Sphingosine 1-phosphate lyase blockade elicits myogenic differentiation of murine myoblasts acting via Spns2/S1P2 receptor axis

The bioactive sphingolipid sphingosine 1-phosphate (S1P) has emerged in the last three decades as main regulator of key cellular processes including cell proliferation, survival, migration and differentiation. A crucial role for this sphingolipid has been recognized in skeletal muscle cell biology both in vitro and in vivo. S1P lyase (SPL) is responsible for the irreversible degradation of S1P and together with sphingosine kinases, the S1P producing enzymes, regulates cellular S1P levels. In this study is clearly showed that the blockade of SPL by pharmacological or RNA interference approaches induces myogenic differentiation of C2C12 myoblasts. Moreover, down-regulation of the specific S1P transporter spinster homolog 2 (Spns2) abrogates myogenic differentiation brought about by SPL inhibition or down-regulation, pointing at a role of extracellular S1P in the pro-myogenic action induced by SPL blockade. Furthermore, also S1P₂ receptor down-regulation was found to abrogate the pro-myogenic effect of SPL blockade. These results provide further proof that inside-out S1P signaling is critically implicated in skeletal muscle biology and provide support to the concept that the specific targeting of SPL could represent an exploitable strategy to treat skeletal muscle disorders.     

Transforming Growth Factor-β1 Induces Transdifferentiation of Myoblasts into Myofibroblasts via Up-Regulation of Sphingosine Kinase-1/S1P3 Axis

The pleiotropic cytokine transforming growth factor (TGF)-β1 is a key player in the onset of skeletal muscle fibrosis, which hampers tissue repair. However, the molecular mechanisms implicated in TGFβ1-dependent transdifferentiation of myoblasts into myofibroblasts are presently unknown. Here, we show that TGFβ1 up-regulates sphingosine kinase (SK)-1 in C2C12 myoblasts in a Smad-dependent manner, and concomitantly modifies the expression of sphingosine 1-phosphate (S1P) receptors (S1PRs). Notably, pharmacological or short interfering RNA-mediated inhibition of SK1 prevented the induction of fibrotic markers by TGFβ1. Moreover, inhibition of S1P₃, which became the highest expressed S1PR after TGFβ1 challenge, strongly attenuated the profibrotic response to TGFβ1. Furthermore, downstream of S1P₃, Rho/Rho kinase signaling was found critically implicated in the profibrotic action of TGFβ1. Importantly, we demonstrate that SK/S1P axis, known to play a key role in myogenesis via S1P₂, consequently to TGFβ1-dependent S1PR pattern remodeling, becomes responsible for transmitting a profibrotic, antidifferentiating action. This study provides new compelling information on the mechanism by which TGFβ1 gives rise to fibrosis in skeletal muscle, opening new perspectives for its pharmacological treatment. Moreover, it highlights the pleiotropic role of SK/S1P axis in skeletal myoblasts that, depending on the expressed S1PR pattern, seems capable of eliciting multiple, even contrasting biological responses.     

Sphingosine kinase-1/S1P1 signalling axis negatively regulates mitogenic response elicited by PDGF in mouse myoblasts

PDGF is known to be critically implicated in skeletal muscle repair; however its molecular mechanism of action has been only marginally investigated. In this study we show that in mouse myoblasts PDGF transactivates S1P₁ receptor via sphingosine kinase (SK)-1 activation and that this molecular event exerts a negative regulation of the mitogenic effect elicited by this growth factor. Indeed, pharmacological inhibition of S1P₁, or its specific silencing increased PDGF-dependent cell proliferation, whereas S1P₁ overexpression diminished the biological effect. Moreover, the mitogenic response to PDGF was enhanced by pharmacological inhibition of SK activity as well as specific silencing of SK1 but not SK2. Furthermore, ERK1/2 signalling pathway was found to be upstream of the observed attenuation of PDGF-induced cell proliferation. Interestingly, PDGF-directed engagement of S1P₁ exerted also a positive modulatory action of the growth factor-dependent cell motility. The here highlighted dual role of S1P₁-mediated signalling in response to myoblast challenge with PDGF is likely important to guarantee the fine control of the biological response to this growth factor, finalized to efficient repopulation of skeletal muscle after damage, where a tight balance between proliferation and migration of tissue progenitor cells is required.     

