A temporal switch from notch to Wnt signaling in muscle stem cells is necessary for normal adult myogenesis

The temporal switch from progenitor cell proliferation to differentiation is essential for effective adult tissue repair. We previously reported the critical role of Notch signaling in the proliferative expansion of myogenic progenitors in mammalian postnatal myogenesis. We now show that the onset of differentiation is due to a transition from Notch signaling to Wnt signaling in myogenic progenitors and is associated with an increased expression of Wnt in the tissue and an increased responsiveness of progenitors to Wnt. Crosstalk between these two pathways occurs via GSK3beta, which is maintained in an active form by Notch but is inhibited by Wnt in the canonical Wnt signaling cascade. These results demonstrate that the temporal balance between Notch and Wnt signaling orchestrates the precise progression of muscle precursor cells along the myogenic lineage pathway, through stages of proliferative expansion and then differentiation, during postnatal myogenesis.     

Inhibition of insulin-like growth factor-1 (IGF-1) expression by prolonged transforming growth factor-β1 (TGF-β1) administration suppresses osteoblast differentiation

TGF-β1 can regulate osteoblast differentiation not only positively but also negatively. However, the mechanisms of negative regulation are not well understood. We previously established the reproducible model for studying the suppression of osteoblast differentiation by repeated or high dose treatment with TGF-β1, although single low dose TGF-β1 strongly induced osteoblast differentiation. The mRNA expression and protein level of insulin-like growth factor-1 (IGF-1) were remarkably decreased by repeated TGF-β1 administration in human periodontal ligament cells, human mesenchymal stem cells, and murine preosteoblast MC3T3-E1 cells. Repeated TGF-β1 administration subsequently decreased alkaline phosphatase (ALP) activity and mRNA expression of osteoblast differentiation marker genes, such as RUNX2, ALP, and bone sialoprotein (BSP). Additionally, repeated administration significantly reduced the downstream signaling pathway of IGF-1, such as Akt phosphorylation in these cells. Surprisingly, exogenous and overexpressed IGF-1 recovered ALP activity and mRNA expression of osteoblast differentiation marker genes even with repeated TGF-β1 administration. These facts indicate that the key mechanism of inhibition of osteoblast differentiation induced by repeated TGF-β1 treatment is simply due to the down-regulation of IGF-1 expression. Inhibition of IGF-1 signaling using small interfering RNA (siRNA) against insulin receptor substrate-1 (IRS-1) suppressed mRNA expression of RUNX2, ALP, BSP, and IGF-1 even with single TGF-β1 administration. This study showed that persistence of TGF-β1 inhibited osteoblast differentiation via suppression of IGF-1 expression and subsequent down-regulation of the PI3K/Akt pathway. We think this fact could open the way to use IGF-1 as a treatment tool for bone regeneration in prolonged inflammatory disease.     

PDGF-PDGFR network differentially regulates the fate,migration, proliferation,and cell cycle progression of myogenic cells

Platelet-derived growth factors (PDGFs) regulate embryonic development, tissue regeneration, and wound healing through their binding to PDGF receptors, PDGFRα and PDGFRβ. However, the role of PDGF signaling in regulating muscle development and regeneration remains elusive, and the cellular and molecular responses of myogenic cells are understudied. Here, we explore the PDGF-PDGFR gene expression changes and their involvement in skeletal muscle myogenesis and myogenic fate. By surveying bulk RNA sequencing and single-cell profiling data of skeletal muscle stem cells, we show that myogenic progenitors and muscle stem cells differentially express PDGF ligands and PDGF receptors during myogenesis. Quiescent adult muscle stem cells and myoblasts preferentially express PDGFRβ over PDGFRα. Remarkably, cell culture- and injury-induced muscle stem cell activation altered PDGF family gene expression. In myoblasts, PDGF-AB and PDGF-BB treatments activate two pro-chemotactic and pro-mitogenic downstream transducers, RAS-ERK1/2 and PI3K-AKT. PDGFRs inhibitor AG1296 inhibited ERK1/2 and AKT activation, myoblast migration, proliferation, and cell cycle progression induced by PDGF-AB and PDGF-BB. We also found that AG1296 causes myoblast G0/G1 cell cycle arrest. Remarkably, PDGF-AA did not promote a noticeable ERK1/2 or AKT activation, myoblast migration, or expansion. Also, myogenic differentiation reduced the expression of both PDGFRα and PDGFRβ, whereas forced PDGFRα expression impaired myogenesis. Thus, our data highlight PDGF signaling pathway to stimulate satellite cell proliferation aiming to enhance skeletal muscle regeneration and provide a deeper understanding of the role of PDGF signaling in non-fibroblastic cells.     

