Yusuke Ono

Muscle satellite cells are a functionally heterogeneous population in both somite-derived and branchiomeric muscles

Skeletal muscles of body and limb are derived from somites, but most head muscles originate from cranial mesoderm. The resident stem cells of muscle are satellite cells, which have the same embryonic origin as the muscle in which they reside. Here, we analysed satellite cells with a different ontology, comparing those of the extensor digitorum longus (EDL) of the limb with satellite cells from the masseter of the head. Satellite cell-derived myoblasts from MAS and EDL muscles had distinct gene expression profiles and masseter cells usually proliferated more and differentiated later than those from EDL. When transplanted, however, masseter-derived satellite cells regenerated limb muscles as efficiently as those from EDL. Clonal analysis showed that functional properties differed markedly between satellite cells: ranging from clones that proliferated extensively and gave rise to both differentiated and self-renewed progeny, to others that divided minimally before differentiating completely. Generally, masseter-derived clones were larger and took longer to differentiate than those from EDL. This distribution in cell properties was preserved in both EDL-derived and masseter-derived satellite cells from old mice, although clones were generally less proliferative. Satellite cells, therefore, are a functionally heterogeneous population, with many occupants of the niche exhibiting stem cell characteristics in both somite-derived and branchiomeric muscles.

BMP signalling permits population expansion by preventing premature myogenic differentiation in muscle satellite cells

Satellite cells are the resident stem cells of adult skeletal muscle, supplying myonuclei for homoeostasis, hypertrophy and repair. In this study, we have examined the role of bone morphogenetic protein (BMP) signalling in regulating satellite cell function. Activated satellite cells expressed BMP receptor type 1A (BMPR-1A/Alk-3) and contained phosphorylated Smad proteins, indicating that BMP signalling is operating during proliferation. Indeed, exogenous BMP4 stimulated satellite cell division and inhibited myogenic differentiation. Conversely, interfering with the interactions between BMPs and their receptors by the addition of either the BMP antagonist Noggin or soluble BMPR-1A fragments, induced precocious differentiation. Similarly, blockade of BMP signalling by siRNA-mediated knockdown of BMPR-1A, disruption of the intracellular pathway by either Smad5 or Smad4 knockdown or inhibition of Smad1/5/8 phosphorylation with Dorsomorphin, also caused premature myogenic differentiation. BMP signalling acted to inhibit the upregulation of genes associated with differentiation, in part, through regulating Id1. As satellite cells differentiated, Noggin levels increased to antagonise BMP signalling, since Noggin knockdown enhanced proliferation and impeded myoblast fusion into large multinucleated myotubes. Finally, interference of normal BMP signalling after muscle damage in vivo perturbed the regenerative process, and resulted in smaller regenerated myofibres. In conclusion, BMP signalling operates during routine satellite cell function to help coordinate the balance between proliferation and differentiation, before Noggin is activated to antagonise BMPs and facilitate terminal differentiation.

Further Characterisation of the Molecular Signature of Quiescent and Activated Mouse Muscle Satellite Cells

Satellite cells are the resident stem cells of adult skeletal muscle. To date though, there is a paucity of native markers that can be used to easily identify quiescent satellite cells, with Pax7 probably being the best that is currently available. Here we have further characterized a number of recently described satellite cell markers, and also describe novel ones. Caveolin-1, integrin α7 and the calcitonin receptor proved reliable markers for quiescent satellite cells, being expressed by all satellite cells identified with Pax7. These three markers remained expressed as satellite cells were activated and underwent proliferation. The nuclear envelope proteins lamin A/C and emerin, mutations in which underlie Emery-Dreifuss muscular dystrophy, were also expressed in both quiescent and proliferating satellite cells. Conversely, Jagged-1, a Notch ligand, was not expressed in quiescent satellite cells but was induced upon activation. These findings further contribute to defining the molecular signature of muscle satellite cells.

β-catenin promotes self-renewal of skeletal-muscle satellite cells

Satellite cells are the resident stem cells of adult skeletal muscle. As with all stem cells, how the choice between self-renewal or differentiation is controlled is central to understanding their function. Here, we have explored the role of β-catenin in determining the fate of myogenic satellite cells. Satellite cells express β-catenin, and expression is maintained as they activate and undergo proliferation. Constitutive retroviral-driven expression of wild-type or stabilised β-catenin results in more satellite cells expressing Pax7 without any MyoD – therefore, adopting the self-renewal pathway, with fewer cells undergoing myogenic differentiation. Similarly, preventing the degradation of endogenous β-catenin by inhibiting GSK3β activity also results in more Pax7-positive–MyoD-negative (Pax7+MyoD–) satellite-cell progeny. Consistent with these observations, downregulation of β-catenin using small interfering RNA (siRNA) reduced the proportion of satellite cells that express Pax7 and augmented myogenic differentiation after mitogen withdrawal. Since a dominant-negative version of β-catenin had the same effect as silencing β-catenin using specific siRNA, β-catenin promotes self-renewal via transcriptional control of target genes. Thus, β-catenin signalling in proliferating satellite cells directs these cells towards the self-renewal pathway and, so, contributes to the maintenance of this stem-cell pool in adult skeletal muscle.