S100B engages RAGE or bFGF/FGFR1 in myoblasts depending on its own concentration and myoblast density. Implications for muscle regeneration

In high-density myoblast cultures S100B enhances basic fibroblast growth factor (bFGF) receptor 1 (FGFR1) signaling via binding to bFGF and blocks its canonical receptor, receptor for advanced glycation end-products (RAGE), thereby stimulating proliferation and inhibiting differentiation. Here we show that upon skeletal muscle injury S100B is released from myofibers with maximum release at day 1 post-injury in coincidence with satellite cell activation and the beginning of the myoblast proliferation phase, and declining release thereafter in coincidence with reduced myoblast proliferation and enhanced differentiation. By contrast, levels of released bFGF are remarkably low at day 1 post-injury, peak around day 5 and decline thereafter. We also show that in low-density myoblast cultures S100B binds RAGE, but not bFGF/FGFR1 thereby simultaneously stimulating proliferation via ERK1/2 and activating the myogenic program via p38 MAPK. Clearance of S100B after a 24-h treatment of low-density myoblasts results in enhanced myotube formation compared with controls as a result of increased cell numbers and activated myogenic program, whereas chronic treatment with S100B results in stimulation of proliferation and inhibition of differentiation due to a switch of the initial low-density culture to a high-density culture. However, at relatively high doses, S100B stimulates the mitogenic bFGF/FGFR1 signaling in low-density myoblasts, provided bFGF is present. We propose that S100B is a danger signal released from injured muscles that participates in skeletal muscle regeneration by activating the promyogenic RAGE or the mitogenic bFGF/FGFR1 depending on its own concentration, the absence or presence of bFGF, and myoblast density.     

ERK1/2 is required for myoblast proliferation but is dispensable for muscle gene expression and cell fusion

Skeletal muscle satellite cells, which are found between the muscle fiber and the basal lamina, remain quiescent and undifferentiated unless stimulated to remodel skeletal muscle or repair injured skeletal muscle tissue. Quiescent satellite cells express c-met and fibroblast growth factor receptors (FGFR) 1 and 4, suggesting these receptors are involved in maintaining the undifferentiated quiescent state or involved in satellite cell activation. Although the signaling pathways involved are poorly understood, the mitogen activated protein kinase (MAPK) cascade has been implicated in the regulation of skeletal muscle growth and differentiation by FGFs. In this study, we investigated if activation of the Raf-MKK1/2-ERK1/2 signaling cascade plays a role in FGF-dependent repression of differentiation and proliferation of MM14 cells, a skeletal muscle satellite cell line. Inactivation ofthe Raf-MKK1/2-ERK1/2 pathway in myoblasts through the overexpression of dominant negative mutants of Raf-1 blocks ERK1/2 activity and prevents myoblast proliferation. Additionally, inhibition of MKK1/2 by treatment with pharmacological inhibitors also blocks FGF-mediated stimulation of ERK1/2 and blocks the G1 to S phase transition of myoblasts. Unexpectedly, we found that inactivation of the Raf-ERK pathway does not activate a muscle reporter, nor does inactivation of this pathway promote myogenic differentiation. We conclude that FGF-stimulated ERK1/2 signaling is required during the G1 phase of the cell cycle for commitment of myoblasts to DNA synthesis but is not required for mitosis once cells have entered the S-phase. Moreover, ERK1/2 signaling is not required either to repress differentiation, to promote skeletal muscle gene expression, or to promote myoblast fusion.     

Down-regulation of EDG5/S1P2 during myogenic differentiation results in the specific uncoupling of sphingosine 1-phosphate signalling to phospholipase D

