Naohiro Hashimoto

Osteogenic properties of human myogenic progenitor cells

Here, we identified human myogenic progenitor cells coexpressing Pax7, a marker of muscle satellite cells and bone-specific alkaline phosphatase, a marker of osteoblasts, in regenerating muscle. To determine whether human myogenic progenitor cells are able to act as osteoprogenitor cells, we cultured both primary and immortalized progenitor cells derived from the healthy muscle of a nondystrophic woman. The undifferentiated myogenic progenitors spontaneously expressed two osteoblast-specific proteins, bone-specific alkaline phosphatase and Runx2, and were able to undergo terminal osteogenic differentiation without exposure to an exogenous inductive agent such as bone morphogenetic proteins. They also expressed the muscle lineage-specific proteins Pax7 and MyoD, and lost their osteogenic characteristics in association with terminal muscle differentiation. Both myoblastic and osteoblastic properties are thus simultaneously expressed in the human myogenic cell lineage prior to commitment to muscle differentiation. In addition, C3 transferase, a specific inhibitor of Rho GTPase, blocked myogenic but not osteogenic differentiation of human myogenic progenitor cells. These data suggest that human myogenic progenitor cells retain the capacity to act as osteoprogenitor cells that form ectopic bone spontaneously, and that Rho signaling is involved in a critical switch between myogenesis and osteogenesis in the human myogenic cell lineage.

Role of c-mos proto-oncogene product in the regulation of mouse oocyte maturation.

The c-mos proto-oncogene product (Mos) is essential for the initiation of oocyte maturation, for the suppression of DNA synthesis during meiosis, and for the second metaphase arrest in Xenopus. To clarify the function of Mos in mice, c-mos-deficient mice were generated by gene targeting. We cultured oocytes from c-mos-deficient females to determine the role of Mos in oocyte maturation. c-mos-deficient oocytes matured normally to the second metaphase, but were activated without fertilization. Thus, prevention of parthenogenetic activation might be an ultimate biological function of Mos in animal oocytes.

Down-regulation of an ankyrin repeat-containing protein, V-1, during skeletal muscle differentiation and its re-expression in the regenerative process of muscular dystrophy

Using Western blot analysis and immunohistochemical methods, we examined the expression of V-1, a member of the ankyrin repeat-containing protein family, during differentiation and regeneration of skeletal muscle. The expression of V-1 was high in cultured myoblasts and decreased during their differentiation into myotubes, while high expression was maintained when muscle differentiation was inhibited by treatment with basic fibroblast growth factor. Down-regulation of V-1 also occurred during in vivo muscle differentiation from embryonic to postnatal stages, reaching an undetectable level in mature skeletal muscle. In contrast, strong V-1 immunoreactivity was detected again in myoblasts and regenerating muscle fibers with a small diameter, which were observed in Duchenne muscular dystrophy and its animal model, mdx mouse. Thus, it seems that V-1 is a good marker for early stage of muscle regeneration and changes of its expression suggest that V-1 plays a role in prenatal muscle differentiation and postnatal muscle regeneration.

Differential contribution of Mr 120 kDa rasGTPase-activating protein and neurofibromatosis type 1 gene product during the transition from growth phase to arrested state in human fibroblasts accompanied by a unique rasGTPase-activating active

Using octyl glucoside‐solubilized cell extracts from human fibroblasts during growth phase to G0/G1 arrest state, we found that while the number of M r, 120 kDa rasGTPase‐activating protein (p120GAP) molecules per cell decreases to half its original levels, the amount of neurofibromatosis type 1 gene product (NF1, neurofibromin) remains constant during the transition. The contribution of pl20GAP to the total rasGTPase‐activating (rasGA) activity in growing cells was found to be larger than that observed in arrested cells (84% vs 53%). On the other hand, NF1 contributes less than 15% of the total rasGA activity in either extract. These results indicate that the qualitative changes occur in the contributors to rasGA activity during transition. They also suggest that a unique rasGA activity exists in the arrested cells, which was obtained separatedly from both pl20GAP and NF1 by heparin‐Sepharose column chromatography.