Slow-dividing satellite cells retain long-term self-renewal ability in adult muscle

Satellite cells are muscle stem cells that have important roles in postnatal muscle growth and adult muscle regeneration. Although fast- and slow-dividing populations in activated satellite cells have been observed, the functional differences between them remain unclear. Here we elucidated the relationship between proliferation behaviour and satellite cell function. To assess the frequency of cell division, satellite cells isolated from mouse EDL muscle were labelled with the fluorescent dye PKH26, stimulated to proliferate and then sorted by FACS. The vast majority of activated satellite cells were PKH26low fast-dividing cells, whereas PKH26high slow-dividing cells were observed as a minority population. The fast-dividing cells generated a higher number of differentiated and self-renewed cells compared with the slow-dividing cells. However, cells derived from the slow-dividing population formed secondary myogenic colonies when passaged, whereas those from the fast-dividing population rapidly underwent myogenic differentiation without producing self-renewing cells after a few rounds of cell division. Furthermore, slow-dividing cells transplanted into injured muscle extensively contributed to muscle regeneration in vivo. Id1, a HLH protein, was expressed by all activated satellite cells, but the expression level varied within the slow-dividing cell population. We show that the slow-dividing cells retaining long-term self-renewal ability are restricted to an undifferentiated population that express high levels of Id1 protein (PKH26highId1high population). Finally, genome-wide gene expression analysis described the molecular characteristics of the PKH26highId1high population. Taken together, our results indicate that undifferentiated slow-dividing satellite cells retain stemness for generating progeny capable of long-term self-renewal, and so might be essential for muscle homeostasis throughout life.

Constitutively and highly expressed Oryza sativa polyamine oxidases localize in peroxisomes and catalyze polyamine back conversion.

Polyamine oxidases (PAOs) are FAD-dependent enzymes involved in polyamine (PA) catabolism. Recent studies have revealed that plant PAOs are not only active in the terminal catabolism of PAs as demonstrated for maize apoplastic PAO but also in a polyamine back-conversion pathway as shown for most Arabidopsis PAOs. We have characterized Oryza sativaPAOs at molecular and biochemical levels. The rice genome contains 7 PAO isoforms that are termed OsPAO1 to OsPAO7. Of the seven PAOs, OsPAO3, OsPAO4, and OsPAO5 transcripts were most abundant in 2-week-old seedlings and mature plants, while OsPAO1, OsPAO2, OsPAO6, and OsPAO7 were expressed at very low levels with different tissue specificities. The more abundantly expressed PAOs—OsPAO3, OsPAO4, and OsPAO5—were cloned, and their gene products were produced in Escherichia coli. The enzymatic activities of the purified OsPAO3 to OsPAO5 proteins were examined. OsPAO3 favored spermidine (Spd) as substrate followed by thermospermine (T-Spm) and spermine (Spm) and showed a full PA back-conversion activity. OsPAO4 substrate specificity was similar to that of OsPAO5 preferring Spm and T-Spm but not Spd. Those enzymes also converted Spm and T-Spm to Spd, again indicative of PA back-conversion activities. Lastly, we show that OsPAO3, OsPAO4, and OsPAO5 are localized in peroxisomes. Together, these data revealed that constitutively and highly expressed O. sativa PAOs are localized in peroxisomes and catalyze PA back-conversion processes.

Notch2 negatively regulates myofibroblastic differentiation of myoblasts.

Myofibroblasts are one of the key cellular components involved in fibrosis of skeletal muscle as well as in other tissues. Transforming growth factor‐β1 (TGF‐β1) stimulates differentiation of mesenchymal cells into myofibroblasts, but little is known about the regulatory mechanisms of myofibroblastic differentiation. Since Notch2 was shown to be downregulated in TGF‐β1‐induced non‐muscle fibrogenic tissue, we investigated whether Notch2 also has a distinctive role in myofibroblastic differentiation of myogenic cells induced by TGF‐β1. TGF‐β1 treatment of C2C12 myoblasts led to expression of myofibroblastic marker α‐smooth muscle actin (α‐SMA) and collagen I with concomitant downregulation of Notch2 expression. Overexpression of active Notch2 inhibited TGF‐β1‐induced expression of α‐SMA and collagen I. Interestingly, transient knockdown of Notch2 by siRNA in C2C12 myoblasts and primary cultured muscle‐derived progenitor cells resulted in differentiation into myofibroblastic cells expressing α‐SMA and collagen I without TGF‐β1 treatment. Furthermore, we found Notch3 was counter‐regulated by Notch2 in C2C12 cells. These findings suggest that Notch2 is inhibiting differentiation of myoblasts into myofibroblasts with downregulation of Notch3 expression. J. Cell. Physiol. 210: 358–369, 2007.