The bioactive lipid sphingosine 1-phosphate (S1P) is known to exert powerful biological effects through the interaction with various members of the endothelial differentiation gene (EDG) receptor family, recently renamed S1P receptors. In the present study, evidence is provided that differentiation of C2C12 myoblasts into myotubes was accompanied by profound changes of EDG/S1P receptor expression. Indeed, in differentiated cells a significant increase of EDG3/S1P3 together with a large decrease of EDG5/S1P2 expression at mRNA as well as protein level was detected. Moreover, S1P was capable to initiate the signalling pathways downstream to cytosolic Ca(2+) increase in myotubes, similarly to that observed in myoblasts, whereas the signalling of the bioactive lipid to phospholipase D (PLD), but not that of bradykinin (BK) or lysophosphatidic acid (LPA), was found impaired in differentiated cells. Intriguingly, overexpression of EDG5/S1P2, but not EDG1/S1P1 or EDG3/S1P3, potentiated the efficacy of S1P to stimulate PLD, strongly suggesting a role for EDG5/S1P2 in the signalling to PLD. This view was also supported by the marked reduction of S1P-induced PLD activity in myoblasts loaded with antisense oligodeoxyribonucleotides (ODN) to EDG5/S1P2. Furthermore, overexpression of EDG5/S1P2 rescued the coupling of S1P signalling to PLD in C2C12 myotubes. Experimental evidence here provided supports the notion that EDG5/S1P2 plays a dominant role in the coupling of S1P to PLD in myoblasts and that the down-regulation of the receptor subtype is responsible for the specific uncoupling of S1P signalling to PLD in myotubes.     

Amphoterin stimulates myogenesis and counteracts the antimyogenic factors basic fibroblast growth factor and S100B via RAGE binding

The receptor for advanced glycation end products (RAGE), a multiligand receptor of the immunoglobulin superfamily, has been implicated in the inflammatory response, diabetic angiopathy and neuropathy, neurodegeneration, cell migration, tumor growth, neuroprotection, and neuronal differentiation. We show here that (i) RAGE is expressed in skeletal muscle tissue and its expression is developmentally regulated and (ii) RAGE engagement by amphoterin (HMGB1), a RAGE ligand, in rat L6 myoblasts results in stimulation of myogenic differentiation via activation of p38 mitogen-activated protein kinase (MAPK), up-regulation of myogenin and myosin heavy chain expression, and induction of muscle creatine kinase. No such effects were detected in myoblasts transfected with a RAGE mutant lacking the transducing domain or myoblasts transfected with a constitutively inactive form of the p38 MAPK upstream kinase, MAPK kinase 6, Cdc42, or Rac-1. Moreover, amphoterin counteracted the antimyogenic activity of the Ca(2+)-modulated protein S100B, which was reported to inhibit myogenic differentiation via inactivation of p38 MAPK, and basic fibroblast growth factor (bFGF), a known inhibitor of myogenic differentiation, in a manner that was inversely related to the S100B or bFGF concentration and directly related to the extent of RAGE expression. These data suggest that RAGE and amphoterin might play an important role in myogenesis, accelerating myogenic differentiation via Cdc42-Rac-1-MAPK kinase 6-p38 MAPK.     

Interaction of scaffolding adaptor protein Gab1 with tyrosine phosphatase SHP2 negatively regulates IGF-I-dependent myogenic differentiation via the ERK1/2 signaling pathway

Grb2-associated binder 1 (Gab1) coordinates various receptor tyrosine kinase signaling pathways. Although skeletal muscle differentiation is regulated by some growth factors, it remains elusive whether Gab1 coordinates myogenic signals. Here, we examined the molecular mechanism of insulin-like growth factor-I (IGF-I)-mediated myogenic differentiation, focusing on Gab1 and its downstream signaling. Gab1 underwent tyrosine phosphorylation and subsequent complex formation with protein-tyrosine phosphatase SHP2 upon IGF-I stimulation in C2C12 myoblasts. On the other hand, Gab1 constitutively associated with phosphatidylinositol 3-kinase regulatory subunit p85. To delineate the role of Gab1 in IGF-I-dependent signaling, we examined the effect of adenovirus-mediated forced expression of wild-type Gab1 (Gab1(WT)), mutated Gab1 that is unable to bind SHP2 (Gab1(DeltaSHP2)), or mutated Gab1 that is unable to bind p85 (Gab1(Deltap85)), on the differentiation of C2C12 myoblasts. IGF-I-induced myogenic differentiation was enhanced in myoblasts overexpressing Gab1(DeltaSHP2), but inhibited in those overexpressing either Gab1(WT) or Gab1(Deltap85). Conversely, IGF-I-induced extracellular signal-regulated kinase 1/2 (ERK1/2) activation was significantly repressed in myoblasts overexpressing Gab1(DeltaSHP2) but enhanced in those overexpressing either Gab1(WT) or Gab1(Deltap85). Furthermore, small interference RNA-mediated Gab1 knockdown enhanced myogenic differentiation. Overexpression of catalytic-inactive SHP2 modulated IGF-I-induced myogenic differentiation and ERK1/2 activation similarly to that of Gab1(DeltaSHP2), suggesting that Gab1-SHP2 complex inhibits IGF-I-dependent myogenesis through ERK1/2. Consistently, the blockade of ERK1/2 pathway reversed the inhibitory effect of Gab1(WT) overexpression on myogenic differentiation, and constitutive activation of the ERK1/2 pathway suppressed the enhanced myogenic differentiation by overexpression of Gab1(DeltaSHP2). Collectively, these data suggest that the Gab1-SHP2-ERK1/2 signaling pathway comprises an inhibitory axis for IGF-I-dependent myogenic differentiation.     