Colonization of the satellite cell niche by skeletal muscle progenitor cells depends on notch signals

Skeletal muscle growth and regeneration rely on myogenic progenitor and satellite cells, the stem cells of postnatal muscle. Elimination of Notch signals during mouse development results in premature differentiation of myogenic progenitors and formation of very small muscle groups. Here we show that this drastic effect is rescued by mutation of the muscle differentiation factor MyoD. However, rescued myogenic progenitors do not assume?a satellite cell position and contribute poorly to myofiber growth. The disrupted homing is due to a deficit in basal lamina assembly around emerging satellite cells and to their impaired adhesion to myofibers. On a molecular level, emerging satellite cells deregulate the expression of basal lamina components and adhesion molecules like integrin α7, collagen XVIIIα1, Megf10, and Mcam. We conclude that Notch signals control homing of satellite cells, stimulating them to?contribute to their own microenvironment and to adhere to myofibers.     

Wnt/beta-catenin pathway activation and myogenic differentiation are induced by cholesterol depletion

Myogenic differentiation is a multistep process that begins with the commitment of mononucleated precursors that withdraw from cell cycle. These myoblasts elongate while aligning to each other, guided by the recognition between their membranes. This step is followed by cell fusion and the formation of long and striated multinucleated myotubes. We have recently shown that cholesterol depletion by methyl-beta-cyclodextrin (MbetaCD) induces myogenic differentiation by enhancing myoblast recognition and fusion. Here, we further studied the signaling pathways responsible for early steps of myogenesis. As it is known that Wnt plays a role in muscle differentiation, we used the chemical MbetaCD to deplete membrane cholesterol and investigate the involvement of the Wnt/beta-catenin pathway during myogenesis. We show that cholesterol depletion promoted a significant increase in expression of beta-catenin, its nuclear translocation and activation of the Wnt pathway. Moreover, we show that the activation of the Wnt pathway after cholesterol depletion can be inhibited by the soluble protein Frzb-1. Our data suggest that membrane cholesterol is involved in Wnt/beta-catenin signaling in the early steps of myogenic differentiation.     

Emerin expression at the early stages of myogenic differentiation

Emerin is an ubiquitous protein localized at the nuclear membrane of most cell types including muscle cells. The protein is absent in most patients affected by the X-linked form of Emery-Dreifuss muscular dystrophy, a disease characterized by slowly progressive muscle wasting and weakness, early contractures of the elbows, Achilles tendons, and post-cervical muscles, and cardiomyopathy. Besides the nuclear localization, emerin cytoplasmic distribution has been suggested in several cell types. We studied the expression and the subcellular distribution of emerin in mouse cultured C2C12 myoblasts and in primary cultures of human myoblasts induced to differentiate or spontaneously differentiating in the culture medium. In differentiating myoblasts transiently transfected with a cDNA encoding the complete emerin sequence, the protein localized at the nuclear rim of all transfected cells and also in the cytoplasm of some myoblasts and myotubes. Cytoplasmic emerin was also observed in detergent-treated myotubes, as determined by electron microscopy observation. Both immunofluorescence and biochemical analysis showed, that upon differentiation of C2C12 cells, emerin expression was decreased in the resting myoblasts but the protein was highly represented in the developing myotubes at the early stage of cell fusion. Labeling with specific markers of myogenesis such as troponin-T and myogenin permitted the correlation of increased emerin expression with the onset of muscle differentiation. These data suggest a role for emerin during proliferation of activated satellite cells and at the early stages of differentiation.     

Wnt10b deficiency promotes coexpression of myogenic and adipogenic programs in myoblasts

Adult myoblasts retain plasticity in developmental potential and can be induced to undergo myogenic, adipogenic, or osteoblastogenic differentiation in vitro. In this report, we show that the balance between myogenic and adipogenic potential in myoblasts is controlled by Wnt signaling. Furthermore, this balance is altered during aging such that aspects of both differentiation programs are coexpressed in myoblasts due to decreased Wnt10b abundance. Mimicking Wnt signaling in aged myoblasts through inhibition of glycogen synthase kinase or through overexpression of Wnt10b resulted in inhibition of adipogenic gene expression and sustained or enhanced myogenic differentiation. On the other hand, myoblasts isolated from Wnt10b null mice showed increased adipogenic potential, likely contributing to excessive lipid accumulation in actively regenerating myofibers in vivo in Wnt10b-/- mice. Whereas Wnt10b deficiency contributed to increased adipogenic potential in myoblasts, the augmented myogenic differentiation potential observed is likely the result of a compensatory increase in Wnt7b during differentiation of Wnt10b-/- myoblasts. No such compensation was apparent in aged myoblasts and in fact, both Wnt5b and Wnt10b were down-regulated. Thus, alteration in Wnt signaling in myoblasts with age may contribute to impaired muscle regenerative capacity and to increased muscle adiposity, both characteristic of aged muscle.     