Dnmt3a Regulates Proliferation of Muscle Satellite Cells via p57Kip2.

Cell differentiation status is defined by the gene expression profile, which is coordinately controlled by epigenetic mechanisms. Cell type-specific DNA methylation patterns are established by chromatin modifiers including de novo DNA methyltransferases, such as Dnmt3a and Dnmt3b. Since the discovery of the myogenic master gene MyoD, myogenic differentiation has been utilized as a model system to study tissue differentiation. Although knowledge about myogenic gene networks is accumulating, there is only a limited understanding of how DNA methylation controls the myogenic gene program. With an aim to elucidate the role of DNA methylation in muscle development and regeneration, we investigate the consequences of mutating Dnmt3a in muscle precursor cells in mice. Pax3 promoter-driven Dnmt3a-conditional knockout (cKO) mice exhibit decreased organ mass in the skeletal muscles, and attenuated regeneration after cardiotoxin-induced muscle injury. In addition, Dnmt3a-null satellite cells (SCs) exhibit a striking loss of proliferation in culture. Transcriptome analysis reveals dysregulated expression of p57Kip2, a member of the Cip/Kip family of cyclin-dependent kinase inhibitors (CDKIs), in the Dnmt3a-KO SCs. Moreover, RNAi-mediated depletion of p57Kip2 replenishes the proliferation activity of the SCs, thus establishing a role for the Dnmt3a-p57Kip2 axis in the regulation of SC proliferation. Consistent with these findings, Dnmt3a-cKO muscles exhibit fewer Pax7+ SCs, which show increased expression of p57Kip2 protein. Thus, Dnmt3a is found to maintain muscle homeostasis by epigenetically regulating the proliferation of SCs through p57Kip2.

Proteolytic processing regulates pathological accumulation in dentatorubral‐pallidoluysian atrophy

Dentatorubral‐pallidoluysian atrophy is caused by polyglutamine (polyQ) expansion in atrophin‐1 (ATN1). Recent studies have shown that nuclear accumulation of ATN1 and cleaved fragments with expanded polyQ is the pathological process underlying neurodegeneration in dentatorubral‐pallidoluysian atrophy. However, the mechanism underlying the proteolytic processing of ATN1 remains unclear. In the present study, we examined the proteolytic processing of ATN1 aiming to understand the mechanisms of ATN1 accumulation with polyQ expansion. Using COS‐7 and Neuro2a cells that express the ATN1 gene, in which ATN1 was accumulated by increasing the number of polyQs, we identified a novel C‐terminal fragment containing a polyQ tract. The mutant C‐terminal fragment with expanded polyQ selectively accumulated in the cells, and this was also demonstrated in the brain tissues of patients with dentatorubral‐pallidoluysian atrophy. Immunocytochemical and biochemical studies revealed that full‐length ATN1 and C‐terminal fragments displayed individual localization. The mutant C‐terminal fragment was preferentially found in the cytoplasmic membrane/organelle and insoluble fractions. Accordingly, it is assumed that the proteolytic processing of ATN1 regulates the localization of C‐terminal fragments. Accumulation of the C‐terminal fragment was enhanced by inhibition of caspases in the cytoplasm of COS‐7 cells. Collectively, these results suggest that the C‐terminal fragment plays a principal role in the pathological accumulation of ATN1 in dentatorubral‐pallidoluysian atrophy.

Differential impact of the Bisphosphonate Alendronate on undifferentiated and terminally differentiated human myogenic cells

Alendronate, a nitrogen‐containing bisphosphonate, is well established as a treatment for osteoporosis through regulation of osteoclast activity. Previously, the pharmacological effects of bisphosphonates on cells outside the bone environment have been considered irrelevant because of the bone‐targeting property of bisphosphonates. However, the chronic effects of bisphosphonates on tissue‐neighbouring bone, in particular skeletal muscles, should not be ignored because patients are treated with bisphosphonates for long periods.