Skeletal muscle cell activation by low-energy laser irradiation: a role for the MAPK/ERK pathway

Low-energy laser irradiation (LELI) has been shown to promote skeletal muscle regeneration in vivo and to activate skeletal muscle satellite cells, enhance their proliferation and inhibit differentiation in vitro. In the present study, LELI, as well as the addition of serum to serum-starved myoblasts, restored their proliferation, whereas myogenic differentiation remained low. LELI induced mitogen-activated protein kinase/extracellular signal-regulated protein kinase (MAPK/ERK) phosphorylation with no effect on its expression in serum-starved myoblasts. Moreover, a specific MAPK kinase inhibitor (PD098059) inhibited the LELI- and 10% serummediated ERK1/2 activation. However, LELI did not affect Jun N-terminal kinase (JNK) or p38 MAPK phosphorylation or protein expression. Whereas a 3-sec irradiation induced ERK1/2 phosphorylation, a 12-sec irradiation reduced it, again with no effect on JNK or p38. Moreover, LELI had distinct effects on receptor phosphorylation: it caused phosphorylation of the hepatocyte growth factor (HGF) receptor, previously shown to activate the MAPK/ERK pathway, whereas no effect was observed on tumor suppressor necrosis alpha (TNF-alpha) receptor which activates the p38 and JNK pathways. Therefore, by specifically activating MAPK/ERK, but not JNK and p38 MAPK enzymes, probably by specific receptor phosphorylation, LELI induces the activation and proliferation of quiescent satellite cells and delays their differentiation.     

Effects of S1P on skeletal muscle repair/regeneration during eccentric contraction

Skeletal muscle regeneration is severely compromised in the case of extended damage. The current challenge is to find factors capable of limiting muscle degeneration and/or potentiating the inherent regenerative program mediated by a specific type of myoblastic cells, the satellite cells. Recent studies from our groups and others have shown that the bioactive lipid, sphingosine 1-phosphate (S1P), promotes myoblast differentiation and exerts a trophic action on denervated skeletal muscle fibres. In the present study, we examined the effects of S1P on eccentric contraction (EC)-injured extensor digitorum longus muscle fibres and resident satellite cells. After EC, skeletal muscle showed evidence of structural and biochemical damage along with significant electrophysiological changes, i.e. reduced plasma membrane resistance and resting membrane potential and altered Na(+) and Ca(2+) current amplitude and kinetics. Treatment with exogenous S1P attenuated the EC-induced tissue damage, protecting skeletal muscle fibre from apoptosis, preserving satellite cell viability and affecting extracellular matrix remodelling, through the up-regulation of matrix metalloproteinase 9 (MMP-9) expression. S1P also promoted satellite cell renewal and differentiation in the damaged muscle. Notably, EC was associated with the activation of sphingosine kinase 1 (SphK1) and with increased endogenous S1P synthesis, further stressing the relevance of S1P in skeletal muscle protection and repair/regeneration. In line with this, the treatment with a selective SphK1 inhibitor during EC, caused an exacerbation of the muscle damage and attenuated MMP-9 expression. Together, these findings are in favour for a role of S1P in skeletal muscle healing and offer new clues for the identification of novel therapeutic approaches to counteract skeletal muscle damage and disease.     