Menin expression modulates mesenchymal cell commitment to the myogenic and osteogenic lineages

Menin plays an established role in the differentiation of mesenchymal cells to the osteogenic lineage. Conversely, whether Menin influences the commitment of mesenschymal cells to the myogenic lineage, despite expression in the developing somite was previously unclear. We observed that Menin is down-regulated in C2C12 and C3H10T1/2 mesenchymal cells when muscle differentiation is induced. Moreover, maintenance of Menin expression by constitutive ectopic expression inhibited muscle cell differentiation. Reduction of Menin expression by siRNA technology results in precocious muscle differentiation and concomitantly attenuates BMP-2 induced osteogenesis. Reduced Menin expression antagonizes BMP-2 and TGF-β1 mediated inhibition of myogenesis. Furthermore, Menin was found to directly interact with and potentiate the transactivation properties of Smad3 in response to TGF-β1. Finally in concert with these observations, tissue-specific inactivation of Men1 in Pax3-expressing somite precursor cells leads to a patterning defect of rib formation and increased muscle mass in the intercostal region. These data invoke a pivotal role for Menin in the competence of mesenchymal cells to respond to TGF-β1 and BMP-2 signals. Thus, by modulating cytokine responsiveness Menin functions to alter the balance of multipotent mesenchymal cell commitment to the osteogenic or myogenic lineages.     

Hedgehog and Wnt coordinate signaling in myogenic progenitors and regulate limb regeneration

Amphibians have a remarkable capacity for limb regeneration. Following a severe injury, there is complete regeneration with restoration of the patterning and cellular architecture of the amputated limb. While studies have focused on the structural anatomical changes during amphibian limb regeneration, the signaling mechanisms that govern cellular dedifferentiation and blastemal progenitors are unknown. Here, we demonstrate the temporal and spatial requirement for hedgehog (Hh) signaling and its hierarchical correlation with respect to Wnt signaling during newt limb regeneration. While the dedifferentiation process of mature lineages does not depend on Hh signaling, the proliferation and the migration of the dedifferentiated cells are dependent on Hh signaling. Temporally controlled chemical inactivation of the Hh pathway indicates that Hh-mediated antero-posterior (AP) specification occurs early during limb regeneration and that Hh is subsequently required for expansion of the blastemal progenitors. Inhibition of Hh signaling results in G0/G1 arrest with a concomitant reduction in S-phase and G2/M population in myogenic progenitors. Furthermore, Hh inhibition leads to reduced Pax7-positive cells and fewer regenerating fibers relative to control tissue. We demonstrate that activation of Wnt signaling rescues the inhibition of Hh pathway mainly by enhancing proliferative signals, possibly mediated through TCF4 activity. Collectively, our results demonstrate coordinated signaling of Hh and Wnt activities in regulating blastemal progenitors and their hierarchical positioning during limb regeneration.     

Changes at P183 of emerin weaken its protein-protein interactions resulting in X-linked Emery-Dreifuss muscular dystrophy

Emery-Dreifuss muscular dystrophy (EDMD) is an X-linked recessive muscular dystrophy characterized by early contractures of the elbows, Achilles tendons and spine, slowly progressive muscle wasting and weakness, and cardiomyopathy associated with cardiac conduction defects. The emerin gene has been mapped to Xq28 and encodes a 34-kDa serine-rich protein, emerin, which has been localized to the nuclear envelope in a wide variety of tissues, including skeletal and cardiac muscle. Mutations spanning the emerin gene have been identified in patients with EDMD. We present here the effect, on emerin protein expression, of two missense mutations identified in unrelated EDMD patients. These alterations predict the replacement of a proline residue at position 183 with either a histidine or a threonine. Biochemical analysis has demonstrated that the mobility and expression levels of the mutant forms of emerin are indistinguishable from that of wild-type emerin, but that they have weakened interactions with nuclear lamina components. In comparison with the usual EDMD phenotype, patients with P183 missense mutations have a later age at onset of first symptoms, elbow contractures, ankle contractures, upper limb weakness and lower limb weakness, but there is no difference for the age at onset of cardiac involvement. This is the first report of protein studies on patients with missense mutations resulting in the clinical features of EDMD. These studies demonstrate the importance of proline 183 for the proper structure/function of emerin. Received: 29 September 1998 / Accepted: 14 January 1999     