Pro‐Insulin‐Like Growth Factor‐II Ameliorates Age‐Related Inefficient Regenerative Response by Orchestrating Self‐Reinforcement Mechanism of Muscle Regeneration

Sarcopenia, age‐related muscle weakness, increases the frequency of falls and fractures in elderly people, which can trigger severe muscle injury. Rapid and successful recovery from muscle injury is essential not to cause further frailty and loss of independence. In fact, we showed insufficient muscle regeneration in aged mice. Although the number of satellite cells, muscle stem cells, decreases with age, the remaining satellite cells maintain the myogenic capacity equivalent to young mice. Transplantation of young green fluorescent protein (GFP)‐Tg mice‐derived satellite cells into young and aged mice revealed that age‐related deterioration of the muscle environment contributes to the decline in regenerative capacity of satellite cells. Thus, extrinsic changes rather than intrinsic changes in satellite cells appear to be a major determinant of inefficient muscle regeneration with age. Comprehensive protein expression analysis identified a decrease in insulin‐like growth factor‐II (IGF‐II) level in regenerating muscle of aged mice. We found that pro‐ and big‐IGF‐II but not mature IGF‐II specifically express during muscle regeneration and the expressions are not only delayed but also decreased in absolute quantity with age. Supplementation of pro‐IGF‐II in aged mice ameliorated the inefficient regenerative response by promoting proliferation of satellite cells, angiogenesis, and suppressing adipogenic differentiation of platelet derived growth factor receptor (PDGFR)α+ mesenchymal progenitors. We further revealed that pro‐IGF‐II but not mature IGF‐II specifically inhibits the pathological adipogenesis of PDGFRα+ cells. Together, these results uncovered a distinctive pro‐IGF‐II‐mediated self‐reinforcement mechanism of muscle regeneration and suggest that supplementation of pro‐IGF‐II could be one of the most effective therapeutic approaches for muscle injury in elderly people. Stem Cells 2015;33:2456—2468

Cancer cachexia causes skeletal muscle damage via transient receptor potential vanilloid 2-independent mechanisms, unlike muscular dystrophy

Muscle wasting during cancer cachexia contributes to patient morbidity. Cachexia‐induced muscle damage may be understood by comparing its symptoms with those of other skeletal muscle diseases, but currently available data are limited.

Expression of an Acyl-CoA Synthetase, Lipidosin, in Astrocytes of the Murine Brain and Its Up-Regulation During Remyelination Following Cuprizone-Induced Demyelination

Lipidosin is an 80‐kDa protein with long‐chain acyl‐CoA synthetase activity expressed in the brain, adrenal gland, testis, and ovary, which are selectively damaged in X‐linked adrenoleukodystrophy (X‐ALD). Western blot analysis of the cerebrum and cerebellum revealed a gradual increase in the expression of lipidosin postnatally. Light microscopic immunohistochemistry using a panel of specific monoclonal antibodies showed that the lipidosin‐immunopositive cells were ubiquitously distributed in the brain and were denser in the gray matter than in the white matter. Lipidosin immunoreactivity was colocalized with GFAP immunoreactivity but not with ubiquitin C‐terminal hydrolase 1 (= PGP9.5) immunoreactivity, a neuronal marker, and lipidosin‐producing cells detected by an antisense probe specific for lipidosin mRNA were also GFAP immunopositive. These data together with Western blot analysis of primary cultured astrocytes indicate that lipidosin is expressed in astrocytes. Immunoelectron microscopic analysis revealed that lipidosin immunoreactivity was widely distributed from perivascular endfeet to perisynaptic processes without being limited to peroxisomes. Lipidosin immunoreactivity was greatly increased in astrocytes in the area of remyelination following experimental demyelination induced by the administration of cuprizone to mice. These data suggest that lipidosin was involved in fatty acid metabolism during reconstruction of the myelin sheath.