JAK2/STAT2/STAT3 are required for myogenic differentiation

Skeletal muscle satellite cell-derived myoblasts are mainly responsible for postnatal muscle growth and injury-induced regeneration. However, the cellular signaling pathways that control proliferation and differentiation of myoblasts remain poorly defined. Recently, we found that JAK1/STAT1/STAT3 not only participate in myoblast proliferation but also actively prevent them from premature differentiation. Unexpectedly, we found that a related pathway consisting of JAK2, STAT2, and STAT3 is required for early myogenic differentiation. Interference of this pathway by either a small molecule inhibitor or small interfering RNA inhibits myogenic differentiation. Consistently, all three molecules are activated upon differentiation. The pro-differentiation effect of JAK2/STAT2/STAT3 is partially mediated by MyoD and MEF2. Interestingly, the expression of the IGF2 gene and the HGF gene is also regulated by JAK2/STAT2/STAT3, suggesting that this pathway could also promote differentiation by regulating signaling molecules known to be involved in myogenic differentiation. In summary, our current study reveals a novel role for the JAK2/STAT2/STAT3 pathway in myogenic differentiation.     

Ceramide 1-phosphate stimulates proliferation of C2C12 myoblasts

Recent studies have established specific cellular functions for different bioactive sphingolipids in skeletal muscle cells. Ceramide 1-phosphate (C1P) is an important bioactive sphingolipid that has been involved in cell growth and survival. However its possible role in the regulation of muscle cell homeostasis has not been so far investigated. In this study, we show that C1P stimulates myoblast proliferation, as determined by measuring the incorporation of tritiated thymidine into DNA, and progression of the myoblasts through the cell cycle. C1P induced phosphorylation of glycogen synthase kinase-3β and the product of retinoblastoma gene, and enhanced cyclin D1 protein levels. The mitogenic action of C1P also involved activation of phosphatidylinositol 3-kinase/Akt, ERK1/2 and the mammalian target of rapamycin. These effects of C1P were independent of interaction with a putative G(i)-coupled C1P receptor as pertussis toxin, which maintains G(i) protein in the inactive form, did not affect C1P-stimulated myoblast proliferation. By contrast, C1P was unable to inhibit serum starvation- or staurosporine-induced apoptosis in the myoblasts, and did not affect myogenic differentiation. Collectively, these results add up to the current knowledge on cell types targeted by C1P, which so far has been mainly confined to fibroblasts and macrophages, and extend on the mechanisms by which C1P exerts its mitogenic effects. Moreover, the biological activities of C1P described in this report establish that this phosphosphingolipid may be a relevant cue in the regulation of skeletal muscle regeneration, and that C1P-metabolizing enzymes might be important targets for developing cellular therapies for treatment of skeletal muscle degenerative diseases, or tissue injury.     

TRAF6 regulates proliferation and differentiation of skeletal myoblasts

We could recently demonstrate an important role of receptor interacting protein-2 (RIP2), an activator of nuclear factor kappa B (NF-κB) and a target of activated receptors of the tumor necrosis factor receptor (TNFR) type, in myogenic differentiation and regeneration. Here, we analyze a potential role of TNFR associated factor 6 (TRAF6), which also associates with the cytoplasmic domain of TNFR type, but also IL-1-R and TLR type receptors, and activates NF-κB, in these processes. Specifically, we show that during myogenic differentiation in vitro, traf6 gene expression is downregulated in normal myoblasts, but not in rhabdomyosarcoma cells, suggesting a role of the TRAF6 protein in this process. Inhibition of traf6 expression using specific siRNAs led to an inhibition of both myoblast proliferation and differentiation, whereas inhibition of the TRAF6 effector NF-κB alone in our system only blocked proliferation. Finally, we demonstrate that the traf6 gene is downregulated in skeletal muscle tissue of the dystrophic mdx mouse. Taken together, these data argue for a role of TRAF6 in the regulation of skeletal muscle differentiation and regeneration.     