Notch signalling acts in postmitotic avian myogenic cells to control MyoD activation

During Drosophila myogenesis, Notch signalling acts at multiple steps of the muscle differentiation process. In vertebrates, Notch activation has been shown to block MyoD activation and muscle differentiation in vitro, suggesting that this pathway may act to maintain the cells in an undifferentiated proliferative state. In this paper, we address the role of Notch signalling in vivo during chick myogenesis. We first demonstrate that the Notch1 receptor is expressed in postmitotic cells of the myotome and that the Notch ligands Delta1 and Serrate2 are detected in subsets of differentiating myogenic cells and are thus in position to signal to Notch1 during myogenic differentiation. We also reinvestigate the expression of MyoD and Myf5 during avian myogenesis, and observe that Myf5 is expressed earlier than MyoD, consistent with previous results in the mouse. We then show that forced expression of the Notch ligand, Delta1, during early myogenesis, using a retroviral system, has no effect on the expression of the early myogenic markers Pax3 and Myf5, but causes strong down-regulation of MyoD in infected somites. Although Delta1 overexpression results in the complete lack of differentiated muscles, detailed examination of the infected embryos shows that initial formation of a myotome is not prevented, indicating that exit from the cell cycle has not been blocked. These results suggest that Notch signalling acts in postmitotic myogenic cells to control a critical step of muscle differentiation.     

Role of integrin-β3 protein in macrophage polarization and regeneration of injured muscle

Following injury, skeletal muscle achieves repair by a highly coordinated, dynamic process resulting from interplay among numerous inflammatory, growth factors and myogenic regulators. To identify genes involved in muscle regeneration, we used a microarray analysis; there was a significant increase in the expression of a group of integrin genes. To verify these results, we used RT-PCR and Western blotting and found that 12 integrins were up-regulated from 3 h to 15 days following injury. Following muscle injury, integrin-β3 was initially expressed, mainly in macrophages. In integrin-β3 global KO mice, the expression of myogenic genes was decreased and muscle regeneration was impaired, whereas fibrosis was enhanced versus events in wild type (WT) mice. The mechanism for these responses in integrin-β3 KO mice included an infiltration of macrophages that were polarized into the M2 phenotype. These macrophages produced more TGF-β1 and increased TGF-β1/Smad signaling. In vitro, we confirmed that M2 macrophages lacking integrin-β3 produced more TGF-β1. Furthermore, transplantation of bone marrow cells from integrin-β3 KO mice into WT mice led to suppression of the infiltration and accumulation of macrophages into injured muscles. There was also impaired muscle regeneration with an increase in muscle fibrosis. Our results demonstrate that integrin-β3 plays a fundamental role in muscle regeneration through a regulation of macrophage infiltration and polarization leading to suppressed TGF-β1 production. This promotes efficient muscle regeneration. Thus, an improvement in integrin-β3 function could stimulate muscle regeneration.     

TGF-β mediated FGF10 signaling in cranial neural crest cells controls development of myogenic progenitor cells through tissue-tissue interactions during tongue morphogenesis

Skeletal muscles are formed from two cell lineages, myogenic and fibroblastic. Mesoderm-derived myogenic progenitors form muscle cells whereas fibroblastic cells give rise to the supportive connective tissue of skeletal muscles, such as the tendons and perimysium. It remains unknown how myogenic and fibroblastic cell-cell interactions affect cell fate determination and the organization of skeletal muscle. In the present study, we investigated the functional significance of cell-cell interactions in regulating skeletal muscle development. Our study shows that cranial neural crest (CNC) cells give rise to the fibroblastic cells of the tongue skeletal muscle in mice. Loss of Tgfbr2 in CNC cells (Wnt1-Cre;Tgfbr2^flox/flox) results in microglossia with reduced Scleraxis and Fgf10 expression as well as decreased myogenic cell proliferation, reduced cell number and disorganized tongue muscles. Furthermore, TGF-β2 beads induced the expression of Scleraxis in tongue explant cultures. The addition of FGF10 rescued the muscle cell number in Wnt1-Cre;Tgfbr2^flox/flox mice. Thus, TGF-β induced FGF10 signaling has a critical function in regulating tissue-tissue interaction during tongue skeletal muscle development.