Six family genes control the proliferation and differentiation of muscle satellite cells.

Muscle satellite cells are essential for muscle growth and regeneration and their morphology, behavior and gene expression have been extensively studied. However, the mechanisms involved in their proliferation and differentiation remain elusive. Six1 and Six4 proteins were expressed in the nuclei of myofibers of adult mice and the numbers of myoblasts positive for Six1 and Six4 increased during regeneration of skeletal muscles. Six1 and Six4 were expressed in quiescent, activated and differentiated muscle satellite cells isolated from adult skeletal muscle. Overexpression of Six4 and Six5 repressed the proliferation and differentiation of satellite cells. Conversely, knockdown of Six5 resulted in augmented proliferation, and that of Six4 inhibited differentiation. Muscle satellite cells isolated from Six4(+/-)Six5(-/-) mice proliferated to higher cell density though their differentiation was not altered. Meanwhile, overproduction of Six1 repressed proliferation and promoted differentiation of satellite cells. In addition, Six4 and Six5 repressed, while Six1 activated myogenin expression, suggesting that the differential regulation of myogenin expression is responsible for the differential effects of Six genes. The results indicated the involvement of Six genes in the behavior of satellite cells and identified Six genes as potential target for manipulation of proliferation and differentiation of muscle satellite cells for therapeutic applications.

Knockdown of hypoxia-inducible factor-1α by siRNA inhibits C2C12 myoblast differentiation

We analyzed the role of Hypoxia‐inducible factor (HIF)‐1α in myoblast differentiation by examining the expression and regulation of HIF‐1α in proliferating and differentiating C2C12 myoblast, and by knocking down HIF‐1α of C2C12 myoblasts with small interfering RNA (siRNA), given that HIF‐1α has been shown to be involved in differentiative process in non‐muscle tissues. Although HIF‐1α mRNA was constantly expressed in C2C12 myoblasts both under growth and differentiating phase, HIF‐1α protein was hardly detectable in the growth phase but became detectable only during myogenic differentiation even under normoxia. During early stage of C2C12 myogenesis, HIF‐1α accumulated in the nuclei of myogenin‐positive myoblasts. The inhibition of proteasome in the growth phase led to HIF‐1α protein accumulation, whereas in the differentiation phase the inhibition of Hsp90, which stabilizes HIF‐1α, suppressed HIF‐1α accumulation. Therefore, we suggest that the level of HIF‐1α protein expression is regulated by a proteasome‐and chaperon‐dependent pathway in C2C12 myoblast. Knockdown of HIF‐1α effectively blocked myotube formation and myosin heavy chain (MHC) expression. Finally, HIF‐1α expression in vivo was confirmed in the regenerative muscle tissue of mice after eccentric exercise. We conclude that HIF‐1α is required for C2C12 myogenesis in vitro, and suggest that HIF‐1α may have an essential role in regenerative muscle tissue in vivo. J. Cell. Biochem. 98: 642–649, 2006.

Suppression of BMP-Smad signaling axis-induced osteoblastic differentiation by small C-terminal domain phosphatase 1, a Smad phosphatase.

Bone morphogenetic proteins (BMPs) induce osteoblastic differentiation in myogenic cells via the phosphorylation of Smads. Two types of Smad phosphatases--small C-terminal domain phosphatase 1 (SCP1) and protein phosphatase magnesium-dependent 1A--have been shown to inhibit BMP activity. Here, we report that SCP1 inhibits the osteoblastic differentiation induced by BMP-4, a constitutively active BMP receptor, and a constitutively active form of Smad1. The phosphatase activity of SCP1 was required for this suppression, and the knockdown of SCP1 in myoblasts stimulated the osteoblastic differentiation induced by BMP signaling. In contrast to protein phosphatase magnesium-dependent 1A, SCP1 did not reduce the protein levels of Smad1 and failed to suppress expression of the Id1, Id2, and Id3 genes. Runx2-induced osteoblastic differentiation was suppressed by SCP1 without affecting the transcriptional activity or phosphorylation levels of Runx2. Taken together, these findings suggest that SCP1 may inhibit the osteoblastic differentiation induced by the BMP-Smad axis via Runx2 by suppressing downstream effector(s).