Ebp1 regulates myogenic differentiation of myoblast cells via SMAD2/3 signaling pathway

Regulation of skeletal muscle development requires many of the regulatory networks that are fundamental to developmental myogenesis. ErbB3 binding protein‐1 (Ebp1) is involved in the control of myoblasts development in chicken. However, the expression and biological functions of Ebp1 in the progress of myogenesis are unclear. This study focused on determining the effect of Ebp1 on myogenic proliferation and differentiation using a primary myoblasts culture model. Ebp1 was found to upregulate in proliferating myoblasts and decrease at the early stage of myogenic differentiation. The level of endogenous Ebp1 increased from E9 to E20 chicken leg muscles. Knockdown of Ebp1 had no effect on myoblasts proliferation. However, myogenic differentiation into multinucleated myotubes was significantly reduced. The mRNA and protein expression of MRFs was decreased when Ebp1 was knocked down. Downregulation of Ebp1, accompanied by elevated levels of pSMAD2/3, suggests that Ebp1 is involved in regulating myogenic differentiation via SMAD2/3 inhibition. The phosphorylation of SMAD2/3 was activated and the expression of MYOD and MYOG was reduced in Ebp1 knockdown myoblasts, but addition of LY2109761 (an inhibitor specified to SMAD2/3) blocked these effects. Collectively, these results indicate that Ebp1 promotes myoblast differentiation by inhibition of SMAD2/3 signaling pathway during chicken myogenesis. These data provide new insights into the biological role of Ebp1 in embryonic chicken skeletal muscle development.     

Sphingosine kinase activity is required for myogenic differentiation of C2C12 myoblasts

Sphingosine kinase (SphK) is a conserved lipid kinase that catalyzes the formation of sphingosine 1-phosphate (S1P), an important lipid mediator, which regulates fundamental biological processes. Here, we provide evidence that SphK is required for the achievement of cell growth arrest as well as myogenic differentiation of C2C12 myoblasts. Indeed, SphK activity, SphK1 protein content and S1P formation were found to be enhanced in myoblasts that became confluent as well as in differentiating cells. Enforced expression of SphK1 reduced the myoblast proliferation rate, enhanced the expression of myogenic differentiation markers and anticipated the onset of differentiated muscle phenotype. Conversely, down-regulation of SphK1 by specific silencing by RNA interference or overexpression of the catalytically inactive SphK1, significantly increased cell growth and delayed the beginning of myogenesis; noticeably, exogenous addition of S1P rescued the biological processes. Importantly, stimulation of myogenesis in SphK1-overexpressing myoblasts was abrogated by treatment with short interfering RNA specific for S1P(2) receptor. This is the first report of the role of endogenous SphK1 in myoblast growth arrest and stimulation of myogenesis through the formation of S1P that acts as morphogenic factor via the engagement of S1P(2).     

Trpc1 ion channel modulates phosphatidylinositol 3-kinase/Akt pathway during myoblast differentiation and muscle regeneration

We previously showed in vitro that calcium entry through Trpc1 ion channels regulates myoblast migration and differentiation. In the present work, we used primary cell cultures and isolated muscles from Trpc1(-/-) and Trpc1(+/+) murine model to investigate the role of Trpc1 in myoblast differentiation and in muscle regeneration. In these models, we studied regeneration consecutive to cardiotoxin-induced muscle injury and observed a significant hypotrophy and a delayed regeneration in Trpc1(-/-) muscles consisting in smaller fiber size and increased proportion of centrally nucleated fibers. This was accompanied by a decreased expression of myogenic factors such as MyoD, Myf5, and myogenin and of one of their targets, the developmental MHC (MHCd). Consequently, muscle tension was systematically lower in muscles from Trpc1(-/-) mice. Importantly, the PI3K/Akt/mTOR/p70S6K pathway, which plays a crucial role in muscle growth and regeneration, was down-regulated in regenerating Trpc1(-/-) muscles. Indeed, phosphorylation of both Akt and p70S6K proteins was decreased as well as the activation of PI3K, the main upstream regulator of the Akt. This effect was independent of insulin-like growth factor expression. Akt phosphorylation also was reduced in Trpc1(-/-) primary myoblasts and in control myoblasts differentiated in the absence of extracellular Ca(2+) or pretreated with EGTA-AM or wortmannin, suggesting that the entry of Ca(2+) through Trpc1 channels enhanced the activity of PI3K. Our results emphasize the involvement of Trpc1 channels in skeletal muscle development in vitro and in vivo, and identify a Ca(2+)-dependent activation of the PI3K/Akt/mTOR/p70S6K pathway during myoblast differentiation and muscle regeneration.