unchanged ace‐1 mRNA level and enzymatic activity similar to class A AChE.

Rabbit slow and fast skeletal muscle-derived satellite myoblast phenotypes do not involve constitutive differences in the components of the insulin-like growth factor system.

The insulin‐like growth factor (IGF) system is actively involved in the control of proliferation and differentiation of several myogenic cell lines, and phenotypic differences between myoblasts are associated with modifications of the equilibrium of the components of the IGF system. To determine whether this observation is a physiologic feature that also concerns the phenotypes of ex vivo adult satellite myoblasts in primary cell culture, we investigated the IGF system in rabbit slow‐twitch muscle‐derived satellite myoblasts (SSM), which differ phenotypically from fast‐twitch muscle‐derived satellite myoblasts (FSM) by their proliferation and differentiation kinetics in vitro. The expression of IGF‐I and IGF‐II were similar in SSM and FSM as well as their concentrations measured in cell‐conditioned media. Ligand blotting of conditioned media samples indicated the presence of five IGF binding protein (IGFBP) species of Mr 37–40, 32, 30–31, 28, and 24 kDa. The 30–31 kDa doublet was visible in SSM‐conditioned medium only and associated with the presence of a 22‐kDa protein, which may represent a proteolytic fragment. In contrast, the 32‐kDa band was observed in FSM‐conditioned medium only. The other IGFBP moieties were present in both SSM‐ and FSM‐conditioned media. Cross‐linking experiments revealed the presence of the M6P/IGF‐II receptor on both SSM and FSM membranes. We also observed an IGF‐I receptor form bearing unusual high affinity for IGF‐II: the binding of [125I]IGF‐I on this receptor was preferentially displaced by IGF‐I but that of [125I]IGF‐II was mostly inhibited by IGF‐II, suggesting that the two tracers did not bind on the same epitopes. [125I]IGF‐II binding to this receptor was greater on SSM than on FSM membranes. Autophosphorylation of WGA‐purified receptors revealed an ∼400‐kDa band after SDS‐PAGE under nonreducing conditions, which corresponded to the α2β2 form of the IGF‐I receptor, and two β subunit moieties of Mr 101 and 105 kDa under reducing conditions in both SSM and FSM extracts. Phosphorylation of the 105‐kDa moiety was more intensively increased than that of the 101‐kDa protein after growth factor stimulation. Basal phosphorylation state of the two β subunits was similarly stimulated by IGF‐I and IGF‐II and less by insulin. Since both insulin and IGF‐I receptors were expressed in FSM and SSM, one of the two β subunits may actually correspond to that of the insulin receptor. We conclude that the IGF system is not considerably affected by the phenotypes of SSM and FSM. The differences observed, which mostly concern IGFBP species, more likely appear as regulatory adaptations than as phenotypic changes targeting the components of the IGF system.

The atypical α2β2 IGF receptor expressed in inducible c2.7 myoblasts is derived from post-translational modifications of the mouse IGF-I receptor

OBJECTIVE Unlike parental permissive C2.7 myoblasts, inducible C2.7 myoblasts require IGF-I or IGF-II to differentiate and expression of MyoD is not constitutive. Our previous studies indicated that inducible myoblasts express an atypical alpha2beta2 IGF receptor that differs from the classical IGF-I receptor by its higher affinity for IGF-II compared with IGF-I and the higher molecular weight of its alpha and beta subunits. Expression of this atypical IGF-I receptor is developmentally regulated; hence this receptor is lost upon terminal differentiation. Muscle cell differentiation is a system in which IGF-II plays an essential role and developmentally regulated atypical IGF-I receptor may represent a candidate for mediating differentiation signals provided by IGF-II. To further understand the structure and the role of the atypical IGF-I receptor, (i) we investigated for a putative IGF-I receptor transcript polymorphism by extensive sequencing of RT-PCR products; (ii) we overexpressed cloned mouse IGF-I receptor in permissive and inducible C2.7 myoblasts and characterized the binding and structural properties of overexpressed IGF-I receptor and (iii) we analysed the effects of this overexpression on myoblasts differentiation. DESIGN Cultured mouse myoblasts C2.7 and subclone variant inducible C2.7 cell lines were used. Mouse IGF-I receptor cDNA was cloned by cDNA library screening. Gene expression was measured by semi-quantitative RT-PCR analysis and receptor affinity by ligand binding. Receptor protein autophosphorylation of IGF-IR was analysed by immunoprecipitation and Western blot. Myoblastic differentiation was accessed by myogenic factors expression and immunofluorescence study. RESULTS Atypical IGF-I receptor may correspond to a new receptor belonging to the insulin/IGF-I receptor family, or it may also derive from alternate splicing of the gene of the insulin/IGF-I receptors and/or post-translational modifications of the insulin/IGF-I receptors. Our results exclude the existence of a polymorphism of the IGF-I receptor transcripts in inducible and permissive myoblasts. In embryo and cancer cells IGF-II binds to insulin receptor (IR) isoform A, RT-PCR experiments show that IR is expressed in permissive but not in inducible myoblasts. We demonstrated here that post-translational processing of the mouse IGF-I receptor is responsible for the existence of the mouse atypical IGF-I receptor in inducible myoblasts. Overexpressed mouse IGF-I receptor in permissive myoblasts has the same biochemical and binding characteristics as the classical IGF-I receptor whereas in inducible myoblasts, overexpressed mouse IGF-I receptor has the biochemical, binding and functional characteristics of the atypical IGF-I receptor. CONCLUSIONS Our results provide experimental evidence that the atypical IGF-I receptor variant expressed in subclone inducible C2.7 is issued from a post-translational processing of mouse IGF-I receptor. We show that this post-translational modification is closely associated with the cell lines indeed permissive C2.7 myoblasts process mouse cDNA IGF-I receptor as a classical IGF-I receptor whereas inducible C2.7 myoblasts process mouse cDNA IGF-I receptor as an atypical IGF-I receptor. On other hand, we show that overexpression of mouse IGF-I receptor in inducible myoblasts does not abrogate IGF-I or IGF-II requirement to differentiate.

Role and Function of Wnts in the Regulation of Myogenesis: When Wnt Meets Myostatin

Wnt glycolipoproteins are extracellular ligands that can be found in many species, ranging from the sea anemone to human [1]. Wnts are signaling factors regulating several key developmental processes, such as proliferation, differentiation, asymmetric division, patterning and cell fate determination [2,3]. The Wnt family consists of 19 lipid modified secreted glycoproteins that are primarily divided into two main categories based on their role in cytosolic -catenin stabilization: canonic and non canonic [4,5,6]. During canonical Wnt signaling, binding of Wnt ligands to Frizzled/low-density lipoprotein-related protein (LRP) receptor complexes causes a stabilization of -catenin, which is normally degraded by axin/glycogen synthase kinase-3 (GSK-3)/adenomatous polyposis coli (APC) complexes. Stabilized -catenin is then able to translocate to the nucleus and through interactions with the T-cell factor (Tcf)/ lymphoid enhancer factor 1 (LEF-1), modulates the expression of specific genes [7]. These genes, by regulating cell proliferation, differentiation, adhesion, morphogenesis are involved in various essential physiological and physiopathological processes as embryonic and adult development, cellular and tissular homeostasis, and diseases [8,9,10,11,12]. In contrast, the less-characterized non-canonical Wnt pathways are independent of -catenin and transduce Wnt signals through numerous signaling, including either c-Jun NH2-terminal kinases (JNK)/planar cell polarity or Wnt/calcium pathways [13,14,15,16,17].